CN109464478A - A kind of sea-buckthorn granule, preparation method and its detection method - Google Patents

A kind of sea-buckthorn granule, preparation method and its detection method Download PDF

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Publication number
CN109464478A
CN109464478A CN201811580354.2A CN201811580354A CN109464478A CN 109464478 A CN109464478 A CN 109464478A CN 201811580354 A CN201811580354 A CN 201811580354A CN 109464478 A CN109464478 A CN 109464478A
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buckthorn
sea
granule
solution
reference substance
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陈钟
郭静
王飞
刘红
王世静
侯有玉
邢会香
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Qinghai Pulante Pharmaceutical Co Ltd
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Qinghai Pulante Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • 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
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—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
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

Abstract

The present invention relates to a kind of sea-buckthorn granule, preparation method and its detection methods, the granule is made of hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, auxiliary material, after granule is made in sea-buckthorn by the present invention, doctor is facilitated to carry out diagnosis and treatment, is added and subtracted with card；Sea-buckthorn simple extracts the volatile oil in sea-buckthorn by steam distillation, and volatile oil is included with beta-cyclodextrin coprecipitation, and dregs of a decoction ethyl alcohol extracts, and extracts effective component all sufficiently, pharmacological property is strong, drug effect is high.Quality determining method include check, with control substance of Rutin be control according to UV-VIS spectrophotometry survey general flavone content, with Isorhamnetin reference substance be control high performance liquid chromatography assay, with Isorhamnetin reference substance, Quercetin reference substance be control high performance liquid chromatography identification.

Description

A kind of sea-buckthorn granule, preparation method and its detection method
Technical field
The present invention relates to Chinese medicinal granule, belong to the field of Chinese medicines, and in particular to a kind of sea-buckthorn granule, preparation method and Its detection method.
Background technique
Sea-buckthorn is medicinal and edible plant, the nutrition rich in of the root of sea-buckthorn, stem, leaf, flower, fruit, especially sea buckthorn fruit Substance and bioactive substance can be widely applied to many of the national economy such as food, medicine, light industry, space flight, agriculture and animal husbandry fishery Field.Sea buckthorn fruit is used as medicine with cough-relieving
The effect of resolving sputum, stomach strengthening and digestion promoting, promoting blood circulation to remove blood stasis.Modern medicine study, sea-buckthorn can reduce cholesterol, allevating angina pectoris Breaking-out, there are also the effects for preventing and treating coronary atherosclerotic heart disease.
Single medicinal material granule is to use the traditional Chinese medicine medicine materical crude slice for meeting concocted specification as raw material, through modern pharmaceutical technology It extracts, is concentrated, separates, dry, pelletize, packing refined pure Chinese medicine product line.It ensure that the complete of the former prepared slices of Chinese crude drugs Portion's feature, can satisfy doctor carry out diagnosis and treatment, with card add and subtract, pharmacological property is strong, drug effect is high, simultaneously again have do not need decoct, Directly take after mixing it with water, dose is few, effect is rapid, composition is complete, curative for effect, safety and sanitation, carry save it is convenient, be easy to modulate and Many advantages, such as being suitble to industrialized production.
Sea-buckthorn is the medicinal material that Qinghai Pulante Medicine Co., Ltd's Chinese-caterpillar-fungus lung-clearing capsule uses, and former Chinese patent drug also has preferably Curative effect, but doctor cannot add and subtract with disease, therefore inventor is to preferably utilize sea-buckthorn, and preferably be examined to its quality It surveys, has developed easy to use, the eutherapeutic sea-buckthorn granule of one kind and its detection method.
Summary of the invention
The object of the present invention is to provide a kind of sea-buckthorn granules.
The granule is made of hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, auxiliary material.
Sea-buckthorn granule of the present invention is made of following composition: 120-200 parts of hippophae rhamnoides xeraphium, sand 0.1-0.7 parts of spine volatile oil clathrate compound, 0.5-4 parts of silica, 0.2-0.8 parts of magnesium stearate.
Preferably, sea-buckthorn granule of the present invention is made of following composition: hippophae rhamnoides xeraphium 140-180 Part, 0.2-0.5 parts of sea-buckthorn volatile oil clathrate compound, 0.8-3.5 parts of silica, 0.3-0.7 parts of magnesium stearate.
It is further preferred that sea-buckthorn granule of the present invention is made of following composition: hippophae rhamnoides xeraphium 160 parts, 0.3 part of sea-buckthorn volatile oil clathrate compound, silica 1 part, 0.4 part of magnesium stearate.
It is a further object of the present invention to provide a kind of sea-buckthorn granule preparation methods.
Granule of the present invention is made of following preparation method:
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatilization in sea-buckthorn by steam distillation It is spare to collect volatile oil for oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. the medicinal material 1. step is extracted after is added the ethyl alcohol that 10-15 times is measured 60%-85% and extracts 1-2 times, each 1-2 Hour, extracting solution filters, and filtrate is spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated under reduced pressure into 1.05- at a temperature of 60 DEG C 1.15 clear creams, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165- 195 DEG C, leaving air temp: 80-105 DEG C, obtain hippophae rhamnoides xeraphium；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 5h at 60 DEG C Double sieve methods carry out whole grains, pack to get.
It is a further object of the present invention to provide a kind of detection method of sea-buckthorn granule, the quality of the granule Detection method include check, with control substance of Rutin be control according to UV-VIS spectrophotometry survey general flavone content, with different mouse Li Su reference substance is the high performance liquid chromatography assay of control, is control with Isorhamnetin reference substance, Quercetin reference substance High performance liquid chromatography identify.
UV-VIS spectrophotometry surveys general flavone content method in granule quality determining method of the present invention Are as follows:
The preparation of test solution: taking sea-buckthorn granule 0.8-1.2g, and ethyl alcohol 30ml is added, and is heated to reflux 40-70 points Clock, filtration, residue are added 60% ethyl alcohol 20-30ml, 45-50 DEG C ultrasound 30-40 minutes, filter, residue is added 60% again Ethyl alcohol 20-30ml, 40-50 DEG C ultrasound 30-40 minutes, filtration merges ethanol solution three times, sets in evaporating dish and be evaporated, residual Slag adds methanol to dissolve, and is transferred in 100mL volumetric flask, adds methanol to scale, shakes up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 18-22mg, accurately weighed, sets in 50ml measuring bottle, adds 55%-65% Appropriate amount of ethanol, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55%-65% ethyl alcohol to scale, shakes up；22-27ml is measured, is set In 50ml measuring bottle, it is diluted with water to scale, is shaken up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 25-35% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% nitre Sour aluminum solutions 0.8-12ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 8-12ml, then plus 30% ethyl alcohol to scale, shake It is even, it places 10-20 minutes, using corresponding reagent as blank, according to UV-VIS spectrophotometry, is measured at the wavelength of 500nm Absorbance, using absorbance as ordinate, concentration is abscissa, draws standard curve.
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
In granule quality determining method of the present invention with Isorhamnetin reference substance be control high-efficient liquid phase color Spectrometry content assaying method are as follows:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method difference is accurate to draw reference substance and each 5-10 μ l of test solution, injects liquid chromatograph, measures, i.e., ?.
In granule quality determining method of the present invention with Isorhamnetin reference substance, Quercetin reference substance be control High performance liquid chromatography discrimination method are as follows:
Isorhamnetin reference substance, Quercetin reference substance are taken, adding methanol that every 1ml is made, respectively the mixing containing 0.8-1.2mg is molten Liquid, as reference substance solution；High performance liquid chromatography using claim 8 test solution as test solution, according to claim 8 Condition sample introduction, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
Hippophae rhamnoides xeraphium quality requirement of the present invention is moisture≤5%, should be without breeze and magma.
