CN110196291A - The detection method of 7 kinds of flavones ingredients in a kind of Sabia parviflora Wall.ex Roxb medicinal material - Google Patents
The detection method of 7 kinds of flavones ingredients in a kind of Sabia parviflora Wall.ex Roxb medicinal material Download PDFInfo
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- CN110196291A CN110196291A CN201910361486.4A CN201910361486A CN110196291A CN 110196291 A CN110196291 A CN 110196291A CN 201910361486 A CN201910361486 A CN 201910361486A CN 110196291 A CN110196291 A CN 110196291A
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Abstract
The invention discloses a kind of detection methods of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material.The method is to carry out assay to Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII).The present invention has specificity strong, precision, stability, the advantage that repeatability is good and measurement result is accurate, and each ingredient is in good linear relationship with corresponding peak area value in concentration range.
Description
Technical field
The present invention relates to Sabia parviflora Wall.ex Roxb field, the inspection of 7 kinds of flavones ingredients in especially a kind of Sabia parviflora Wall.ex Roxb medicinal material
Survey method.
Background technique
Sabia parviflora Wall.ex Roxb Sabia parviflora Wall.ex Roxb. is Sabiaceae Sabiaceae fresh breeze Calamus
Sabia plant is the common drug of the national treatment hepatitis such as Miao in Guizhou, Bouyei, rheumatic arthralgia, traumatic injury.The southwest of Guizhou Province
Miao ethnic group be referred to as " Hlat det lod ninx (stupid beans old you) ".Seedling doctor thinks that its pharmacological property is cold, and bitter enters hot warp, has clear
Hot dampness removing detoxifies and dissipates stasis of blood and other effects；The Bouyei is referred to as " Jassy is strong ", is also commonly used for treatment icteric catarrhal jaundice.It is modern
Studies have shown that Sabia parviflora Wall.ex Roxb and belonging to various plants and mainly containing alkaloids, flavonoids and triterpenes components, have significant
Anti-hepatitis virus, liver protection, drop enzyme, it is anti-inflammatory the effects of, especially treatment hepatitis it is curative for effect, toxic side effect is small.Little Hua fresh breeze
Rattan is recorded in version " Guizhou Province's Chinese medicine, Ethnic crude drugs quality standard " in 2003, and only character identification, microscopical characters and physics and chemistry
Identify item；Now preparation " Longhua liver-clearing granule " (flower mast Qinggan Granules), " fresh breeze protect liver are developed by primary raw material of Sabia parviflora Wall.ex Roxb
Tea " etc., sales volume rises year by year in recent years.Although its contain in terms of medicinal and health care huge value of exploiting and utilizing and
Wide application prospect, but the research of quality control and pharmacological effect etc. is also extremely lagged at present, lack quality of medicinal material
The effective ways of control seriously hinder the sound development of related industry.
Summary of the invention
The object of the present invention is to provide a kind of detection methods of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material.This hair
It is bright to Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV),
The content of kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) carries out
Detection, the specificity of detection method is stronger, has the advantages that precision, stability, repeatability are good and measurement result is accurate, and
Each ingredient is in good linear relationship with corresponding peak area value in concentration range.
Technical solution of the present invention: the detection method of 7 kinds of flavones ingredients, the method in a kind of Sabia parviflora Wall.ex Roxb medicinal material
Be to Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV),
Kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) are carried out containing measurement
It is fixed.
