CN106526057B - A kind of thin-layer chromatographic analysis method for detecting flavones ingredient in serpentgrass - Google Patents
A kind of thin-layer chromatographic analysis method for detecting flavones ingredient in serpentgrass Download PDFInfo
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- CN106526057B CN106526057B CN201610841521.9A CN201610841521A CN106526057B CN 106526057 B CN106526057 B CN 106526057B CN 201610841521 A CN201610841521 A CN 201610841521A CN 106526057 B CN106526057 B CN 106526057B
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- 235000012804 Polygonum viviparum Nutrition 0.000 title claims abstract description 37
- 229930003944 flavone Natural products 0.000 title claims abstract description 23
- 235000011949 flavones Nutrition 0.000 title claims abstract description 23
- 150000002213 flavones Chemical class 0.000 title claims abstract description 22
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000004615 ingredient Substances 0.000 title claims abstract description 18
- 241000118324 Bistorta vivipara Species 0.000 title claims abstract 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 57
- 239000013558 reference substance Substances 0.000 claims abstract description 46
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims abstract description 35
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims abstract description 32
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000012360 testing method Methods 0.000 claims abstract description 28
- GQODBWLKUWYOFX-UHFFFAOYSA-N Isorhamnetin Natural products C1=C(O)C(C)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 GQODBWLKUWYOFX-UHFFFAOYSA-N 0.000 claims abstract description 18
- IZQSVPBOUDKVDZ-UHFFFAOYSA-N isorhamnetin Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 IZQSVPBOUDKVDZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 235000008800 isorhamnetin Nutrition 0.000 claims abstract description 18
- 235000005875 quercetin Nutrition 0.000 claims abstract description 18
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims abstract description 17
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229960001285 quercetin Drugs 0.000 claims abstract description 17
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000008777 kaempferol Nutrition 0.000 claims abstract description 15
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims abstract description 11
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 230000007062 hydrolysis Effects 0.000 claims abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 3
- -1 methylene chloride acetone methanol formic acid Chemical compound 0.000 claims abstract description 3
- 244000228295 Polygonum viviparum Species 0.000 claims description 29
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 238000011161 development Methods 0.000 claims description 11
- 238000004458 analytical method Methods 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 230000001174 ascending effect Effects 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims description 2
- 125000003963 dichloro group Chemical group Cl* 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 8
- 239000000741 silica gel Substances 0.000 abstract description 8
- 229910002027 silica gel Inorganic materials 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 2
- 238000013441 quality evaluation Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 21
- BDAGIHXWWSANSR-UHFFFAOYSA-N formic acid Substances OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 9
- 239000003814 drug Substances 0.000 description 5
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000219050 Polygonaceae Species 0.000 description 1
- 244000292697 Polygonum aviculare Species 0.000 description 1
- 235000006386 Polygonum aviculare Nutrition 0.000 description 1
- 235000014258 Polygonum bistorta Nutrition 0.000 description 1
- 244000233952 Polygonum bistorta Species 0.000 description 1
- CVBNMWXECPZOLM-UHFFFAOYSA-N Rhamnetin Natural products COc1cc(O)c2C(=O)C(=C(Oc2c1)c3ccc(O)c(O)c3O)O CVBNMWXECPZOLM-UHFFFAOYSA-N 0.000 description 1
- JMFSHKGXVSAJFY-UHFFFAOYSA-N Saponaretin Natural products OCC(O)C1OC(Oc2c(O)cc(O)c3C(=O)C=C(Oc23)c4ccc(O)cc4)C(O)C1O JMFSHKGXVSAJFY-UHFFFAOYSA-N 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- MOZJVOCOKZLBQB-UHFFFAOYSA-N Vitexin Natural products OCC1OC(Oc2c(O)c(O)cc3C(=O)C=C(Oc23)c4ccc(O)cc4)C(O)C(O)C1O MOZJVOCOKZLBQB-UHFFFAOYSA-N 0.000 description 1
- 230000000879 anti-atherosclerotic effect Effects 0.000 description 1
- 230000001142 anti-diarrhea Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000002225 anti-radical effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- FDSGHYHRLSWSLQ-UHFFFAOYSA-N dichloromethane;propan-2-one Chemical compound ClCCl.CC(C)=O FDSGHYHRLSWSLQ-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 229930182486 flavonoid glycoside Natural products 0.000 description 1
- 150000007955 flavonoid glycosides Chemical class 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- JGUZGNYPMHHYRK-UHFFFAOYSA-N rhamnetin Chemical compound C=1C(OC)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 JGUZGNYPMHHYRK-UHFFFAOYSA-N 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- SGEWCQFRYRRZDC-VPRICQMDSA-N vitexin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O SGEWCQFRYRRZDC-VPRICQMDSA-N 0.000 description 1
- PZKISQRTNNHUGF-UHFFFAOYSA-N vitexine Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O PZKISQRTNNHUGF-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Plant Substances (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to a kind of thin-layer chromatographic analysis method for detecting flavones ingredient in serpentgrass.This method comprises the following steps：(a) preparation of need testing solution：After the hydrolysis extraction of serpentgrass medicinal material hydrochloric acid methanol, as serpentgrass need testing solution.(b) preparation of reference substance solution：Dissolve 3 kinds of Quercetin, Isorhamnetin, Kaempferol reference substances respectively with methanol, reference substance solution is made.(c) thin-layer chromatography detects：Using silica gel thin-layer chromatography plate, test sample and reference substance solution are deployed using methylene chloride acetone methanol formic acid system as solvent；Then AlCl is sprayed3Chromophoric solution, you can the fluorescence viewing thin-layer chromatography spot under uviol lamp.The inventive method checks that efficiency high, cost are low, and practical value is high, differentiates for serpentgrass medicinal material and quality evaluation provides a kind of effective approach.