The preferred detection method of the present invention is as follows:
The quality determining method of granule of the present invention include check, with control substance of Rutin be control according to purple Outside-visible spectrophotometry survey general flavone content, with Isorhamnetin reference substance be compare high performance liquid chromatography assay, Identified with the high performance liquid chromatography that Isorhamnetin reference substance, Quercetin reference substance are control.
UV-VIS spectrophotometry surveys general flavone content method in granule quality determining method of the present invention Are as follows:
The preparation of test solution: taking sea-buckthorn granule powder 1g, and ethyl alcohol 30ml is added, is heated to reflux 2 hours, filters Cross, residue is added 60% ethyl alcohol 30ml, 50 DEG C ultrasound 30 minutes, filtration, residue is added 60% ethyl alcohol 30ml again, 50 DEG C Ultrasound 30 minutes, filtration, merges ethanol solution three times, sets in evaporating dish and be evaporated, residue adds methanol to dissolve, and is transferred to 10mL amount In bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 18-22mg, accurately weighed, sets in 50ml measuring bottle, adds 55%-65% Appropriate amount of ethanol, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55%-65% ethyl alcohol to scale, shakes up；22-27ml is measured, is set In 50ml measuring bottle, it is diluted with water to scale, is shaken up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 30% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 6 minutes, then plus 10% aluminum nitrate Solution 1ml, shakes up, and places 6 minutes；Adding sodium hydroxide test solution 10ml, then plus 30% ethyl alcohol to scale, shake up, place 15 minutes, Using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured at the wavelength of 500nm, is vertical with absorbance Coordinate, concentration are abscissa, draw standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
In granule quality determining method of the present invention with Isorhamnetin reference substance be control high-efficient liquid phase color Spectrometry content assaying method are as follows:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 10 μ l of test solution, injects liquid chromatograph, measurement to get；
In granule quality determining method of the present invention with Isorhamnetin reference substance, Quercetin reference substance be control High performance liquid chromatography discrimination method are as follows:
Isorhamnetin reference substance, Quercetin reference substance are taken, adds methanol that every 1ml is made respectively containing the mixed solution of 1mg, as right According to product solution；Using claim 8 test solution as test solution, high-efficient liquid phase chromatogram condition sample introduction according to claim 8, In test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
Hippophae rhamnoides xeraphium quality requirement of the present invention is moisture≤5%, should be without breeze and magma.
" part " of the present invention can be the unit well known in the art such as kg, g.
The invention has the following advantages that
1, sea-buckthorn is the medicinal material that uses of Qinghai Pulante Medicine Co., Ltd's Chinese-caterpillar-fungus lung-clearing capsule, former Chinese patent drug also have compared with Good curative effect, but doctor cannot add and subtract with disease, after being made into granule, doctor is facilitated to carry out diagnosis and treatment, added and subtracted with card；
2, the present invention extracts sea-buckthorn simple by spray drying, and volatile oil takes co-precipitation legal system with beta-cyclodextrin At effective component is extracted sufficiently, and pharmacological property is strong, drug effect is high.
3, the quality determining method of the granule of the invention described in granule is pair including checking, with control substance of Rutin According to according to UV-VIS spectrophotometry survey general flavone content, with Isorhamnetin reference substance be compare high performance liquid chromatography Assay is identified with the high performance liquid chromatography that Isorhamnetin reference substance, Quercetin reference substance are control, can be convenient and efficient, Efficiently comprehensively control the quality of granule.
4, the quality determining method of granule described in sea-buckthorn granule of the present invention, optimizes in existing quality standard About the method for determination of total flavonoids, the new method for exploring a kind of test sample processing is simple, and this method is easy to operate, at A test solution is managed, is able to satisfy that flavones content is fixed, identification of Isorhamnetin assay and Isorhamnetin, Quercetin.
5, general flavone (in terms of rutin) the content method verifying in this method detection sea-buckthorn granule: rutin recurrence side Journey: y=0.0253x-0.0004, R2=1, the range of linearity are as follows: 0~48.264 μ g/ml；Instrument precision is good, RSD%= 0.9776%；The good RSD%=1.5679% of repeatability；Rutin is relatively stable in for 24 hours, and stability RSD% is 1.2565%, Show that the measuring method of this rutin is good, this method meets the verifying of drug standard analysis method and requires.
6, using detection method of the invention, Isorhamnetin compares linear equation are as follows: Y=2.0598X+0.826, R2=1； Isorhamnetin sample volume is in good linear relationship in 50.07~1001.4 μ g ranges, and accuracy of instrument is high, and RSD% value is 1.454%, repeated RSD value be 1.9196%, use the method for the present invention handle test solution stability RSD% for 1.7351%, high using the response of the method for the present invention test sample, as a result accurately and reliably, this method is easy, reproducible, can have The quality of effect ground control sea-buckthorn granule.
Detailed description of the invention:
The canonical plotting of Fig. 1 rutin control
The canonical plotting of Fig. 2 Isorhamnetin control
Experimental example 1
(1) sea-buckthorn granule is compared with sea-buckthorn medicine materical crude slice thin-layer chromatography
Sea-buckthorn granule 1g is taken to set in stuffed conical flask, ethyl alcohol 50ml is added in precision, and it is small to be heated to reflux 1 for weighed weight When, it lets cool, then weighed weight, shakes up, filter.Precision measures subsequent filtrate 25ml, sets in stuffed conical flask, adds hydrochloric acid 3.5ml, Heating hydrolysis 1 hour, cools down immediately, is transferred in 50ml measuring bottle, wash container with ethanol in proper amount, washing lotion is incorporated in 75 DEG C of water-baths In same measuring bottle, adds ethyl alcohol to scale, shake up, filter, take subsequent filtrate to get water 20ml is added, low-grade fever makes to dissolve, filtration, filtrate It is extracted 2 times, each 20ml with ether shaking, merges ether extracted liquid, volatilize ether, residue adds ethyl acetate 1ml to make to dissolve, and makees For test solution.Separately take with sea-buckthorn medicine materical crude slice 5g, be made in the same way of contrast solution.According to thin-layered chromatography (" Chinese Pharmacopoeia " 2015 One annex of version) test, each 2 μ l of above two solution is drawn, is put respectively in the same silica G for containing the preparation of 3% sodium acetate solution On lamellae, with toluene-ethyl acetate-formic acid (5: 2: 1) for solvent, it is unfolded, takes out, dry, spray is tried with alchlor Liquid is set and is inspected under ultraviolet lamp (365nm).In sample chromatogram, at the position corresponding to the chromatogram of the reference substance, same color is shown Fluorescence spot.
(2) sea-buckthorn granule is compared with sea-buckthorn medicine materical crude slice thin-layer sample infrared finger print atlas
It takes sea-buckthorn granule and prepares the sea-buckthorn medicine materical crude slice of sea-buckthorn granule same batch, crush, sieving carries out infrared It detects, as a result the sample infrared finger print atlas peak shape of sea-buckthorn granule and sea-buckthorn medicine materical crude slice, peak position, relative peak intensities basic one It causes.
The results show that sea-buckthorn granule of the present invention is consistent with sea-buckthorn medicine materical crude slice ingredient, present invention process is reasonable.
