CN107764911A - Herbal mixture has the sample construction method of chemical composition content difference - Google Patents
Herbal mixture has the sample construction method of chemical composition content difference Download PDFInfo
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- 239000000203 mixture Substances 0.000 title claims abstract description 45
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- 238000010276 construction Methods 0.000 title claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 37
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- 239000000463 material Substances 0.000 claims abstract description 23
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- 239000009277 Panax notoginseng extract Substances 0.000 claims abstract description 10
- 239000002775 capsule Substances 0.000 claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 7
- 239000008187 granular material Substances 0.000 claims abstract description 6
- 239000009683 kouyanqing Substances 0.000 claims abstract description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 238000013461 design Methods 0.000 claims description 20
- 239000012071 phase Substances 0.000 claims description 20
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 16
- 239000004615 ingredient Substances 0.000 claims description 9
- 244000163122 Curcuma domestica Species 0.000 claims description 8
- 235000003392 Curcuma domestica Nutrition 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 8
- 125000003118 aryl group Chemical group 0.000 claims description 8
- 238000007621 cluster analysis Methods 0.000 claims description 8
- 235000003373 curcuma longa Nutrition 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 235000013976 turmeric Nutrition 0.000 claims description 8
- 241001074093 Echinopsis Species 0.000 claims description 7
- 239000009636 Huang Qi Substances 0.000 claims description 7
- 244000131316 Panax pseudoginseng Species 0.000 claims description 7
- 235000003181 Panax pseudoginseng Nutrition 0.000 claims description 7
- 240000007164 Salvia officinalis Species 0.000 claims description 7
- 240000001398 Typha domingensis Species 0.000 claims description 7
- 235000005412 red sage Nutrition 0.000 claims description 7
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- 240000002948 Ophiopogon intermedius Species 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 241000432824 Asparagus densiflorus Species 0.000 claims description 5
- 239000007791 liquid phase Substances 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 108010074864 Factor XI Proteins 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 108010076282 Factor IX Proteins 0.000 claims description 2
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 2
- 230000001419 dependent effect Effects 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims 3
- 238000002474 experimental method Methods 0.000 claims 1
- 238000007689 inspection Methods 0.000 claims 1
- 230000003595 spectral effect Effects 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
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- 239000000284 extract Substances 0.000 description 12
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- 238000004458 analytical method Methods 0.000 description 7
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- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 5
- 235000021050 feed intake Nutrition 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
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- 238000000605 extraction Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
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- 235000008216 herbs Nutrition 0.000 description 3
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- 238000010992 reflux Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- -1 filtration Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 241000432767 Asparagus setaceus Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- AZHSSKPUVBVXLK-UHFFFAOYSA-N ethane-1,1-diol Chemical compound CC(O)O AZHSSKPUVBVXLK-UHFFFAOYSA-N 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000002803 maceration Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 208000003265 stomatitis Diseases 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000004724 ultra fast liquid chromatography Methods 0.000 description 1
Classifications
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- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
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Abstract
The invention discloses the sample construction method that a kind of herbal mixture has chemical composition content difference.And the structure and detection method of FUFANG XUESHUANTONG JIAONANG, Naoshuantong capsule and kouyanqing granules difference sample are further disclosed.This method comprises the following steps：With herbal mixture each component separately as experimental factor, the excursion of negated monarch drug in a prescription component is 2 times of 0% to its prescription content, and the excursion of monarch drug in a prescription medicinal material subtracts non-monarch drug in a prescription component content sum for 100%, and determines suitable number of levels within the range.Each component and its inventory are uniformly arranged；Multigroup difference sample is prepared according to standard process flows；And the finger-print of each group difference sample is made, the otherness of difference sample room is verified using clustering method.
Description
Technical field
The present invention relates to the construction method of herbal mixture difference sample.