It is described to Quercetin -3-O- rough gentian in Sabia parviflora Wall.ex Roxb medicinal material above-mentioned in the detection method of 7 kinds of flavones ingredients
Disaccharide glycosides (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside
(V), the method that Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) carry out assay includes following step
It is rapid:
(1) Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), reed the preparation of reference substance solution: are taken
Fourth (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin-
3-O- rutinoside (VII), respectively plus methanol solution dissolves and constant volume scale, shakes up, obtains each single reference substance stock solution；Divide again
It does not take each single reference substance stock solution to be placed in volumetric flask, adds methanol solution dissolution and constant volume scale molten to get mixing reference substance
Liquid；
(2) prepared by test solution: Sabia parviflora Wall.ex Roxb powder is taken, it is accurately weighed, and it is placed in conical flask, methanol is added in precision
Solution dissolution, weighed weight are cooled to room temperature after water-bath reflux, and again with methanol solution supplies the weight of less loss, shakes up, and are filtered,
Up to test solution；
(3) chromatographic condition: chromatographic column: Thermo Accucore-C18, 4.6 × 150mm, 2.6 μm；Detection wavelength:
360nm；Column temperature: 30 DEG C；Sample volume: 10 μ L；Flow velocity: 1.0mLmin-1；Mobile phase: 30% tetrahydrofurfuryl carbinol solution is A
Phase, is B phase acetonitrile, and 0.1% phosphate aqueous solution is C phase, gradient elution；
(4) measuring method: it is accurate respectively to draw reference substance solution and test solution, under above-mentioned chromatographic condition, respectively
Record Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV),
The peak area of kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII),
Calculate corresponding content to get.
In Sabia parviflora Wall.ex Roxb medicinal material above-mentioned in the detection method of 7 kinds of flavones ingredients, the preparation of the reference substance solution
Methanol solution used is 80% methanol solution in step；Volumetric flask is 10mL volumetric flask.
In Sabia parviflora Wall.ex Roxb medicinal material above-mentioned in the detection method of 7 kinds of flavones ingredients, each single reference substance deposit
Liquid is placed in volumetric flask, after adding methanol solution dissolution and constant volume scale, Quercetin -3-O- gentiobioside with cape jasmine (I),
Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), little Hua are clear
The mass concentration of wind rattan glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) is respectively 0.06250,0.2500,0.2500,
0.06750,0.06250,0.06250 and 0.05000mgmL-1。
In Sabia parviflora Wall.ex Roxb medicinal material above-mentioned in the detection method of 7 kinds of flavones ingredients, the test solution preparation
Step are as follows: precision weighs Sabia parviflora Wall.ex Roxb 0.9995~1.0005g of powder, is placed in conical flask, and 80% methanol is added in precision
Solution 20mL, weighed weight, water-bath refluxing extraction 1h are cooled to room temperature, the weight of less loss are supplied with 80% methanol solution, is shaken
Even, filtering takes subsequent filtrate to cross 0.45 μm of miillpore filter to get test solution.
In Sabia parviflora Wall.ex Roxb medicinal material above-mentioned in the detection method of 7 kinds of flavones ingredients, the program of the gradient elution is:
0-5min:5% → 12%A and 3% → 6%B；5-15min:12% → 10%A and 6% → 8%B；15-20min:10% →
20%A and 8% → 10%B；20-35min:20% → 53%A and 10% → 28%B；35-45min:53%A and 28%B；45-
50min:53% → 70%A and 28% → 30%B；50-60min:70% → 65%A and 30% → 35%B.
Inventor has carried out a large amount of experiment, is the research to detection method of the present invention below
Experimental example: detection method research
1. instrument and material
1.1 instrument
ThermoUltimate-3000 type high performance liquid chromatograph (German Thermo company)；Ten a ten thousandth of AG135 type
Electronic analytical balance (Mettler-Toledo company, Switzerland)；Water-bath (DK-98II, the limited public affairs of Tianjin Stettlen instrument
Department)；KQ-500DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；Chromatographic column: ThermoHypersil-
C18(4.6×150mm,2.6μm)。
1.2 reagent
Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A
(IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII), it is above
Reference substance is the monomeric compound voluntarily separated from Sabia parviflora Wall.ex Roxb cauline leaf, in conjunction with1H-NMR and13The spectral datas such as C-NMR
It identifies each compound structure, is calculated using HPLC areas of peak normalization method, mass fraction is all larger than 98%, for containing measurement
It is fixed；Methanol (U.S. world Co., Ltd, chromatographically pure；Sinopharm Chemical Reagent Co., Ltd. analyzes pure)；Acetonitrile (the U.S. day
Ground Co., Ltd, chromatographically pure)；Tetrahydrofuran (Tianjin great Mao chemical reagent factory, chromatographically pure)；Phosphoric acid is that analysis is pure；Experiment is used
Water is double distilled water.