Experimental example 2
1, experiment purpose: research sea-buckthorn medicine materical crude slice decocting liquid and granule general flavone content gap, so that it is determined that quality testing Feasibility, the consistency of method.
2, method:
[determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 0.8-1.2g, and ethyl alcohol 30ml is added, and is heated to reflux 40-70 points Clock, filtration, residue are added 60% ethyl alcohol 20-30ml, 45-50 DEG C ultrasound 30-40 minutes, filter, residue is added 60% again Ethyl alcohol 20-30ml, 40-50 DEG C ultrasound 30-40 minutes, filtration merges ethanol solution three times, sets in evaporating dish and be evaporated, residual Slag adds methanol to dissolve, and is transferred in 100mL volumetric flask, adds methanol to scale, shakes up, take subsequent filtrate, take as test solution Sea-buckthorn medicine materical crude slice decocting liquid micrometric measurement after concentration, with legal system available test sample solution；
The preparation of reference substance solution takes control substance of Rutin 18-22mg, accurately weighed, sets in 50ml measuring bottle, adds 55%-65% Appropriate amount of ethanol, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55%-65% ethyl alcohol to scale, shakes up；22-27ml is measured, is set In 50ml measuring bottle, it is diluted with water to scale, is shaken up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 25-35% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% nitre Sour aluminum solutions 0.8-12ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 8-12ml, then plus 30% ethyl alcohol to scale, shake It is even, it places 10-20 minutes, using corresponding reagent as blank, according to UV-VIS spectrophotometry, is measured at the wavelength of 500nm Absorbance, using absorbance as ordinate, concentration is abscissa, draws standard curve；
Measuring method precision measures above two each 3ml of test solution, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, Method under sighting target directrix curve preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test sample molten Liquid 3ml, in addition to sodium hydroxide test solution is not added, remaining is ibid operated, and as blank, is read in test solution from standard curve Weight containing rutin, calculate to get；
It the results are shown in Table 1:
Table 1: sea-buckthorn medicine materical crude slice and sea-buckthorn granule assay result table
Sample Content of the general flavone in terms of rutin
Sea-buckthorn medicine materical crude slice 1.82%
Sea-buckthorn granule 16.0864%
It is found that sea-buckthorn medicine materical crude slice assay result from table are as follows: 1.82%；Sea-buckthorn granule assay result is 16.0864%；Containing general flavone in terms of rutin (C27H30O16), sea-buckthorn granule 1g is equivalent to the equivalent of sea-buckthorn medicine materical crude slice 8.8g, Meet the equivalent that sea-buckthorn granule 1g is equivalent to sea-buckthorn medicine materical crude slice 7-12g, production technology is reasonable, while illustrating the present invention General flavone detection method it is feasible.
Experimental example 3
1, experiment purpose: research sea-buckthorn medicine materical crude slice decocting liquid and granule Isorhamnetin content difference are away from so that it is determined that quality is examined Feasibility, the consistency of survey method.
2, method:
[Isorhamnetin assay]
Isorhamnetin is measured according to " high performance liquid chromatography "；
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 0.4% phosphoric acid solution of methanol-is mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 10 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, must not be less than 0.70% containing Isorhamnetin (C16H12O7).
It the results are shown in Table 2:
Table 2: sea-buckthorn medicine materical crude slice and sea-buckthorn granule assay result table
Sample The content of Isorhamnetin
Sea-buckthorn medicine materical crude slice 0.14%
Sea-buckthorn granule 1.19%
It is found that sea-buckthorn medicine materical crude slice assay result from table are as follows: 1.82%；Sea-buckthorn granule assay result is 16.0864%；Containing Isorhamnetin (C16H12O7), sea-buckthorn granule 1g is equivalent to the equivalent of sea-buckthorn medicine materical crude slice 8.5g, meets husky Spine granule 1g is equivalent to the equivalent of sea-buckthorn medicine materical crude slice 7-12g, and production technology is reasonable, while illustrating total Huang of the invention The detection method of ketone is feasible.
The following experiment that is obtained by of optimal technical solution of the present invention obtains, and is described as follows:
Experimental material used in the present invention is the sea-buckthorn granule produced by Qinghai Pulante Medicine Co., Ltd；
Instrument includes:
High performance liquid chromatograph: (model: Waters e2695, producer: U.S. Waters is public for Waters e2695 series Department), including PDA diode array detector (model: Waters 2998, producer: Waters, US) and Empower work It stands (Waters, US)；
Chromatographic column: green hundred grass Ecosil 120-5-C18AQ Plus chromatographic column (4.6mm × 250mm, 5 μm)；
Balance: a ten thousandth balance (Sartorius BS224S)；
Ultrapure water system: (U.S. Millipore (Mi Libo) company)；
It is that domestic analysis is pure that agents useful for same of the present invention, which includes: methanol, ethyl acetate, phosphoric acid etc. with reagent,；HPLC first Alcohol, acetonitrile are chromatographically pure (German Merck company, Darmstadt, Gemany)；Water is ultrapure water (resistivity 8.2m Ω .cm)。
1, the preparation of test solution
(1) selection of Extraction solvent
By literature search, the Extraction solvent of sea-buckthorn often uses methanol, ethyl acetate, and the research of granule finger-print is common Solvent is ethyl alcohol, methanol, water.Therefore methanol, ethyl alcohol, water is respectively adopted as Extraction solvent to mention sea-buckthorn granule It takes, long due to being heated to reflux the time, sample dissolution is insufficient, therefore is ultrasonically treated again after using alcohol reflux.Sea-buckthorn is matched Square particle is to be concentrated by sea-buckthorn Aqueous extracts, therefore water solubility is strong, therefore reduce concentration of alcohol, is used as extracting solution using 60%, leads to More various solvent extractable matter chromatograms are crossed, the main chromatographic peak information of water extract is more, therefore selects 60% ethyl alcohol, 40-50 DEG C of ultrasound is that sea-buckthorn granule is fully dissolved out, and is studied liposoluble constituent therein.
Sample treatment: taking sea-buckthorn granule 0.8-1.2g, and ethyl alcohol 30ml is added, is heated to reflux 40-70 minutes, filters, Residue is added 60% ethyl alcohol 20-30ml, 45-50 DEG C ultrasound 30-40 minutes, filter, 60% ethyl alcohol 20- is added in residue again 30ml, 40-50 DEG C ultrasound 30-40 minutes, filtration, merge ethanol solution three times, set in evaporating dish and be evaporated, residue adds methanol molten Solution, is transferred in 100mL volumetric flask, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
(2) final extraction scheme
Sea-buckthorn granule 1g is taken, ethyl alcohol 30ml is added, is heated to reflux 50 minutes, is filtered, 60% ethyl alcohol is added in residue 30ml, 50 DEG C ultrasound 35 minutes, filtration, residue is added 60% ethyl alcohol 30ml again, 50 DEG C ultrasound 35 minutes, filter, merge Ethanol solution three times, sets in evaporating dish and is evaporated, and residue adds methanol to dissolve, and is transferred in 100mL volumetric flask, adds methanol to quarter Degree, shakes up, takes subsequent filtrate, as test solution；
2, the determination of assay analysis condition
2.1 determination of total flavonoids are measured according to " UV-VIS spectrophotometry "
The preparation of test solution: taking sea-buckthorn granule powder 0.8-1.2g, and ethyl alcohol 30ml is added, is heated to reflux 40- 70 minutes, filtration, residue was added 60% ethyl alcohol 20-30ml, 45-50 DEG C ultrasound 30-40 minutes, filter, residue is added again 60% ethyl alcohol 20-30ml, 40-50 DEG C ultrasound 30-40 minutes, filtration merges ethanol solution three times, sets in evaporating dish and steam Dry, residue adds methanol to dissolve, and is transferred in 100mL volumetric flask, and methanol is added to shake up, take subsequent filtrate to scale, molten as test sample Liquid；
The preparation of reference substance solution takes control substance of Rutin 18-22mg, accurately weighed, sets in 50ml measuring bottle, adds 55%-65% Appropriate amount of ethanol, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55%-65% ethyl alcohol to scale, shakes up；22-27ml is measured, is set In 50ml measuring bottle, it is diluted with water to scale, is shaken up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 25-35% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% nitre Sour aluminum solutions 0.8-12ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 8-12ml, then plus 30% ethyl alcohol to scale, shake It is even, it places 10-20 minutes, using corresponding reagent as blank, according to UV-VIS spectrophotometry, is measured at the wavelength of 500nm Absorbance, using absorbance as ordinate, concentration is abscissa, draws standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
Two parts of Duplicate Samples are done, deviation must not cross 3.0%.