Background technology
The performance of herbal mixture entirety curative effect is the result of effective component group and Mutiple Targets interphase interaction.Work as active ingredient
When group changes, its drug effect can also change therewith.On this basis, according to correlation between composition and drug effect, can distinguish
Analyse the effective component group in Chinese medicine.The relevance changed by the changes of contents of herbal mixture ingredients with its bioactivity,
Can clear contribution of its ingredients to drug effect, effect and between interaction, be capable of the group of science parsing herbal mixture
Square intension.
In order to preferably study change and the correlation between drug effect of herbal mixture Chinese medicine, composition, its prescription section is specified
Intension and core active components group are learned, the structure with medicinal material, the herbal mixture sample of chemical composition content difference, is to solve to ask
The key point of topic.In traditional Chemistry for Chinese Traditional Medicine research, separated one by one using chemical composition, prepare sample by the way of recombinant
Product, but on the one hand this method realizes that difficulty is big, working strength is high, on the other hand do not meet the Combination, globality, system of Chinese medicine
One property feature.
The content of the invention
It is an object of the invention to：Establishing herbal mixture has the sample construction method of chemical composition content difference, for solution
Certainly the identification problem of the parsing of herbal mixture prescription intension and active ingredient group provides innovative approach support.
The technical solution adopted by the present invention is as follows：Herbal mixture ingredients component is individually regarded as experimental factor, according to
The inventory excursion of original prescription ratio-dependent ingredients, and suitable number of levels is determined within the range；Using uniform
Design method, the uniform arrangement of suitable uniform designs table progress medicinal material and its inventory is selected according to factor number, number of levels；Press
Multigroup difference sample is prepared according to standard process flows, and makes the monitoring of its finger-print, difference is verified using clustering method
The otherness of sample room, that is, complete the structure of the difference sample.The herbal mixture of this method 3 to 6 kinds of Chinese medicines of preferred component.
The experimental factor number is the component number of herbal mixture.The preferred 9-11 of test level number is horizontal.
The inventory excursion design method of each component is：The excursion of non-monarch drug in a prescription component contains for 0% to its prescription
2 times of amount, the excursion of monarch drug in a prescription medicinal material subtract non-monarch drug in a prescription component content sum for 100%.
The outstanding advantages of the present invention：Compared with prior art, the beneficial effects of the present invention are make public for the first time in compound
Medical instrument has the sample construction method of chemical composition content difference, and this method is especially suitable for the herbal mixture of 3 to 6 taste medicinal materials composition.
This method chemical composition different from the past is separated one by one, the mode of recombinant, but the medicinal material of herbal mixture is entered from entirety
Row is readjusted, combined, and is had and is realized the advantages of difficulty is small, applicability is high, meets the Combination, globality, uniformity of Chinese medicine
The characteristics of.For the clinical practice of Compound Xueshuantong preparation, further exploitation provides innovative support.
Brief description of the drawings
Fig. 1：The FUFANG XUESHUANTONG JIAONANG difference sample HPLC chromatogram that the embodiment of the present invention 1 provides.
Fig. 2：The FUFANG XUESHUANTONG JIAONANG difference sample clustering analysis result figure that the embodiment of the present invention 1 provides.
Fig. 3：The Naoshuantong capsule difference sample HPLC chromatogram that the embodiment of the present invention 2 provides.
Fig. 4：The Naoshuantong capsule difference sample clustering analysis result that the embodiment of the present invention 2 provides.
Fig. 5：The kouyanqing granules difference sample LC-MS/MS total ion current figures that the embodiment of the present invention 3 provides.
Fig. 6：The kouyanqing granules difference sample clustering analysis result that the embodiment of the present invention 3 provides.
Embodiment
Construction method of the present invention is described in further detail with reference to specific embodiment.