1.3 sample source
Experiment is all from Guizhou university of TCM with sample and introduces a fine variety, and awards and be accredited as through Guizhou university of TCM Sun Qing culture and education
Sabiaceae plant Sabia parviflora Wall.ex Roxb (Sabia parviflora Wall.ex Roxb.) crushed No. 6 after 50 DEG C dry
Sieve, is stored in spare in drier；Plant voucher specimen is stored in Guizhou university of TCM crude drug laboratory, and sample details are shown in
Table 1.
1 Sabia parviflora Wall.ex Roxb sample source information of table
2, measuring method
The preparation of 2.1 reference substance solutions
Respectively precision weigh Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III),
Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rue
Fragrant glucosides (VII) reference substance 10.00mg adds the dissolution of 80% methanol solution and constant volume scale, shakes up, obtain each single reference substance deposit
Liquid；It takes appropriate single reference substance solution to be placed in 10mL volumetric flask respectively again, adds the dissolution of 80% methanol solution and constant volume scale, i.e.,
Obtaining mass concentration is respectively 0.06250,0.2500,0.2500,0.06750,0.06250,0.06250,0.05000mgmL-1
Mixed reference substance solution, it is spare.
2.2 sample solution preparation method
Medicinal powder about 1.0g is taken, it is accurately weighed, it is placed in conical flask, 80% methanol solution 20mL is added in precision, claims
Determine weight, water-bath refluxing extraction 1h is cooled to room temperature, the weight of less loss is supplied with 80% methanol solution, is shaken up, and filtering takes
Subsequent filtrate cross 0.45 μm of miillpore filter to get.
2.3 chromatographic condition
Chromatographic column: Thermo Accucore-C18(4.6 × 150mm, 2.6 μm)；Detection wavelength: 360nm；Column temperature: 30 DEG C；
Sample volume: 10 μ L；Flow velocity: 1.0mLmin-1；Mobile phase: 30% tetrahydrofurfuryl carbinol solution (A)--0.1% phosphoric acid of acetonitrile (B)
Aqueous solution (C) gradient elution, elution program are 0-5min:A (5% → 12%) and B (3% → 6%)；5-15min:A (12% →
And B (6% → 8%) 10%)；15-20min:A (10% → 20%) and B (8% → 10%)；20-35min:A (20% →
And B (10% → 28%) 53%)；35-45min:A (53%) and B (28%)；45-50min:A (53% → 70%) and B (28%
→ 30%)；50-60min:A (70% → 65%) and B (30% → 35%).
3, methodological study
3.1 system suitabilities and specificity test
Precision draws above-mentioned mixed reference substance solution, test solution and blank solution, under " 2.3 " item chromatographic condition into
Sample measurement, the results show that blank solution has no chromatographic peak in corresponding position, illustrates that blank solution is noiseless；Test solution
The retention time presented 1 in chromatogram, in the retention time and mixed reference substance solution at 2,3,4,5,6 and No. 7 peaks is consistent,
Its purity factor is followed successively by 998.98,999.87,999.88,998.69,998.97,999.98,999.98, and peak purity is good,
Separating degree is all larger than 1.5, sees Fig. 1；And UV absorption spectrum curve figure is more consistent, sees Fig. 2, shows that this method specificity is good.
The investigation of 3.2 linear relationships
Accurate " 2.1 " item reference substance that places an order of drawing is diluted to various concentration respectively, chemical compounds I (0.1250,0.09380,
0.06250,0.03130,0.01000,0.001000mg/mL), compound ii (0.5000,0.3750,0.2500,0.1250,
0.01000,0.001000mg/mL), compound III (1.000,0.7500,0.5000,0.2500,0.01000,
0.001000mg/mL), compounds Ⅳ (0.2500,0.1875,0.1250,0.06250,0.001000,0.0005000mg/
ML), compound V (0.2500,0.1880,0.1250,0.06250,0.05000,0.01000mg/mL), compound VI
(0.1020,0.07500,0.05000,0.02500,0.01000,0.001000mg/mL) and compound VII (0.4000,
0.3000,0.2000,0.1000,0.05000,0.001000mg/mL), peak area is measured under " 2.3 " item chromatographic condition, with
Peak area value is ordinate (Y), and each composition quality concentration is that abscissa (X) draws standard curve, and carries out linear regression analysis,
Obtain regression equation and related coefficient.Table 2 is the results show that 7 kinds of reference substances are in good with peak area value in respective concentration range
Linear relationship.