This product is calculated by dry product, containing general flavone in terms of rutin (C27H30O16), must not be less than 10.5%.
2.1 Isorhamnetins are measured according to " high performance liquid chromatography ".
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method difference is accurate to draw reference substance and each 5-10 μ l of test solution, injects liquid chromatograph, measures, i.e., ?；
Two parts of Duplicate Samples are done, deviation must not cross 2.0%.
This product is calculated by dry product, must not be less than 0.70% containing Isorhamnetin (C16H12O7).
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Embodiment 1
Ingredient: 120 parts of hippophae rhamnoides xeraphium, 0.1 part of sea-buckthorn volatile oil clathrate compound, 0.5 part of silica, stearic acid 0.2 part of magnesium.
The ingredient of embodiment 1 is made by following any preparation method.
Preparation method (one):
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, leaches and be evaporated under reduced pressure using 50~60 DEG C, by water Vapor distillation method extracts the volatile oil in sea-buckthorn, and it is spare to collect volatile oil；Aqueous solution in extractor pours into containers for future use；It mentions Medicinal material after taking is spare；
2. ethyl alcohol extraction 1 time of medicinal material 10 times of amounts 60% of addition 1. step is extracted after, 1 hour every time, extracting solution mistake Filter, filtrate are spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 4h at 55 DEG C Double sieve methods carry out whole grains, pack to get.Preparation method (two):
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatilization in sea-buckthorn by steam distillation It is spare to collect volatile oil for oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. ethyl alcohol extraction 2 times of medicinal material 12 times of amounts 85% of addition 1. step is extracted after, 2 hours every time, extracting solution mistake Filter, filtrate are spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 5h at 60 DEG C Double sieve methods carry out whole grains, pack to get.
Preparation method (three):
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatilization in sea-buckthorn by steam distillation It is spare to collect volatile oil for oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. the ethyl alcohol that the medicinal material 1. step is extracted after is added 15 times 85% extracts 2 times, 1 hour every time, extracting solution was filtered, Filtrate is spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 6h at 65 DEG C Double sieve methods carry out whole grains, pack to get.
The granule of embodiment 1 is by following any detection method detection.
Detection method (one)
1, [determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 0.8g, and ethyl alcohol 30ml is added, is heated to reflux 40 minutes, filters, Residue is added 60% ethyl alcohol 20ml, 45 DEG C ultrasound 30 minutes, 60% ethyl alcohol 20ml is added in filtration, residue again, and 40 DEG C are super Sound 30 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, and residue adds methanol to dissolve, and was transferred to 100mL appearance In measuring bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 20mg, accurately weighed, sets in 50ml measuring bottle, adds 60% ethyl alcohol suitable Amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 60% ethyl alcohol to scale, shakes up；25ml is measured, sets in 50ml measuring bottle, adds water dilute It releases to scale, shakes up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 25% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, and places 4 minutes, then plus 10% aluminum nitrate it is molten Liquid 0.8ml, shakes up, and places 4 minutes；Adding sodium hydroxide test solution 8ml, then plus 30% ethyl alcohol to scale, shake up, place 10 minutes, Using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured at the wavelength of 500nm, is vertical with absorbance Coordinate, concentration are abscissa, draw standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
This product is calculated by dry product, containing general flavone in terms of rutin C27H30O16, must not be less than 10.5%；
2, Isorhamnetin shines high effective liquid chromatography for measuring:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 5 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, (the C containing Isorhamnetin16H12O7) 0.70% must not be less than.
3, [identify item]
Isorhamnetin reference substance, Quercetin reference substance are taken, adds methanol that every 1ml is made respectively containing the mixed solution of 0.8mg, makees For reference substance solution；High performance liquid chromatography item using test solution in claim 8 as test solution, according to claim 8 Part sample introduction, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
4, [inspection]
Granularity: being detected using double sieve methods, result 8-12%；
Melting: taking granule 10g, observes immediately after adding 200mL hot water stirs 5min, find be in after it is dissolved in hot water Slight turbid shape；
Detection method (two)
1, [determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 1g, and ethyl alcohol 30ml is added, is heated to reflux 45 minutes, filters, Residue is added 60% ethyl alcohol 20ml, 46 DEG C ultrasound 35 minutes, 60% ethyl alcohol 20ml is added in filtration, residue again, and 45 DEG C are super Sound 30 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, and residue adds methanol to dissolve, and was transferred to 100mL appearance In measuring bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 20mg, accurately weighed, sets in 50ml measuring bottle, adds 60% ethyl alcohol suitable Amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 60% ethyl alcohol to scale, shakes up；25ml is measured, sets in 50ml measuring bottle, adds water dilute It releases to scale, shakes up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 20% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4.5 minutes, then plus 10% aluminum nitrate Solution 1ml, shakes up, and places 5 minutes；Adding sodium hydroxide test solution 10ml, then plus 30% ethyl alcohol to scale, shake up, place 15 minutes, Using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured at the wavelength of 500nm, is vertical with absorbance Coordinate, concentration are abscissa, draw standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
This product is calculated by dry product, containing general flavone in terms of rutin C27H30O16, must not be less than 10.5%；
2, Isorhamnetin shines high effective liquid chromatography for measuring:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 5 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, (the C containing Isorhamnetin16H12O7) 0.70% must not be less than.
3, [identify item]
Isorhamnetin reference substance, Quercetin reference substance are taken, adds methanol that every 1ml is made respectively containing the mixed solution of 1mg, as right According to product solution；Using test solution in claim 8 as test solution, high-efficient liquid phase chromatogram condition according to claim 8 into Sample, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
4, [inspection]
Granularity: being detected using double sieve methods, result 8-12%；
Melting: taking granule 10g, observes immediately after adding 200mL hot water stirs 5min, find be in after it is dissolved in hot water Slight turbid shape；
Detection method (three)
1, [determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 1g, and ethyl alcohol 30ml is added, is heated to reflux 50 minutes, filters, Residue is added 60% ethyl alcohol 20ml, 48 DEG C ultrasound 35 minutes, 60% ethyl alcohol 20ml is added in filtration, residue again, and 50 DEG C are super Sound 30 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, and residue adds methanol to dissolve, and was transferred to 100mL appearance In measuring bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 20mg, accurately weighed, sets in 50ml measuring bottle, adds 60% ethyl alcohol suitable Amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 60% ethyl alcohol to scale, shakes up；25ml is measured, sets in 50ml measuring bottle, adds water dilute It releases to scale, shakes up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 20% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4.5 minutes, then plus 10% aluminum nitrate Solution 1ml, shakes up, and places 5 minutes；Adding sodium hydroxide test solution 10ml, then plus 30% ethyl alcohol to scale, shake up, place 15 minutes, Using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured at the wavelength of 500nm, is vertical with absorbance Coordinate, concentration are abscissa, draw standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
This product is calculated by dry product, containing general flavone in terms of rutin C27H30O16, must not be less than 10.5%；
2, Isorhamnetin shines high effective liquid chromatography for measuring:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 5 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, (the C containing Isorhamnetin16H12O7) 0.70% must not be less than.