Embodiment 1：The FUFANG XUESHUANTONG JIAONANG being made up of pseudo-ginseng, the red sage root, the Radix Astragali, radix scrophulariae four traditional Chinese medicine material has chemical composition
The sample structure of content difference
1. material
(1) instrument, reagent：Numerical control ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd, KQ-250DE), vortex oscillation
Device (Scientific Industries Vortex-Genie 2), rotary evaporator (Shanghai Yarong Biochemical Instrument Plant, RE-
5205/RE-2000B), baking oven (Millipore, UFB400), Superpure water machine (Millipore Simplicity), electronic analysis
The glass apparatus such as balance (Sartorius), refrigerator (Siemens), beaker, conical flask, cucurbit；Methanol, acetonitrile, phosphoric acid (color
Compose pure), ultra-pure water.
(2) medicinal material：Pseudo-ginseng, lot number：161110；The red sage root, lot number：161110；The Radix Astragali, lot number：161110；Radix scrophulariae, lot number：
161110, provided by Guangdong Zhongsheng Pharmaceutical Co., Ltd.
2. method
(1) pseudo-ginseng, the red sage root, the Radix Astragali, radix scrophulariae feed intake design
In the original prescription ratio of FUFANG XUESHUANTONG JIAONANG pseudo-ginseng, the red sage root, the Radix Astragali, radix scrophulariae proportion be respectively 54%,
10%th, 18%, 18%.In uniform design, red sage root even variation between 0%-20%, the Radix Astragali uniformly becomes between 0%-36%
Change, radix scrophulariae even variation between 0%-36%, monarch drug in a prescription pseudo-ginseng even variation between 33.2%-74.8%.According to four factors 11
Horizontal homogeneous design table is fed intake, and is shown in Table 1.
The medicinal material inventory and its ratio of the FUFANG XUESHUANTONG JIAONANG difference sample of table 1
(2) preparation of difference sample
According to《Pharmacopoeia of People's Republic of China》Version FUFANG XUESHUANTONG JIAONANG standard process flows in 2015, are carried out by table 1
Medicinal material feeds intake：Pseudo-ginseng is extracted 2 times after crushing with 50% alcohol dipping, and maceration extract filtration, filtrate merges, and is reclaimed ethanol and is condensed into
Medicinal extract；The red sage root, the Radix Astragali, the taste medicinal material of radix scrophulariae three, with 50% ethanol heating and refluxing extraction 2 times, extract solution filtration, filtrate merges, and returns
Receive ethanol and be condensed into medicinal extract；Above-mentioned two parts of medicinal extract is mixed, produced.
(3) the finger-print monitoring of difference sample
Instrument and reagent：Dionex Ultimate 3000DGLC high performance liquid chromatographs, Dionex
120C18 chromatographic columns (150mm × 4.6mm, 3um), acetonitrile, phosphoric acid (chromatographically pure), ultra-pure water.
It is prepared by test sample：11 groups of difference samples each about 0.3g is taken, it is accurately weighed, put in conical flask with cover, add 70% methanol
20mL, close plug, 60min is ultrasonically treated, filtration, takes filtrate, solvent is recovered under reduced pressure and is done near, adds 50% methanol to make dissolving, it is quantitative
10mL measuring bottles are transferred to, add 50% methanol to shake up to scale, filtered with 0.22um miillpore filter, take subsequent filtrate, produce.
Liquid phase chromatogram condition：Mobile phase A acetonitrile (0-50min, 15% → 34%), the phosphoric acid solution of Mobile phase B 0.05%
(50-59min, 34% → 75%), flow velocity 1.0mL/min, 25 DEG C of column temperature, Detection wavelength 203nm, 270nm.
3. result
Finger-print and its cluster analysis result：The chromatogram of 11 groups of difference samples is shown in Fig. 1.According to 20 shared Feng Feng faces
Product, is conducted into SPSS 21.0 and difference sample S1-S11 is clustered, clustering method Between-groups
Linkage, distance calculating method use Minkowski.As shown in Fig. 2 work as Rescaled Distance Cluster
When Combine is 4, difference sample can be divided into 8 classes, obvious chemical composition content difference be present.It follows that by original place
On square proportional basis, ingredients is allocated by Uniform ity Design Method, can be built with chemical composition content difference
Traditional Chinese medicine sample, the characteristics of meeting Chinese medicine globality.