The regression equation and the range of linearity of 2 Sabia parviflora Wall.ex Roxb medicinal material of table
3.3 precision test
Precision draws the mixed reference substance solution prepared under " 2.1 " item, presses " 2.3 " item chromatographic condition continuous sample introduction 6 times, note
Record Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), mountain
How phenol -3-O- rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) peak area, meter
The RSD value for calculating each Component peak area is respectively 0.80%, 0.83%, 0.66%, 1.1%, 0.82%, 1.4%, 0.96%, table
Bright instrument precision is good, the results are shown in Table 3 and Fig. 3.
3 Precision test result of table (n=6)
3.4 stability test
Take same sample powder about 1.0g, it is accurately weighed, by legal system available test sample solution below " 2.2 " item, respectively 0,
2,4,6,8,12,24,36h is tested by chromatographic condition sample introduction under " 2.3 " item, record Quercetin -3-O- gentiobioside with cape jasmine (I),
Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), little Hua are clear
The peak area of wind rattan glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII), calculate its RSD value be followed successively by 0.55%, 1.0%,
0.75%, 0.80%, 0.52%, 0.23%, 0.23%, respectively less than 2.0%, show that test solution is stablized in 36h, as a result
It is shown in Table 4 and Fig. 4.
4 stability test result (n=8) of table
3.5 repetitive test
Precision weighs 6 parts of same sample powder, every part of 1.0g, and it is molten to prepare 6 parts of test samples in parallel by method under " 2.2 " item
Liquid distinguishes sample introduction measurement under " 2.3 " item chromatographic condition.Record Quercetin -3-O- gentiobioside with cape jasmine (I),
Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), little Hua are clear
The peak area of wind rattan glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII), calculate its average amount be respectively 1.225,
5.134、3.937、1.465、0.6857、0.9620、1.005mg·g-1, RSD value is respectively 0.86%, 0.87%, 1.4%,
0.62%, 1.6%, 1.4%, 0.80%, show that this method repeatability is good, the results are shown in Table 5 and Fig. 5.
5 repeatability of table investigates result (n=6)
3.6 sample-adding recovery tests
Precision weighs 9 parts of sample of known content, and every part of about 0.5g is placed in 50mL conical flask, by 1:0.5,1:1,1:
1.5 ratio is separately added into reference substance, is operated by the method for preparing test sample under " 2.2 " item, and test solution is made, according to
According to being measured under " 2.3 " item chromatographic condition.The result shows that Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside
(II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and different
The average recovery rate of rhamnetin -3-O- rutinoside (VII) is respectively 98.97%, 99.36%, 98.21%, 99.14%,
97.87%, 98.02,98.92%, RSD value is respectively 1.6%, 1.4%, 1.2%, 1.6%, 1.2%, 1.4%, 1.9%,
Show that this method accuracy is good, the results are shown in Table 6.
Table 6 is loaded recovery experiment result
The measurement of 3.7 samples
Sabia parviflora Wall.ex Roxb medicinal powder is taken, by test solution is prepared under " 2.2 " item, by under chromatographic condition under " 2.3 " item
Sample introduction measurement, record Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III),
Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rue
The peak area of fragrant glucosides (VII), and calculate the dry product content of each ingredient；The result is shown in table 7~8, HPLC chromatogram stacking chart sees Fig. 6-
7。
Assay result (the mgg of 7 Sabia parviflora Wall.ex Roxb medicinal material Different plant parts of table-1)
Assay result (the mgg of 8 Sabia parviflora Wall.ex Roxb medicinal material Different Harvesting Time of table-1)
4, data are analyzed
The distribution of the effective component of 4.1 Different plant parts
Active components in medicinal plant is spatially not uniformly distributed in entire plant, and synthesis and storage site tend to vary with
The development of plant constantly synthesize and be transferred to other sites and be further metabolized, store or degrade.Little Hua fresh breeze as shown in Figure 8
7 kinds of flavones ingredient tender leafs of rattan, old leaf and annual stem are just the same, the only difference in amount, fail to detect in old stem
To kaempferol-3-O-rutinoside (V) ingredient, root position fails to detect kaempferol-3-O-rutinoside (V) He little Hua fresh breeze
Rattan glycosides (VI).