3, [identify item]
Isorhamnetin reference substance, Quercetin reference substance are taken, adds methanol that every 1ml is made respectively containing the mixed solution of 1mg, as right According to product solution；Using test solution in claim 8 as test solution, high-efficient liquid phase chromatogram condition according to claim 8 into Sample, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
4, [inspection]
Granularity: being detected using double sieve methods, result 8-12%；
Melting: taking granule 10g, observes immediately after adding 200mL hot water stirs 5min, find be in after it is dissolved in hot water Slight turbid shape；
Embodiment 2
Ingredient: 200 parts of hippophae rhamnoides xeraphium, 0.7 part of sea-buckthorn volatile oil clathrate compound, 4 parts of silica, magnesium stearate 0.8 part.
The ingredient of embodiment 2 is made by following any preparation method.
Preparation method (one):
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatilization in sea-buckthorn by steam distillation It is spare to collect volatile oil for oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. ethyl alcohol extraction 1 time of medicinal material 10 times of amounts 60% of addition 1. step is extracted after, 1 hour every time, extracting solution mistake Filter, filtrate are spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 4h at 55 DEG C Double sieve methods carry out whole grains, pack to get.
Preparation method (two):
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatilization in sea-buckthorn by steam distillation It is spare to collect volatile oil for oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. ethyl alcohol extraction 2 times of medicinal material 12 times of amounts 85% of addition 1. step is extracted after, 2 hours every time, extracting solution mistake Filter, filtrate are spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 5h at 60 DEG C Double sieve methods carry out whole grains, pack to get.
Preparation method (three):
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatilization in sea-buckthorn by steam distillation It is spare to collect volatile oil for oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. the ethyl alcohol that the medicinal material 1. step is extracted after is added 85% extracts 2 times, 1 hour every time, extracting solution was filtered, filtrate It is spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, is added appropriate 70% ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, uses after drying 4h at 65 DEG C Double sieve methods carry out whole grains, pack to get.
The granule of embodiment 2 is by following any detection method detection.
Detection method (one)
1, [determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 1g, and ethyl alcohol 30ml is added, is heated to reflux 60 minutes, filters, Residue is added 60% ethyl alcohol 20ml, 50 DEG C ultrasound 30 minutes, 60% ethyl alcohol 20ml is added in filtration, residue again, and 50 DEG C are super Sound 30 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, and residue adds methanol to dissolve, and was transferred to 100mL appearance In measuring bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 22mg, accurately weighed, sets in 50ml measuring bottle, adds 55% ethyl alcohol suitable Amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55% ethyl alcohol to scale, shakes up；25ml is measured, sets in 50ml measuring bottle, adds water dilute It releases to scale, shakes up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 30% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% aluminum nitrate Solution 1ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 10ml, then plus 30% ethyl alcohol to scale, shake up, place 10-20 Minute, using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured, at the wavelength of 500nm with extinction Degree is ordinate, and concentration is abscissa, draws standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
This product is calculated by dry product, containing general flavone in terms of rutin C27H30O16, must not be less than 10.5%；
2, Isorhamnetin shines high effective liquid chromatography for measuring:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 5 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, (the C containing Isorhamnetin16H12O7) 0.70% must not be less than.
3, [identify item]
Isorhamnetin reference substance, Quercetin reference substance are taken, adds methanol that every 1ml is made respectively containing the mixed solution of 0.8mg, makees For reference substance solution；High performance liquid chromatography item using test solution in claim 8 as test solution, according to claim 8 Part sample introduction, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
4, [inspection]
Granularity: being detected using double sieve methods, result 8-12%；
Melting: taking granule 10g, observes immediately after adding 200mL hot water stirs 5min, find be in after it is dissolved in hot water Slight turbid shape；
Detection method (two)
1, [determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 1g, and ethyl alcohol 30ml is added, is heated to reflux 60 minutes, filters, Residue is added 60% ethyl alcohol 25ml, 50 DEG C ultrasound 35 minutes, 60% ethyl alcohol 25ml is added in filtration, residue again, and 50 DEG C are super Sound 35 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, and residue adds methanol to dissolve, and was transferred to 100mL appearance In measuring bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 22mg, accurately weighed, sets in 50ml measuring bottle, adds 55% ethyl alcohol suitable Amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55% ethyl alcohol to scale, shakes up；25ml is measured, sets in 50ml measuring bottle, adds water dilute It releases to scale, shakes up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 30% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% aluminum nitrate Solution 1ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 10ml, then plus 30% ethyl alcohol to scale, shake up, place 10-20 Minute, using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured, at the wavelength of 500nm with extinction Degree is ordinate, and concentration is abscissa, draws standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
This product is calculated by dry product, containing general flavone in terms of rutin C27H30O16, must not be less than 10.5%；
2, Isorhamnetin shines high effective liquid chromatography for measuring:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 10 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, (the C containing Isorhamnetin16H12O7) 0.70% must not be less than.
3, [identify item]
Isorhamnetin reference substance, Quercetin reference substance are taken, adds methanol that every 1ml is made respectively containing the mixed solution of 1mg, as right According to product solution；Using test solution in claim 8 as test solution, high-efficient liquid phase chromatogram condition according to claim 8 into Sample, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
4, [inspection]
Granularity: being detected using double sieve methods, result 8-12%；
Melting: taking granule 10g, observes immediately after adding 200mL hot water stirs 5min, find be in after it is dissolved in hot water Slight turbid shape；
Detection method (three)
1, [determination of total flavonoids]
The preparation of test solution: taking sea-buckthorn granule 1g, and ethyl alcohol 30ml is added, is heated to reflux 60 minutes, filters, Residue is added 60% ethyl alcohol 30ml, 50 DEG C ultrasound 40 minutes, 60% ethyl alcohol 30ml is added in filtration, residue again, and 50 DEG C are super Sound 40 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, and residue adds methanol to dissolve, and was transferred to 100mL appearance In measuring bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 22mg, accurately weighed, sets in 50ml measuring bottle, adds 55% ethyl alcohol suitable Amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55% ethyl alcohol to scale, shakes up；25ml is measured, sets in 50ml measuring bottle, adds water dilute It releases to scale, shakes up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 30% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% aluminum nitrate Solution 1ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 10ml, then plus 30% ethyl alcohol to scale, shake up, place 10-20 Minute, using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured, at the wavelength of 500nm with extinction Degree is ordinate, and concentration is abscissa, draws standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve Method under preparation measures absorbance from adding sodium nitrite solution 1ml in accordance with the law, while taking test solution 3ml, except not Outside adding sodium hydroxide test solution, remaining is ibid operated, as blank, from reading the weight containing rutin in test solution on standard curve Amount, calculate to get；
This product is calculated by dry product, containing general flavone in terms of rutin C27H30O16, must not be less than 10.5%；
2, Isorhamnetin shines high effective liquid chromatography for measuring:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol To scale, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 10 μ l of test solution, injects liquid chromatograph, measurement to get；
This product is calculated by dry product, (the C containing Isorhamnetin16H12O7) 0.70% must not be less than.