Embodiment 2：The Naoshuantong capsule being made up of cattail pollen, the radix paeoniae rubrathe, root tuber of aromatic turmeric, rhizoma Gastrodiae, Radix Rhapontici seu Radix Echinopsis gomi herbs have chemistry into
Divide the sample structure of content difference
1. material
(1) instrument, reagent：Numerical control ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd, KQ-250DE), vortex oscillation
Device (Scientific Industries Vortex-Genie 2), rotary evaporator (Shanghai Yarong Biochemical Instrument Plant, RE-
5205/RE-2000B), baking oven (Millipore, UFB400), Superpure water machine (Millipore Simplicity), electronic analysis
The glass apparatus such as balance (Sartorius), refrigerator (Siemens), beaker, conical flask, cucurbit；Methanol, acetonitrile, tetrahydrochysene furan
Mutter, phosphoric acid (chromatographically pure), ultra-pure water.
(2) medicinal material：Cattail pollen, lot number：160604；The radix paeoniae rubrathe, lot number：160604；Root tuber of aromatic turmeric, lot number：160604；Rhizoma Gastrodiae, lot number：
160604；Radix Rhapontici seu Radix Echinopsis, lot number：160604.There is provided by GuangDong HuaNan Pharmacy Group Co., Ltd.
2. method
(1) cattail pollen, the radix paeoniae rubrathe, root tuber of aromatic turmeric, rhizoma Gastrodiae, Radix Rhapontici seu Radix Echinopsis feed intake design
In the original prescription ratio of Naoshuantong capsule cattail pollen, the radix paeoniae rubrathe, root tuber of aromatic turmeric, rhizoma Gastrodiae, Radix Rhapontici seu Radix Echinopsis accounting example be respectively 33%,
24%th, 19%, 10%, 14%.In uniform design, radix paeoniae rubrathe even variation between 0%-48%, root tuber of aromatic turmeric between 0%-38%
Even change, rhizoma Gastrodiae even variation between 0%-20%, Radix Rhapontici seu Radix Echinopsis change between 0%-28%, and monarch drug in a prescription cattail pollen is in 18.5%-
47.5% even variation.Fed intake according to the horizontal homogeneous design table of five factor nine, be shown in Table 2.
The brain bolt of table 2 leads to the medicinal material inventory and its ratio of difference sample
(2) difference sample preparation
According to《Pharmacopoeia of People's Republic of China》Version Naoshuantong capsule standard process flows in 2015, medicinal material is carried out by table 2
Feed intake：The radix paeoniae rubrathe adds 70% ethanol, and heating and refluxing extraction 2 times, 1 hour every time, filtrate recycling ethanol was simultaneously condensed into medicinal extract；Root tuber of aromatic turmeric adds
80% ethanol, heating and refluxing extraction 2 times, 1 hour every time, merge extract solution, filtration, filtrate is standby, the dregs of a decoction and cattail pollen, rhizoma Gastrodiae,
Radix Rhapontici seu Radix Echinopsis adds water to cook 2 times, and 1 hour every time, collecting decoction, filtration, it was 1.04~1.10 (40 DEG C) that filtrate, which is concentrated into relative density,
Medicinal extract, add ethanol alcohol content is reached 70%, refrigerate 48 hours, take supernatant to merge with the alcohol extract of root tuber of aromatic turmeric, recovery ethanol
And it is concentrated into medicinal extract；Above-mentioned two parts of medicinal extract is mixed, produced.
(3) the finger-print monitoring of difference sample
Instrument and reagent：Dionex Ultimate 3000DGLC high performance liquid chromatographs, Dionex
120C18 chromatographic columns (150mm × 4.6mm, 3um), acetonitrile, tetrahydrofuran, phosphoric acid (chromatographically pure), ultra-pure water.