The chemical component accumulation dynamic analysis of different growing stages in 4.2 leaves
Leaf is that plant carries out photosynthetic major organs, is played an important role to the vital movement of plant, plant is developing
Early stage secondary metabolite just will appear, and the critical stage that early period is plant life be developed, because plant must be at this
Period establishes the ecosystem of oneself.It can be obtained by Fig. 9-11, chemical compounds I is in bimodal trend, respectively 3.340 Hes in 2 and August
1.348mg·g-1, occurring lowest trough June is 0.7401mgg-1.There is quintet in 2,5,8,10 and December in compound ii,
With postponing for phenological period, peak is gradually reduced, wherein 2 months contents are up to 8.716mgg-1, reaching lowest trough June is
2.029mg·g-1, it is equivalent to nearly 4 times of ingredient degradation.Compound III content to maximum value is in fast enriching in 1~April
9.100mg·g-1Although occurring peak again in September in 4~12 months, whole content, which is appointed, is in downward trend, in 10 and 11
There is most ebb, respectively 1.785 and 1.554mgg in the moon-1.Compounds Ⅳ in addition to 6,7 and September it is relatively low other than, remaining is all
Variation is little.It is 4.581mgg that compound V, which is top in April,-1, it is November nearly 27 times of lowest trough, is presented in 6 and September
Bimodal trend, respectively 2.319 and 1.884mgg-1.Top is presented in July in compound VI, is 1.004mgg-1, present
It is intermediate high, the low trend in both sides.Compound VII is progressively enriched in 1~April, and April reaches top, is 1.917mgg-1, 4~
Degradation trend is integrally presented in December.
Using 7 software of GraphPadPrism to 7 kinds of flavonoids in the Sabia parviflora Wall.ex Roxb leaf site acquired the 1-12 month
The analysis of object row is reference with the thermal map band on the right by Figure 12 heat at Tu Ke get, as the increase and color change of number can be seen
The height of content out, thermal map longitudinal direction colour band analyze the accumulated state of 7 kinds of flavones ingredients in every month:
1~March is VII > compound of compound ii > compound III > compounds Ⅳ > chemical compounds I > compound, V >
Compound VI, April are V > compound ii > chemical compounds I > compounds Ⅳ > compound of compound III > compound, VII > chemical combination
Object VI, May are V > compound of compound ii > compound III > compounds Ⅳ > chemical compounds I > compound, VII > compound
VI, June be V > compound ii > compounds Ⅳ > compound of compound III > compound, VII > chemical compounds I > compound VI, 7
The moon is VII > compound of compound ii > compound III > compounds Ⅳ > chemical compounds I > compound, VII > compound V, and August is
VII > compound of compound ii > compounds Ⅳ > compound III > chemical compounds I > compound, VI > compound V, September is chemical combination
III V > compound of > compound ii > compounds Ⅳ > compound of object, VII > chemical compounds I > compound VI, 10~November is
VII > compound of compound ii > compounds Ⅳ > compound III > chemical compounds I > compound, VI > compound V, December are to change
Close II VII > compound of > compound III > compounds Ⅳ > chemical compounds I > compound of object, V > compound VI.
Beneficial effects of the present invention: compared with prior art, the present invention to Quercetin -3-O- gentiobioside with cape jasmine (I),
Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), little Hua are clear
The content of wind rattan glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) is detected, detection method good, concentration with specificity
Range and chromatographic condition peak area value in good linear relationship, precision is good, stability is good, reproducible and measurement result is quasi-
True advantage.Result of study provides scientific basis for the determination of Sabia parviflora Wall.ex Roxb leaf suitable collection period, also having for its resource
Effect, to excavate the resource utilization value and Developing Prospects on Industrialized Exploitation of Sabia parviflora Wall.ex Roxb, may be used also using data supporting is provided
Theoretical base is established for the control of Sabia parviflora Wall.ex Roxb quality, breed improvement, stock breeding and the resources development and utilization of Sabia parviflora Wall.ex Roxb
Plinth.