3, [identify item]
Isorhamnetin reference substance, Quercetin reference substance are taken, adding methanol that every 1ml is made, respectively the mixing containing 0.8-1.2mg is molten Liquid, as reference substance solution；High-efficient liquid phase color using test solution in claim 8 as test solution, according to claim 8 Spectral condition sample introduction, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
4, [inspection]
Granularity: being detected using double sieve methods, result 8-12%；
Melting: taking granule 10g, observes immediately after adding 200mL hot water stirs 5min, find be in after it is dissolved in hot water Slight turbid shape；
Embodiment 3
Ingredient: 140 parts of hippophae rhamnoides xeraphium, 0.2 part of sea-buckthorn volatile oil clathrate compound, 0.8 part of silica, stearic acid 0.3 part of magnesium.
Preparation method, detection method are the same as embodiment 1
Embodiment 4
Ingredient: 180 parts of hippophae rhamnoides xeraphium, 0.5 part of sea-buckthorn volatile oil clathrate compound, 3.5 parts of silica, stearic acid 0.7 part of magnesium.
Preparation method, detection method are the same as embodiment 1
Embodiment 5
Ingredient: 160 parts of hippophae rhamnoides xeraphium, 0.3 part of sea-buckthorn volatile oil clathrate compound, silica 1 part, magnesium stearate 0.4 part.
Preparation method, detection method are the same as embodiment 1
Experimental example: it to prove the science of detection method and the existing property of conjunction in the present invention, has carried out following methods experiment and has ground Study carefully
Experimental example 1
General flavone content determines methodology validation
1 material, equipment and reagent
1.1 material
Experimental material used in the present invention is the sea-buckthorn granule produced by Qinghai Pulante Medicine Co., Ltd；Lot number: 180601,180602,180603,180604,180605,180606 totally 6 batches
Reference substance: control substance of Rutin
Reference substance source: National Institute for Food and Drugs Control
1.2 equipment
Instrument: the general general T6 ultraviolet-uisible spectrophotometer of analysis in Beijing
KQ-250B supersonic cleaning machine (Kunshan ultrasonic instrument Co., Ltd)
Temperature controlled water bath pot (Nantong Hua Tai laboratory apparatus Co., Ltd)
1.3 reagent
Methanol: Chongqing Chuan Dong chemical industry (group) Co., Ltd
Ethyl alcohol: Chongqing Chuan Dong chemical industry (group) Co., Ltd
Aluminum nitrate: Tianjin Kermel Chemical Reagent Co., Ltd.
Sodium nitrite: Tianjin Kermel Chemical Reagent Co., Ltd.
Sodium hydroxide: Tianjin Kermel Chemical Reagent Co., Ltd.
The preparation of 1.4 test solutions
Method 1 takes sea-buckthorn granule 0.8g, and ethyl alcohol 30ml is added, is heated to reflux 40 minutes, filters, and residue is added 60% Ethyl alcohol 20ml, 45 DEG C ultrasound 30 minutes, filtration, residue is added 60% ethyl alcohol 20ml again, 40 DEG C ultrasound 30 minutes, filter It crosses, merges ethanol solution three times, set in evaporating dish and be evaporated, residue adds methanol to dissolve, and is transferred in 100mL volumetric flask, adds first Alcohol shakes up to scale, takes subsequent filtrate, as test solution；
Method 2 (preferred) takes sea-buckthorn granule 1.2g, and ethyl alcohol 30ml is added, is heated to reflux 70 minutes, filters, residue Be added 60% ethyl alcohol 30ml, 50 DEG C ultrasound 40 minutes, 60% ethyl alcohol 30ml, 50 DEG C of ultrasounds 40 are added in filtration, residue again Minute, filtration merges ethanol solution three times, sets in evaporating dish and be evaporated, residue adds methanol to dissolve, and is transferred to 100mL volumetric flask In, add methanol to scale, shakes up, take subsequent filtrate, as test solution；
Method 3 (most preferred method) takes sea-buckthorn granule 1g, and ethyl alcohol 30ml is added, is heated to reflux 50 minutes, filters, residual Slag is added 60% ethyl alcohol 30ml, 50 DEG C ultrasound 30 minutes, 60% ethyl alcohol 30ml, 50 DEG C of ultrasounds are added in filtration, residue again 30 minutes, filtration merged ethanol solution three times, sets in evaporating dish and be evaporated, residue adds methanol to dissolve, and is transferred to 100mL capacity In bottle, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
Method 4 (comparative example)
Method source: the detection method of 2015 editions first sea-buckthorn medicinal materials of Chinese Pharmacopoeia.
This product about 1g is taken, it is accurately weighed, add 60% ethyl alcohol 30ml, be heated to reflux 2 hours and let cool, filter, residue is distinguished again Add 60% ethyl alcohol 25ml, is heated to reflux 2 times, 1 hour every time, filtration, merging filtrate was set in 100ml measuring bottle, 60% second of residue Alcohol washing, washing lotion are incorporated in same measuring bottle, are diluted to scale with 60% ethyl alcohol, shake up.Precision measures 25ml, sets 100ml measuring bottle In, scale is added water to, is shaken up, as test solution.
The preparation of reference substance solution takes control substance of Rutin 20mg, accurately weighed, sets in 50ml measuring bottle, adds 55%-65% second Appropriate alcohol, setting low-grade fever in water-bath makes to dissolve, and lets cool, and adds 55%-65% ethyl alcohol to scale, shakes up；25ml is measured, 50ml measuring bottle is set In, be diluted with water to scale, shake up to get；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, sets 25ml amount respectively In bottle, respectively plus 25-35% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, places 4-5 minutes, then plus 10% nitre Sour aluminum solutions 0.8-12ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 8-12ml, then plus 30% ethyl alcohol to scale, shake It is even, it places 10-20 minutes, using corresponding reagent as blank, according to UV-VIS spectrophotometry, is measured at the wavelength of 500nm Absorbance, using absorbance as ordinate, concentration is abscissa, draws standard curve, and standard curve is shown in attached drawing Fig. 1, absorbance value It is shown in Table 3.
1.2 control substance of Rutin linear relationships
Regression equation: y=0.0253x-0.0004, wherein x is concentration (μ g/ml), and y is absorbance related coefficient: r=1, The range of linearity are as follows: 0~48.264 μ g/ml
The standard curve absorbance value of 3. rutin of table
1.3 precision test
The reference substance solution for being 32.176 μ g/ml with above-mentioned concentration is taken, according to UV-VIS spectrophotometry, at 500nm Absorbance is measured, METHOD FOR CONTINUOUS DETERMINATION 6 times, the results are shown in Table 4, the results showed that instrument precision is good.
4. instrument precision of table investigates data
1.4 stability test
It takes and test solution 1ml is made by 3 preparation method of method, be placed in 50ml volumetric flask, add purifying moisture constant volume, shake Its trap is not measured in different time after even, the results are shown in Table 5, the results showed that sample is stablized in 24 hours.
5. sample stability of table investigates data
1.5 repetitive test
6 parts of test solutions are prepared by 3 preparation method of method, 1ml is taken from every part of test solution, is respectively placed in 6 In 50ml volumetric flask, adds purifying moisture constant volume, measured after shaking up by above-mentioned content assaying method, the results are shown in Table 6, the results showed that sample Product measuring method favorable reproducibility.