Description
Technical field
The present invention relates to Chinese medicine quality testing field, and in particular to a kind of thin layer for detecting flavones ingredient in serpentgrass
Chromatogram analysis method.
Technical background
This product be polygonaceae plant serpentgrass Polygonum viviparum L. rhizome, record in《The Ministry of Public Health issues Tibetan medicine
Standard first》, there is the effect of antidiarrheal, stomach invigorating.Serpentgrass is mainly grown in patana of the height above sea level in 1500-5000m, mountain
The ground such as Gu Xi sides, are distributed widely in the ground such as Tibet, Qinghai, Gansu, the title with " plateau adder-wort ", and abundant tan is contained in serpentgrass
The compositions such as matter, flavones, protein, polysaccharide.Wherein flavone compound is the main secondary metabolite of serpentgrass, is had stronger
Anti-radical action and oxidation resistance, have been reported that and the flavones such as Quercetin, Isorhamnetin, Kaempferol be found that in serpentgrass
Aglycon and corresponding flavonoid glycoside composition, Quercetin, Isorhamnetin, Kaempferol etc. have hypotensive, reducing blood lipid, reduce blood and glue
Denseness, strengthen the function such as blood vessel elasticity, antiatherosclerosis, be cardiovascular and cerebrovascular disease " jinx ".
Serpentgrass is recorded in《Drug Standard of Ministry of Public Health of the Peoples Republic of China Tibetan medicine (first)》, wherein only to medicinal material
Carry out microscopical characters not differentiate active component, be unfavorable for the quality of effectively control serpentgrass.Document is to bulbil at present
The active component quality testing of knotweed mostly using 1-2 organic acid or flavones as index components, is mostly analyzed using HPLC.Such as
Publication《RP-HPLC methods determine the content of Vitexin, quercitin and Quercetin in Tibetan medicine serpentgrass simultaneously》(natural products
Research and development, 2011),《The content of Gallic Acid in Viviparous Bistort Rhizome by HPLC》(China Dispensary, 2005
Year).Above HPLC analysis methods instrument cost is high, and complex operation is also high to experimenter's skill set requirements, and toxic agent dosage
It is more, while the test of single sample can only be carried out every time.
Searching is a kind of simple and easy, and the low method of cost is to active component in serpentgrass --- and flavones ingredient detects
As demand, particularly to being detected while Quercetin, Isorhamnetin, Kaempferol.
The content of the invention
For existing issue, the present invention is analyzed and researched to flavones ingredient property in serpentgrass, there is provided a kind of
Detect the thin-layer chromatographic analysis method of flavones ingredient in serpentgrass, this method have easy to operate, rapid, specificity it is strong, into
The characteristics of this is cheap, reagent toxicity is small, while quality evaluation has been carried out to serpentgrass by 3 kinds of flavones ingredients.