Detailed description of the invention
Fig. 1 is the HPLC chromatogram that specificity is investigated；
Fig. 2 is the UV spectrogram of each ingredient in test sample and reference substance solution；
Fig. 3 is the HPLC chromatogram stacking chart of precision test；
Fig. 4 is the HPLC chromatogram stacking chart of stability test；
Fig. 5 is the HPLC chromatogram stacking chart of repetitive test；
Fig. 6 is the HPLC chromatogram stacking chart of 12 batches of picking time samples；
Fig. 7 is the HPLC stacking chart of Sabia parviflora Wall.ex Roxb Different plant parts；
Fig. 8 is the accumulation histogram of 7 kinds of flavone compounds；
Fig. 9 is Quercetin -3-O- gentiobioside with cape jasmine (I) and the accumulation dynamic stacking figure of Camellianoside (II)
Figure 10 is rutin (III) and the accumulation dynamic stacking figure of Tsubakioside A (IV)；
Figure 11 is kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside
(VII) accumulation dynamic stacks figure；
Figure 12 is the heat of seven kinds of flavones ingredient accumulation dynamics in Sabia parviflora Wall.ex Roxb leaf into figure.
The label in accompanying drawing is: A- mixed reference substance solution in Fig. 1, B- test solution, C- blank solution, 1: quercitrin
Element -3-O- gentiobioside with cape jasmine (I), 2:Camellianoside (II), 3: rutin (III), 4:Tsubakioside A (IV), 5:
Kaempferol-3-O-rutinoside (V), 6: Sabia parviflora Wall.ex Roxb glycosides (VI), 7: Isorhamnetin -3-O- rutinoside (VII).
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawings and examples, but be not intended as to the present invention limit according to
According to.
Embodiment 1.The detection method of 7 kinds of flavones ingredients in a kind of Sabia parviflora Wall.ex Roxb medicinal material, the method is to quercitrin
Element -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), Kaempferol -3-
O- rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) carry out assay.It is specific to survey
Determine method are as follows:
(1) preparation of reference substance solution: respectively precision weigh Quercetin -3-O- gentiobioside with cape jasmine (I),
Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), little Hua are clear
Wind rattan glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) reference substance 10.00mg, add the dissolution of 80% methanol solution and constant volume is carved
Degree, shakes up, obtains each single reference substance stock solution；It takes appropriate single reference substance solution to be placed in 10mL volumetric flask respectively again and adds 80%
Methanol solution dissolution and constant volume scale to get mass concentration be respectively 0.06250,0.2500,0.2500,0.06750,
0.06250、0.06250、0.05000mg·mL-1Mixed reference substance solution, it is spare.
(2) sample solution preparation method: taking medicinal powder about 1.0g, accurately weighed, is placed in conical flask, is added 80%
Methanol solution 20mL, weighed weight, water-bath refluxing extraction 1h is cooled to room temperature, and supplies less loss with 80% methanol solution
Weight shakes up, filtering, take subsequent filtrate cross 0.45 μm of miillpore filter to get.
(3) chromatographic condition: chromatographic column: ThermoAccucore-C18(4.6 × 150mm, 2.6 μm)；Detection wavelength:
360nm；Column temperature: 30 DEG C；Sample volume: 10 μ L；Flow velocity: 1.0mLmin-1；Mobile phase: 30% tetrahydrofurfuryl carbinol solution (A)-
Acetonitrile (B) -0.1% phosphate aqueous solution (C) gradient elution, elution program be 0-5min:A (5% → 12%) and B (3% →
6%)；5-15min:A (12% → 10%) and B (6% → 8%)；15-20min:A (10% → 20%) and B (8% → 10%)；
20-35min:A (20% → 53%) and B (10% → 28%)；35-45min:A (53%) and B (28%)；45-50min:A
(53% → 70%) and B (28% → 30%)；50-60min:A (70% → 65%) and B (30% → 35%).