6. repeatability of table investigates data
The measurement comparison of 1.6 sample sizes
It takes with a collection of sea-buckthorn granule, respectively according to the above method 1, method 2, the system of 3. method of method, 4 test solution Preparation Method respectively prepares 3 parts of parallel test solutions, takes 1ml from every part of test solution, be respectively placed in 50ml volumetric flask, Add purifying moisture constant volume, is measured after shaking up by above-mentioned content assaying method, calculate the content of rutin, the results are shown in Table 7.
7. sample size comparing result of table
The measurement of 1.7 sample sizes
It takes with 5 batches of sea-buckthorn granules, the preparation method of 3 test solutions, each to prepare 3 parts in parallel for examination according to the above method Product solution takes 1ml from every part of test solution, is respectively placed in 50ml volumetric flask, adds purifying moisture constant volume, by upper after shaking up Content assaying method measurement is stated, the content of rutin is calculated, the results are shown in Table 8.
8. sample size measurement result of table
Conclusion: general flavone (in terms of rutin) the content method verifying in this method detection sea-buckthorn granule: rutin returns Return equation: y=0.0253x-0.0004, R2=1, the range of linearity are as follows: 0~48.264 μ g/ml；Instrument precision is good, RSD% =0.9776%；The good RSD%=1.5679% of repeatability；Rutin is relatively stable in for 24 hours, and stability RSD% is 1.2565%, show that the measuring method of this rutin is good, this method meets the verifying of drug standard analysis method and requires.
Experimental example 2
Isorhamnetin content determines methodology validation
1 material, equipment and reagent
1.1 material
Experimental material used in the present invention is the sea-buckthorn granule produced by Qinghai Pulante Medicine Co., Ltd；Lot number: 180601,180602,180603,180604,180605,180606,180607,180608,180609,180610 totally 10 batches.
Reference substance: Isorhamnetin reference substance
Reference substance source: National Institute for Food and Drugs Control
1.2 instruments include:
High performance liquid chromatograph: (model: Waters e2695, producer: U.S. Waters is public for Waters e2695 series Department), including PDA diode array detector (model: Waters 2998, producer: Waters, US) and Empower work It stands (Waters, US)；
Chromatographic column: green hundred grass Ecosil 120-5-C18AQ Plus chromatographic column (4.6mm × 250mm, 5 μm)；
Balance: a ten thousandth balance (Sartorius BS224S)；
Ultrapure water system: U.S. Millipore (Mi Libo) company；
It is that domestic analysis is pure that agents useful for same of the present invention, which includes: methanol, phosphoric acid etc. with reagent,；HPLC methanol, acetonitrile are equal For chromatographically pure (German Merck company, Darmstadt, Gemany)；Water is ultrapure water (resistivity 8.2m Ω .cm).
The preparation of 1.3 test solutions
Method 1: the test solution 2.5ml handled under determination of total flavonoids item by method 3 is taken, 25ml is placed in, adds second Alcohol shakes up to scale, is filtered with 0.45um filter membrane, take subsequent filtrate to get；
Method 2 (comparative example): method source: the method for inspection of 2105 editions first sea-buckthorn medicinal materials of Chinese Pharmacopoeia.Take sea-buckthorn Granule 0.5g, it is accurately weighed, it sets in stuffed conical flask, ethyl alcohol 50ml is added in precision, and weighed weight is heated to reflux 1 hour, It lets cool, then weighed weight, the weight of less loss is supplied with ethyl alcohol, is shaken up, filter.Precision measures subsequent filtrate 2.5ml, sets tool plug taper In bottle, add hydrochloric acid 3.5ml, heating hydrolysis 1 hour, cools down, be transferred in 25ml measuring bottle, immediately with appropriate in 75 DEG C of water-baths Ethanol washing container in washing lotion and the same measuring bottle of people, adds ethyl alcohol to shake up, filter to scale, take subsequent filtrate to get.
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing 10 μ g's Solution to get；
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；With ratio for 58: 42 - 0.4% phosphoric acid solution of methanol be mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
Measuring method difference is accurate to draw reference substance and each 5-10 μ l of test solution, injects liquid chromatograph, measures, i.e., ?；
1.3.1 linear test
Precision draws Isorhamnetin reference substance solution (10.014 μ g/ml) 5 μ l, 10 μ l and Isorhamnetin reference substance solution (100.14 μ g/ml) 2 μ l, 5 μ l, 10 μ l inject liquid chromatograph, peak area are measured by above-mentioned chromatographic condition, with peak area (Y) Linear regression, linear equation are as follows: Y=2.0598X+0.826, R are carried out to sample volume (X)2=1；Show Isorhamnetin sample volume It is in good linear relationship in 50.07~1001.4 μ g ranges.It the results are shown in Table 9.
9 Isorhamnetin linear test result of table
1.3.2 instrument precision is tested
The reference substance solution prepared under 1.3 is taken, under above-mentioned chromatographic condition, repeats sample introduction 6 times, measures Isorhamnetin peak Area, calculating RSD% (Isorhamnetin) value is 1.454%, shows that instrument precision is good, measurement result is shown in Table 10.
10 instrument precision test result of table
1.3.3 repetitive test
Precision weighs same batch of sample (lot number 180605), while preparing by the preparation method 1 of above-mentioned 1.3 test solution 6 parts of test solution, method 2 prepare 3 parts of test solution, according to the above method measure sample in Isorhamnetin content, 1.3 1 average content of method is 1.0861 (%), and it is good heavy to show that the measuring method of this Isorhamnetin has for RSD value 1.9196% Renaturation；4.2 method, two response is low, and content is relatively low, the results are shown in Table 11.
11 repetitive test result of table
1.3.4 stability test
By 1.3 lower methods 1 and method 2 respectively prepare test solution portion, be placed at room temperature for, by draft under chromatographic condition in 0,2,4,8,12, sample introduction respectively, measures peak area for 24 hours.1 test solution RSD% of method is 1.7351%, 2 test sample of method Solution RSD% is 3.1587%, shows that 1 Isorhamnetin of method is relatively stable in 24 hours.2 Isorhamnetins of method exist Internal stability is poor for 24 hours, and stability test the results are shown in Table 12.
12 stability test result of table
1.3.5 sample size measures
Assay is carried out to ten batches of samples by the method for the method of 1.3 lower methods 1, while comparing 1.3 lower methods 2 carry out content detection, the results are shown in Table 13.
130 batches of sample content results tables of table
Isorhamnetin reference substance, Quercetin reference substance are taken, adding methanol that every 1ml is made, respectively the mixing containing 0.8-1.2mg is molten Liquid, as reference substance solution；By test solution in 1.3 methods 1 be test solution, by above-mentioned high-efficient liquid phase chromatogram condition into Sample, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
Conclusion: using detection method of the invention, and Isorhamnetin compares linear equation are as follows: Y=2.0598X+0.826, R2 =1；Isorhamnetin sample volume is in good linear relationship in 50.07~1001.4 μ g ranges, and accuracy of instrument is high, RSD% value It is 1.454%, repeated RSD value is 1.9196%, using the stability RSD% for the test solution that the method for the present invention is handled It is 1.7351%, high using the response of the method for the present invention test sample, as a result accurately and reliably, this method is easy, reproducible, can Efficiently control the quality of sea-buckthorn granule.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail State, but on the basis of the present invention, it can be made it is some modify or improve, this is aobvious and easy to those skilled in the art See.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed Range.