To achieve these goals, the present invention provides a kind of thin-layer chromatographic analysis side for detecting flavones ingredient in serpentgrass
Method, it is characterised in that carry out in accordance with the following steps：
A. prepared by need testing solution：Serpentgrass medicinal powder is taken, is hydrolyzed and extracted with hydrochloric acid methanol, filtering, filtrate is evaporated, residual
Slag adds methanol to make dissolving, centrifuging and taking supernatant, solvent evaporated, makes redissolution with methanol, produces serpentgrass need testing solution；
B. prepared by reference substance solution：Quercetin reference substance, Isorhamnetin reference substance and Kaempferol reference substance are taken, uses first respectively
Above-mentioned reference substance solution is made in alcohol dissolving；
C. indentification by TLC：Thin layer is carried out to reference substance solution described in need testing solution described in above-mentioned steps a and step b
Chromatography, analysis condition：GF254Lamellae, 22-25 DEG C of temperature, humidity 60%-80%, solvent：Dichloromethane-acetone-
Methyl alcohol-formic acid=20-30:0.2-1.0:0.5:2, ascending development, after expansion, spray the AlCl with 3%-7%3Solution, put ultraviolet
Contrasting detection under lamp.
In certain embodiments, the hydrochloric acid methanol during prepared by the step a need testing solutions hydrolyzes extracting method：Took
The serpentgrass medicinal powder 10g of No. 3 sieves, is placed in 500mL round-bottomed flasks, adds absolute methanol 250mL, adds mass fraction and is
25%-30% watery hydrochloric acid 60mL, in 80 DEG C of water-baths backflow 2h, cooled down immediately in ice bath after taking-up.
In certain embodiments, the step a further comprises the residue that is evaporated for centrifuging gained supernatant carrying out post
Chromatography, multiple polarity segment components are collected respectively, is evaporated respectively, makes redissolution with methanol respectively, it is molten to produce serpentgrass test sample
Liquid.Polarity section of the present invention refers to, each big due to respective polarity into branch in material when using column chromatography material
Small difference,, in order will elution according to the priority of time or the size of polarity according to being eluted for certain rule order
The eluent to get off is divided into some, and each part is a polarity section.
In certain embodiments, described column chromatography is the silica gel column chromatography of 200-300 mesh, and eluent is dichloromethane：
Methanol=100:5；The multiple polarity section is 3,4 or 5.
In certain embodiments, the preparation method of reference substance solution is in the step b：Precision, which weighs, respectively dries to perseverance
Quercetin, Isorhamnetin, the Kaempferol reference substance of weight are appropriate, and it is respectively 0.4mgmL to add methanol that concentration is made-1、0.3mg·
mL-1、0.4mg·mL-1Reference substance solution.
In certain embodiments, it is characterised in that thin-layer chromatographic analysis are second outspread thin-layered chromatography in step c.
In certain embodiments, the condition of second of expansion is the same as expansion for the first time.
In certain embodiments, dichloromethane-acetone described in step c-methyl alcohol-formic acid development system proportioning composition is
25:0.6:0.5:2。
The positive effect of the present invention includes：
(1) present invention provides this 3 kinds of flavones ingredients of Quercetin, Isorhamnetin, Kaempferol in a kind of serpentgrass first
The thin layer chromatography of analysis simultaneously, can once analyze multiple samples, compensate for a variety of flavones ingredients in serpentgrass medicinal material
The blank of thin-layer chromatographic analysis is carried out, differentiates for its true and false and quality control provides easy, quick, efficient method.
(2) present invention after hydrochloric acid methanol hydrolysis extraction flavones with column chromatography for separation has been carried out, by the way that elution composition is divided
Into opposed polarity section, detected respectively so that disturb and reduce during thin-layer chromatographic analysis, differentiate more accurate, selectivity more
By force.
(3) present invention further uses dichloromethane-acetone-methyl alcohol-formic acid development system, and secondary exhibition is employed to sample
Thin-layered chromatography is opened, greatly improves the separating degree of detection method.
Brief description of the drawings
Fig. 1 serpentgrass medicinal material flavones ingredient thin-layer chromatograms, 1,2,3,4 be respectively test sample polarity A section things in collection of illustrative plates
Matter, Isorhamnetin reference substance, Quercetin reference substance, Kaempferol reference substance.Thin-layer chromatography condition is that temperature is 20 DEG C, and humidity is
60%, solvent is dichloromethane-acetone-methyl alcohol-formic acid=20:0.2:0.5:2.
Fig. 2 serpentgrass medicinal material flavones ingredient thin-layer chromatograms, 1,2,3,4 be respectively test sample polarity A section things in collection of illustrative plates
Matter, Isorhamnetin reference substance, Quercetin reference substance, Kaempferol reference substance.Condition is that temperature is 22 DEG C, humidity 70%, expansion
Agent is dichloromethane-acetone-methyl alcohol-formic acid=25:0.6:0.5:2.
Fig. 3 serpentgrass medicinal material flavones ingredient thin-layer chromatograms, 1,2,3,4 be respectively test sample polarity A section things in collection of illustrative plates
Matter, Isorhamnetin reference substance, Quercetin reference substance, Kaempferol reference substance.Condition is that temperature is 25 DEG C, humidity 80%, expansion
Agent is dichloromethane-acetone-methyl alcohol-formic acid=30:1.0:0.5:2.