It is prepared by test sample：9 groups of difference samples each about 0.4g is taken, it is accurately weighed, conical flask is put, adds 50% methanol 30mL, it is close
Plug, 30min is ultrasonically treated, lets cool, shakes up, filtered, filtrate decompression recycling design, residue is dissolved with 30% methanol, is transferred to
In 10mL measuring bottles, 30% methanol dilution is added to shake up to scale, stand, take supernatant, filtered, taken continuous with 0.45um miillpore filters
Filtrate, produce.
Liquid phase chromatogram condition：Mobile phase A is acetonitrile (0-70min, 2% → 20%), and Mobile phase B is tetrahydrofuran (0-
70min, 0% → 10%), mobile phase C is 0.05% phosphoric acid solution (0-70min, 98% → 70%), flow velocity 1.1mL/min, post
20 DEG C of temperature, Detection wavelength 254nm, 275nm.
3. result
Finger-print and its cluster analysis result：The chromatogram of 9 groups of difference samples is shown in Fig. 3.According to 11 shared Feng Feng faces
Product, is conducted into SPSS 21.0 and difference sample is clustered, and clustering method is Between-groups linkage, away from
Eulidean is used from computational methods.As shown in figure 4, when Rescaled Distance Cluster Combine are 5,9 groups
Difference sample can be divided into 7 classes, obvious chemical composition content difference be present.It follows that by original prescription proportional basis,
Ingredients is allocated by Uniform ity Design Method, the traditional Chinese medicine sample with chemical composition content difference can be built, is accorded with
The characteristics of closing Chinese medicine globality.
Embodiment 3：The kouyanqing granules being made up of Honeysuckle flower, radix scrophulariae, asparagus fern, the tuber of dwarf lilyturf, radix glycyrrhizae gomi herbs has chemistry
The sample structure of component content difference
1. material
(1) instrument, reagent：Numerical control ultrasonic cleaner (Kunshan ultrasonic instrument Co., Ltd, KQ-250DE), vortex oscillation
Device (Scientific Industries Vortex-Genie 2), rotary evaporator (Shanghai Yarong Biochemical Instrument Plant, RE-
5205/RE-2000B), baking oven (Millipore, UFB400), Superpure water machine (Millipore Simplicity), electronic analysis
The glass apparatus such as balance (Sartorius), refrigerator (Siemens), beaker, conical flask, cucurbit；Methanol, acetonitrile, formic acid (matter
Compose pure), ultra-pure water.
(2) medicinal material：Honeysuckle flower, lot number：160815；Radix scrophulariae, lot number：160815；Asparagus fern, lot number：160815；The tuber of dwarf lilyturf, batch
Number：160815；Radix glycyrrhizae, lot number：160815.Above-mentioned medicinal material provides by Chinese medicine Co., Ltd of Guangzhou Hutchison China Trade Holdings.
2. method
(1) Honeysuckle flower, radix scrophulariae, asparagus fern, the tuber of dwarf lilyturf, radix glycyrrhizae feed intake design
Honeysuckle flower in the original prescription ratio of kouyanqing granules, radix scrophulariae, asparagus fern, the tuber of dwarf lilyturf, radix glycyrrhizae accounting example be respectively 26%,
21%th, 21%, 21%, 11%.In uniform design, radix scrophulariae even variation between 0%-42%, asparagus fern between 0%-42%
Even change, tuber of dwarf lilyturf even variation between 0%-42%, radix glycyrrhizae change between 0%-22%, and monarch drug in a prescription Honeysuckle flower is in 16%-36%
Between even variation.Fed intake according to the horizontal homogeneous design table of five factor 11, be shown in Table 3.
The medicinal material inventory and its ratio of the clear difference sample of the stomatitis of table 3
(2) difference sample preparation
According to《Pharmacopoeia of People's Republic of China》The scorching clear particulate level technological process of type page in 2015, medicinal material is carried out by table 3
Feed intake：Gomi herbs adds water to cook 2 times, 2 hours for the first time, second 1.5 hours, collecting decoction, filtration, and filtrate is concentrated into phase
It is 1.26~1.29 (80 DEG C) to density, adding ethanol makes alcohol content reach 50%, is sufficiently stirred, and stands 12 hours, takes supernatant
Liquid, filtration, filtrate recycling ethanol are simultaneously condensed into medicinal extract, produced.