Claims (10)

1. a kind of sea-buckthorn granule, it is characterised in that: the granule is by hippophae rhamnoides xeraphium, sea-buckthorn inclusion essential oil Object, auxiliary material composition.
2. granule according to claim 1, it is characterised in that: the sea-buckthorn granule is by following composition group At: 120-200 parts of hippophae rhamnoides xeraphium, 0.1-0.7 parts of sea-buckthorn volatile oil clathrate compound, 0.5-4 parts of silica, stearic acid 0.2-0.8 parts of magnesium.
3. granule according to claim 2, it is characterised in that: the sea-buckthorn granule is by below at square group At: 140-180 parts of hippophae rhamnoides xeraphium, 0.2-0.5 parts of sea-buckthorn volatile oil clathrate compound, 0.8-3.5 parts of silica, tristearin Sour magnesium 0.3-0.7 parts.
4. granule according to claim 3, it is characterised in that: the sea-buckthorn granule is by below at square group At: 160 parts of hippophae rhamnoides xeraphium, 0.3 part of sea-buckthorn volatile oil clathrate compound, silica 1 part, 0.4 part of magnesium stearate.
5. granule according to claim 1-4, it is characterised in that: the granule is by following preparation side Method is made:
1) preparation of hippophae rhamnoides xeraphium
1. being cleaned after taking sea-buckthorn decontamination, it is put into water and mentions in extractor, extracts the volatile oil in sea-buckthorn by steam distillation, receive It is spare to collect volatile oil；Aqueous solution in extractor pours into containers for future use；Medicinal material after extraction is spare；
2. the medicinal material 1. step is extracted after is added the ethyl alcohol that 10-15 times is measured 60%-85% and extracts 1-2 times, 1-2 hours each, Extracting solution filtering, filtrate are spare；
3. by step, 1. spare aqueous solution merges with the filtrate of step 2., is concentrated under reduced pressure into 1.05-1.15 at a temperature of 60 DEG C Clear cream, clear cream sieve with 100 mesh sieve, and clear cream, which enters in spray tower, to be spray-dried, and technological parameter is inlet air temperature: 165-195 DEG C, Leaving air temp: 80-105 DEG C, hippophae rhamnoides xeraphium is obtained；
2) sea-buckthorn volatile oil clathrate compound
By step, 1. spare volatile oil takes coprecipitation that sea-buckthorn volatile oil clathrate compound is made with beta-cyclodextrin；
3) it pelletizes
It by hippophae rhamnoides xeraphium, sea-buckthorn volatile oil clathrate compound, silica, magnesium stearate, is mixed, adds appropriate 70% Ethyl alcohol prepares the softwood of " holding is agglomerating, and flicking is to dissipate ", crosses No. 14 sieves and carries out extruding granulation, adopts after drying 4-6h at 55-65 DEG C Carry out whole grain with double sieve methods, pack to get.
6. granule according to claim 1, which is characterized in that the quality determining method of the granule includes inspection It looks into, be that the photograph UV-VIS spectrophotometry compareed surveys general flavone content, is pair with Isorhamnetin reference substance with control substance of Rutin According to high performance liquid chromatography assay, with Isorhamnetin reference substance, Quercetin reference substance be control high performance liquid chromatography Method identifies.
7. granule according to claim 6, which is characterized in that ultraviolet in the granule quality determining method- Visible spectrophotometry surveys general flavone content method are as follows:
The preparation of test solution: taking sea-buckthorn granule 0.8-1.2g, and ethyl alcohol 30ml is added, is heated to reflux 40-70 minutes, filters Cross, residue is added 60% ethyl alcohol 20-30ml, 45-50 DEG C ultrasound 30-40 minutes, filter, 60% ethyl alcohol is added in residue again 20-30ml, 40-50 DEG C ultrasound 30-40 minutes, filtration, merge ethanol solution three times, set in evaporating dish and be evaporated, residue adds first Alcohol dissolution, is transferred in 100mL volumetric flask, adds methanol to scale, shake up, take subsequent filtrate, as test solution；
The preparation of reference substance solution takes control substance of Rutin 18-22mg, accurately weighed, sets in 50ml measuring bottle, adds 55%-65% ethyl alcohol In right amount, setting low-grade fever in water-bath makes to dissolve, and lets cool, adds 55%-65% ethyl alcohol to scale, shake up；22-27ml is measured, 50ml amount is set In bottle, it is diluted with water to scale, is shaken up, i.e.,；
The preparation precision of standard curve measures reference substance solution 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, is set in 25ml measuring bottle respectively, Respectively plus 25-35% ethyl alcohol is to 6.0ml, adds 5% sodium nitrite solution 1ml, mixes, and places 4-5 minutes, then plus 10% aluminum nitrate it is molten Liquid 0.8-12ml, shakes up, and places 4-5 minutes；Adding sodium hydroxide test solution 8-12ml, then plus 30% ethyl alcohol to scale, shake up, place 10-20 minutes, using corresponding reagent as blank, according to UV-VIS spectrophotometry, absorbance is measured at the wavelength of 500nm, with Absorbance is ordinate, and concentration is abscissa, draws standard curve；
Measuring method precision measures test solution 3ml, sets in 25ml measuring bottle, adds 30% ethyl alcohol to 6ml, sighting target directrix curve preparation Under method measure absorbance in accordance with the law from adding sodium nitrite solution 1ml, while test solution 3ml is taken, except hydrogen-oxygen is not added Change outside sodium test solution, remaining is ibid operated, and is counted as blank from the weight containing rutin in test solution is read on standard curve Calculate to get.
8. granule according to claim 6, which is characterized in that with different mouse in the granule quality determining method Li Su reference substance is the high performance liquid chromatography content assaying method of control are as follows:
Chromatographic condition and system suitability are using octadecylsilane chemically bonded silica as filler；The first for being 58: 42 with ratio - 0.4% phosphoric acid solution of alcohol is mobile phase；Detection wavelength is 370nm；Number of theoretical plate should be not less than by the calculating of Isorhamnetin peak 3000；
The preparation of reference substance solution takes Isorhamnetin reference substance appropriate, accurately weighed, adds methanol that every 1ml is made containing the molten of 10 μ g Liquid to get；
The preparation of test solution takes the test solution 2.5ml under determination of total flavonoids item, is placed in 25ml, adds ethyl alcohol to quarter Degree, shake up, with 0.45um filter membrane filter, take subsequent filtrate to get；
Measuring method is accurate respectively to draw reference substance and each 5-10 μ l of test solution, injects liquid chromatograph, measurement to get.
9. granule according to claim 6, which is characterized in that with different mouse in the granule quality determining method Li Su reference substance, Quercetin reference substance are the high performance liquid chromatography discrimination method of control are as follows:
Isorhamnetin reference substance, Quercetin reference substance are taken, methanol is added every 1ml to be made respectively containing the mixed solution of 0.8-1.2mg, as Reference substance solution；High-efficient liquid phase chromatogram condition using test solution in claim 8 as test solution, according to claim 8 Sample introduction, in test sample map, appearance is answered in position corresponding with Isorhamnetin, Quercetin in reference substance map.
10. granule according to claim 1-4, which is characterized in that this granule character: for light brown Particle, mildly bitter flavor；Granularity: being detected using double sieve methods, result 8-12%；Melting: granule 10g is taken, is added It is observed immediately after 200mL hot water stirs 5min, it is found that after it is dissolved in hot water be in slight turbid shape；Containing general flavone with rutin C27H30O16 meter must not be less than 10.5%；C16H12O7 containing Isorhamnetin must not be less than 0.70%.
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Application publication date: 20190315