Embodiment
The present invention is described in further detail with reference to embodiment.
Embodiment 1：
It is prepared by need testing solution：Take sample 10g to be placed in 500mL round-bottomed flasks, add absolute methanol 250mL, add
Mass fraction is 25%-30% watery hydrochloric acid 60mL, in 80 DEG C of water-baths backflow 2h, cools down, filters in ice bath immediately after taking-up,
Take filtrate in Rotary Evaporators evaporated under reduced pressure to remove solvent, residue adds 1mL methanol to make dissolving, centrifuging and taking supernatant, used
The silica gel mixed sample of 2g200-300 mesh, it is standby.
The silica gel 25g of 200-300 mesh is weighed, is stirred with dichloromethane to bubble-free, post is filled, column volume 70mL, uses
Dichloromethane：Methanol=100:5 elutions, polarity A sections, polarity B sections and the polarity C section eluents of silica gel post separation are collected respectively,
Solvent evaporated, respectively plus 2mL methanol redissolves, and obtains 3 parts of need testing solutions.
The preparation of reference substance solution：Accurate weigh is dried to the Quercetin of constant weight, Isorhamnetin, Kaempferol reference substance respectively
In right amount, it is respectively 0.4mgmL to add methanol that concentration is made-1、0.3mg·mL-1、0.4mg·mL-1Reference substance solution.
Thin-layer chromatography：Using silica gel thin-layer chromatography plate, take above-mentioned need testing solution and each 10 μ L of reference substance solution, put in away from
At the 10mm of silica gel g thin-layer plate base, using volume ratio as 20:0.2:0.5:2 dichloromethane-acetone-methyl alcohol-formic acid system conduct
Solvent, it is 20 DEG C in temperature, humidity is pre- flat in the side of the double flute expansion cylinder above-mentioned development system of addition under conditions of being 60%
Weigh 10min, ascending development, opens up away from for 10cm, carries out second outspread.
Colour developing：Spray is with 3% AlCl on to the lamellae volatilized3Test solution, dry, put fluorescence viewing thin layer color under uviol lamp
Spectrum, obtains serpentgrass medicinal material flavones ingredient collection of illustrative plates, sees Fig. 1.1,2,3,4 be respectively test sample polarity A sections material, different in collection of illustrative plates
Rhamnetin reference substance, Quercetin reference substance, Kaempferol reference substance, test sample polarity B sections and polarity C sections eluent are under uviol lamp
Immaculate.The R of Isorhamnetin in collection of illustrative platesfIt is worth for 0.8, the R of QuercetinfIt is worth for 0.35, the R of KaempferolfFor 0.70.
Embodiment 2：
As different from Example 1, the condition of tlc analysis is as follows, takes need testing solution and each 10 μ L of reference substance solution,
Point is 25 with volume ratio at away from silica gel g thin-layer plate base 10mm:0.6:0.5:2 dichloromethane-acetone-methyl alcohol-formic acid system
System be used as solvent, is 22 DEG C in temperature, humidity be 70% under conditions of in the side of the double flute expansion cylinder above-mentioned expansion system of addition
Unite pre-equilibration 10min, ascending development, opens up away from for 10cm, carries out second outspread.The condition of colour developing is to be sprayed on the lamellae volatilized
With 5% AlCl3, dry, put fluorescence viewing thin-layer chromatography spot under uviol lamp, obtain serpentgrass medicinal material flavones ingredient figure
Spectrum, is shown in Fig. 2.1,2,3,4 be respectively test sample polarity A sections material, Isorhamnetin reference substance, Quercetin reference substance, mountain in collection of illustrative plates
How phenol reference substance, test sample polarity B sections and polarity C sections the eluent immaculate under uviol lamp.The R of Isorhamnetin in collection of illustrative platesfValue
For 0.6, the R of QuercetinfIt is worth for 0.25, the R of KaempferolfFor 0.5.