(3) the finger-print monitoring of difference sample
Instrument and reagent：Supper-fast high performance liquid chromatograph (SHIMADZU, Prominence UFLC), high-resolution series connection
Mass spectrum (AB Sciex, TripleTOF 5600+), Dionex Bonded Silica C18 chromatographic columns (150mm × 4.6mm,
3um), acetonitrile, formic acid (mass spectrum is pure), ultra-pure water.
It is prepared by test sample：Take 11 groups of difference samples each appropriate, it is accurately weighed, put in conical flask, add 50% methanol 10mL, it is close
Plug, weighed weight, 30min is ultrasonically treated, is settled to 10mL with 50% methanol supplement, filters, produce through 0.22um miillpore filters.
Chromatographic condition：Mobile phase A be 0.1% formic acid acetonitrile solution (0-10min, 2% → 10%；10-85min, 10% →
50%；85-115min, 50% → 100%), Mobile phase B be 0.1% aqueous formic acid (0-10min, 98% → 90%；10-
85min, 90% → 50%；85-115min, 50% → 0%), flow velocity 0.3mL/min, 40 DEG C of column temperature.
Mass Spectrometry Conditions：ESI electric spray ion sources, positive ion mode.ion spray voltage：5500V, ion
source gas 1：55psi, ion source gas 2：55psi, ion source heater temperature：550
DEG C, curtain gas：35psi, collision gas pressure：10psi.
3. result
Liquid matter chromatogram and its cluster analysis result：The chromatogram of 11 groups of difference samples is shown in Fig. 5.According to 38 shared peak peaks
Area, importing in SPSS 21.0 and cluster analysis is carried out to difference sample, clustering method is Between-groups linkage,
Distance calculating method uses Pearson correlation.As shown in fig. 6, work as Rescaled Distance Cluster
When Combine is 5, difference sample can be divided into 8 classes, obvious chemical composition content difference be present.It follows that by original place
On square proportional basis, ingredients is allocated by Uniform ity Design Method, can be built with chemical composition content difference
Traditional Chinese medicine sample, the characteristics of meeting Chinese medicine globality.
Claims (8)
1. herbal mixture has the sample construction method of chemical composition content difference, described construction method comprises the following steps：
(1) with herbal mixture ingredients separately as experimental factor；
(2) it is according to the inventory excursion of herbal mixture prescription ratio-dependent ingredients：The change model of non-monarch drug in a prescription component
2 times for 0% to its prescription content are enclosed, the excursion of monarch drug in a prescription medicinal material subtracts non-monarch drug in a prescription component content sum for 100%；And
Suitable number of levels is determined in the range of being somebody's turn to do；
(3) Uniform ity Design Method is used, each component and its inventory are uniformly arranged；Multigroup difference is prepared according to standard process flows
Peculiar product；
(4) finger-print of each group difference sample is made, the otherness of difference sample room is verified using clustering method.
2. according to the method for claim 1, it is characterised in that：Experimental factor number is each taste of herbal mixture in step (1)
Medicinal material number.
3. according to the method for claim 1, it is characterised in that：Experiment number of levels is defined as 9-11 levels in step (2).
4. according to the method for claim 1, it is characterised in that：The specific method of step (4) is, according to the mark of herbal mixture
Quasi- technological process prepares more parts of difference samples, makes its finger-print respectively, using the color of cluster analysis each sample
Otherness between spectral peak peak area.
5. the method according to claim 1 to 4, it is characterised in that：The component of described herbal mixture is 3 to 6 parts.