(4) measuring method: it is accurate respectively to draw reference substance solution and test solution, under above-mentioned chromatographic condition, respectively
Measure Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV),
The peak area of kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII),
Calculate its corresponding content to get.
Claims (6)
1. the detection method of 7 kinds of flavones ingredients in a kind of Sabia parviflora Wall.ex Roxb medicinal material, it is characterised in that: the method is to quercitrin
Element -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), Kaempferol -3-
O- rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) carry out assay.
2. the detection method of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material according to claim 1, it is characterised in that:
It is described to Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A
(IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) carry out
The method of assay includes the following steps:
(1) Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin the preparation of reference substance solution: are taken
(III), Tsubakioside A (IV), kaempferol-3-O-rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-
O- rutinoside (VII), respectively plus methanol solution dissolves and constant volume scale, shakes up, obtains each single reference substance stock solution；Distinguish again
It takes each single reference substance stock solution to be placed in volumetric flask, adds methanol solution dissolution and constant volume scale is to get mixed reference substance solution；
(2) prepared by test solution: Sabia parviflora Wall.ex Roxb powder is taken, it is accurately weighed, and it is placed in conical flask, methanol solution is added in precision
Dissolution, weighed weight, water-bath reflux after be cooled to room temperature, again with methanol solution supplies the weight of less loss, shake up, filtering to get
Test solution；
(3) chromatographic condition: chromatographic column: Thermo Accucore-C18, 4.6 × 150mm, 2.6 μm；Detection wavelength: 360nm；Column
Temperature: 30 DEG C；Sample volume: 10 μ L；Flow velocity: 1.0mLmin-1；Mobile phase: 30% tetrahydrofurfuryl carbinol solution is A phase, is B phase second
Nitrile, 0.1% phosphate aqueous solution are C phase, gradient elution；
(4) measuring method: accurate absorption reference substance solution and test solution record respectively under above-mentioned chromatographic condition respectively
Quercetin -3-O- gentiobioside with cape jasmine (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), kaempferia galamga
The peak area of phenol -3-O- rutinoside (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) calculates
Its corresponding content to get.
3. the detection method of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material according to claim 2, it is characterised in that:
Methanol solution used is 80% methanol solution in the preparation step of the reference substance solution；Volumetric flask is 10mL volumetric flask.
4. the detection method of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material according to claim 2, it is characterised in that:
Each single reference substance stock solution is placed in volumetric flask, after adding methanol solution dissolution and constant volume scale, obtains Quercetin -3-O- dragon
Gallbladder disaccharide glycosides (I), Camellianoside (II), rutin (III), Tsubakioside A (IV), Kaempferol -3-O- rutinose
The mass concentration of glycosides (V), Sabia parviflora Wall.ex Roxb glycosides (VI) and Isorhamnetin -3-O- rutinoside (VII) is respectively 0.06250,
0.2500,0.2500,0.06750,0.06250,0.06250 and 0.05000mgmL-1。
5. the detection method of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material according to claim 2, it is characterised in that institute
The step of stating test solution preparation are as follows: precision weighs Sabia parviflora Wall.ex Roxb 0.9995~1.0005g of powder, is placed in conical flask,
80% methanol solution 20mL is added in precision, and weighed weight, water-bath refluxing extraction 1h is cooled to room temperature, molten with 80% methanol
Liquid supplies the weight of less loss, shakes up, and filtering takes subsequent filtrate to cross 0.45 μm of miillpore filter to get test solution.
6. the detection method of 7 kinds of flavones ingredients in Sabia parviflora Wall.ex Roxb medicinal material according to claim 2, it is characterised in that:
The program of the gradient elution is: 0-5min:5% → 12%A and 3% → 6%B；5-15min:12% → 10%A and 6% →
8%B；15-20min:10% → 20%A and 8% → 10%B；20-35min:20% → 53%A and 10% → 28%B；35-
45min:53%A and 28%B；45-50min:53% → 70%A and 28% → 30%B；50-60min:70% → 65%A and
30% → 35%B.
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