Embodiment 3：
As different from Example 1, the condition of tlc analysis is as follows, takes need testing solution and each 10 μ L of reference substance solution,
Point is 30 with volume ratio at away from silica gel g thin-layer plate 10mm:1.0:0.5:2 dichloromethane-acetone-methyl alcohol-formic acid system is made
It it is 25 DEG C in temperature for solvent, humidity is pre- in the side of the double flute expansion cylinder above-mentioned development system of addition under conditions of being 80%
10min is balanced, ascending development, opens up away from for 10cm, carries out second outspread.The condition of colour developing be to spray on the lamellae volatilized with
7% AlCl3Solution, dry, put fluorescence viewing thin-layer chromatography under uviol lamp, obtain serpentgrass medicinal material flavones ingredient collection of illustrative plates,
See Fig. 3.1,2,3,4 be respectively test sample polarity A sections material, Isorhamnetin reference substance, Quercetin reference substance, Kaempferol in collection of illustrative plates
Reference substance, test sample polarity B sections and polarity C sections the eluent immaculate under uviol lamp.The R of Isorhamnetin in collection of illustrative platesfIt is worth and is
0.78, the R of QuercetinfIt is worth for 0.4, the R of KaempferolfFor 0.7.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the feature for combining the embodiment or example description
It is contained at least one embodiment or example of the present invention.In this manual, the schematic representation of above-mentioned term is differed
Surely identical embodiment or example are referred to.Moreover, specific features, structure, material or the feature of description can be any
Combined in an appropriate manner in one or more embodiments or example.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification, the scope of the present invention
Limited by claim and its equivalent.
Claims (6)
- A kind of 1. thin-layer chromatographic analysis method for detecting flavones ingredient in serpentgrass, it is characterised in that enter in accordance with the following steps OK：A. prepared by need testing solution：Serpentgrass medicinal powder is taken, is hydrolyzed and extracted with hydrochloric acid methanol, filtering, filtrate is evaporated, and residue adds Methanol makes dissolving, centrifuging and taking supernatant, solvent evaporated, makes redissolution with methanol, produces serpentgrass need testing solution；B. prepared by reference substance solution：Quercetin reference substance, Isorhamnetin reference substance and Kaempferol reference substance are taken, it is molten with methanol respectively Above-mentioned reference substance solution is made in solution；C. indentification by TLC：Thin-layer chromatography is carried out to reference substance solution described in need testing solution described in above-mentioned steps a and step b Analysis, analysis condition：GF254Lamellae, 22-25 DEG C of temperature, humidity 60%-80%, solvent：Dichloromethane-acetone-methanol- Formic acid=20-30:0.2-1.0:0.5:2, ascending development, after expansion, spray the AlCl with 3%-7%3Solution, it is right under uviol lamp to put Than detection；Wherein, the step a further comprises the residue that is evaporated for centrifuging gained supernatant carrying out column chromatography for separation, receives respectively Collect multiple polarity segment components, be evaporated respectively, make redissolution with methanol respectively, produce serpentgrass need testing solution；Described column chromatography For the silica gel column chromatography of 200-300 mesh, eluent is dichloromethane：Methanol=100:5；The multiple polarity section is 3,4 or 5 It is individual.
- 2. thin-layer chromatographic analysis method according to claim 1, it is characterised in that prepared by the step a need testing solutions In hydrochloric acid methanol hydrolysis extracting method be：The serpentgrass medicinal powder 10g of No. 3 sieves was taken, was placed in 500mL round-bottomed flasks, Absolute methanol 250mL is added, adds the watery hydrochloric acid 60mL that mass fraction is 25%-30%, is flowed back 2h in 80 DEG C of water-baths, after taking-up Cooled down immediately in ice bath.
- 3. thin-layer chromatographic analysis method according to claim 1, it is characterised in that reference substance solution in the step b Preparation method is：Precision weighs dry appropriate to the Quercetin of constant weight, Isorhamnetin, Kaempferol reference substance respectively, adds methanol system It is respectively 0.4mgmL into concentration-1、0.3mg·mL-1、0.4mg·mL-1Reference substance solution.
- 4. thin-layer chromatographic analysis method according to claim 1, it is characterised in that thin-layer chromatographic analysis are two in step c Secondary expansion thin-layered chromatography.
- 5. thin-layer chromatographic analysis method according to claim 4, it is characterised in that the condition of second expansion is the same as the One step development.
- 6. according to the thin-layer chromatographic analysis method described in claim any one of 1-5, it is characterised in that dichloro described in step c Methane-acetone-methanol-formic acid development system proportioning composition is 25:0.6:0.5:2.
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Denomination of invention: A TLC method for the determination of flavonoids in Polygonum viviparum Effective date of registration: 20210630 Granted publication date: 20171205 Pledgee: Bank of Bohai Limited by Share Ltd. Yichang branch|Bank of China Limited by Share Ltd. Three Gorges Branch|Bank of Hubei Limited by Share Ltd. Yichang Nanhu sub branch|Bank of Hankou Limited by Share Ltd. Yichang branch Pledgor: YICHANG SHANCHENGSHUIDU CORDYCEPS Co.,Ltd. Registration number: Y2021420000062 |