6. the sample that Compound Xueshuantong preparation has chemical composition content difference is built and detection method, it is characterised in that including
Following steps：
(1), difference sample inventory：Red sage root even variation between 0%-20%, Radix Astragali even variation between 0%-36%, radix scrophulariae
The even variation between 0%-36%, monarch drug in a prescription pseudo-ginseng even variation between 33.2%-74.8%, according to the horizontal homogeneous of four factor 11
Design table obtains 11 difference sample inventorys；
(2) basis《Pharmacopoeia of People's Republic of China》Version FUFANG XUESHUANTONG JIAONANG standard process flows in 2015, it is poor by step (1)
Peculiar product inventory, which feeds intake, prepares difference sample；
(3) preparation difference sample making finger-print described to step (2), the liquid phase chromatogram condition for making finger-print
For：Using acetonitrile as mobile phase A, 0.05% phosphoric acid solution is Mobile phase B；The gradient elution of mobile phase A, which becomes, to be turned to：0-50min,
15% → 34%；Mobile phase B gradient elution, which becomes, turns to 50-59min, and 34% → 75%；Flow velocity 1.0mL/min, 25 DEG C of column temperature, inspection
Survey wavelength 203nm, 270nm；
(4) using the otherness between the chromatographic peak peak area of cluster analysis step (2) each sample.
7. the sample that Naoshuantong capsule has chemical composition content difference is built and detection method, it is characterised in that including following
Step：
(1) difference sample inventory：Radix paeoniae rubrathe even variation between 0%-48%, root tuber of aromatic turmeric even variation between 0%-38%, rhizoma Gastrodiae
The even variation between 0%-20%, Radix Rhapontici seu Radix Echinopsis change between 0%-28%, and monarch drug in a prescription cattail pollen uniformly becomes between 18.5%-47.5%
Change, nine difference sample inventorys are obtained according to the horizontal homogeneous design table of five factor nine；
(2) basis《Pharmacopoeia of People's Republic of China》Version Naoshuantong capsule standard process flows in 2015, by step (1) difference sample
Product inventory, which feeds intake, prepares difference sample；
(3) preparation difference sample making finger-print described to step (2), the liquid phase chromatogram condition for making finger-print
For：Using acetonitrile as mobile phase A, tetrahydrofuran is Mobile phase B, and 0.05% phosphoric acid solution is mobile phase C；Gradient elution changes：Stream
Dynamic phase A is 0-70min, 2% → 20%, Mobile phase B 0-70min, 0% → 10%, mobile phase C is 0-70min, 98% →
70%；Flow velocity 1.1mL/min, 20 DEG C of column temperature, Detection wavelength 254nm, 275nm；
(4) using the otherness between the chromatographic peak peak area of cluster analysis step (2) each sample.
8. the sample that kouyanqing granules has chemical composition content difference is built and detection method, comprise the following steps：
(1) difference sample inventory：Radix scrophulariae even variation between 0%-42%, asparagus fern even variation between 0%-42%, the tuber of dwarf lilyturf
The even variation between 0%-42%, radix glycyrrhizae change between 0%-22%, and monarch drug in a prescription Honeysuckle flower uniformly becomes between 16%-36%
Change；11 difference sample inventorys are obtained according to the horizontal homogeneous design table of five factor 11；
(2) basis《Pharmacopoeia of People's Republic of China》The scorching clear particulate level technological process of type page in 2015, by step (1) difference sample
Product inventory, which feeds intake, prepares difference sample；
(3) preparation difference sample making finger-print described to step (2), the liquid phase chromatogram condition for making finger-print
For：Mobile phase A is 0.1% formic acid acetonitrile solution, graded 0-10min, 2% → 10%；10-85min, 10% →
50%；85-115min, 50% → 100%；Mobile phase B is 0.1% aqueous formic acid, graded 0-10min, 98% →
90%；10-85min, 90% → 50%；85-115min, 50% → 0%, flow velocity 0.3mL/min, 40 DEG C of column temperature；
(4) using the otherness between the chromatographic peak peak area of cluster analysis step (2) each sample.
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