CN116642988A - Method for measuring content of multiple components in myrobalan and constructing characteristic fingerprint - Google Patents
Method for measuring content of multiple components in myrobalan and constructing characteristic fingerprint Download PDFInfo
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- 235000015489 Emblica officinalis Nutrition 0.000 title claims abstract description 138
- 235000011517 Terminalia chebula Nutrition 0.000 title claims abstract description 138
- 238000000034 method Methods 0.000 title claims abstract description 60
- 244000277583 Terminalia catappa Species 0.000 title claims abstract 29
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims abstract description 50
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 claims abstract description 46
- 229920001864 tannin Polymers 0.000 claims abstract description 46
- 235000018553 tannin Nutrition 0.000 claims abstract description 46
- 239000001648 tannin Substances 0.000 claims abstract description 46
- 229920000241 Punicalagin Polymers 0.000 claims abstract description 43
- ZJVUMAFASBFUBG-OGJBWQGYSA-N punicalagin Chemical compound C([C@H]1O[C@@H]([C@@H]2OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)O[C@H]2[C@@H]1OC(=O)C1=CC(O)=C(O)C(O)=C11)O)OC(=O)C2=CC(O)=C(O)C(O)=C2C2=C(O)C(O)=C(OC3=O)C4=C2C(=O)OC2=C4C3=C1C(O)=C2O ZJVUMAFASBFUBG-OGJBWQGYSA-N 0.000 claims abstract description 43
- LMIBIMUSUFYFJN-RSVYENFWSA-N punicalagin Natural products O[C@@H]1O[C@@H]2COC(=O)c3cc(O)c(O)c(O)c3c4c(O)cc5OC(=O)c6c(c(O)c(O)c7OC(=O)c4c5c67)c8c(O)c(O)c(O)cc8C(=O)O[C@H]2[C@@H]9OC(=O)c%10cc(O)c(O)c(O)c%10c%11c(O)c(O)c(O)cc%11C(=O)O[C@@H]19 LMIBIMUSUFYFJN-RSVYENFWSA-N 0.000 claims abstract description 43
- ZRKSVMFLACVUIU-UHFFFAOYSA-N punicalagin isomer Natural products OC1=C(O)C(=C2C3=4)OC(=O)C=4C4=C(O)C(O)=C3OC(=O)C2=C1C1=C(O)C(O)=C(O)C=C1C(=O)OC1C2OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(O)OC1COC(=O)C1=CC4=C(O)C(O)=C1O ZRKSVMFLACVUIU-UHFFFAOYSA-N 0.000 claims abstract description 43
- 229920002786 Corilagin Polymers 0.000 claims abstract description 28
- TUSDEZXZIZRFGC-XIGLUPEJSA-N corilagin Chemical compound O([C@H]1[C@H](O)[C@H]2OC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC[C@@H](O1)[C@H]2O)C(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-XIGLUPEJSA-N 0.000 claims abstract description 28
- CPWYQGWOJMNXGJ-UHFFFAOYSA-N corilagin Natural products OC1C2COC(=O)c3c(O)c(O)c(O)c(O)c3c4c(O)c(O)c(O)c(O)c4C(=O)OC1C(O)C(OC(=O)c5cc(O)c(O)c(O)c5)O2 CPWYQGWOJMNXGJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229940074391 gallic acid Drugs 0.000 claims abstract description 26
- 235000004515 gallic acid Nutrition 0.000 claims abstract description 25
- AFSDNFLWKVMVRB-UHFFFAOYSA-N Ellagic acid Chemical compound OC1=C(O)C(OC2=O)=C3C4=C2C=C(O)C(O)=C4OC(=O)C3=C1 AFSDNFLWKVMVRB-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229920002079 Ellagic acid Polymers 0.000 claims abstract description 23
- ATJXMQHAMYVHRX-CPCISQLKSA-N Ellagic acid Natural products OC1=C(O)[C@H]2OC(=O)c3cc(O)c(O)c4OC(=O)C(=C1)[C@H]2c34 ATJXMQHAMYVHRX-CPCISQLKSA-N 0.000 claims abstract description 23
- 235000004132 ellagic acid Nutrition 0.000 claims abstract description 23
- 229960002852 ellagic acid Drugs 0.000 claims abstract description 23
- FAARLWTXUUQFSN-UHFFFAOYSA-N methylellagic acid Natural products O1C(=O)C2=CC(O)=C(O)C3=C2C2=C1C(OC)=C(O)C=C2C(=O)O3 FAARLWTXUUQFSN-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000002253 acid Substances 0.000 claims abstract description 22
- RNKMOGIPOMVCHO-SJMVAQJGSA-N 1,3,6-trigalloyl glucose Chemical compound C([C@@H]1[C@H]([C@@H]([C@@H](O)[C@H](OC(=O)C=2C=C(O)C(O)=C(O)C=2)O1)OC(=O)C=1C=C(O)C(O)=C(O)C=1)O)OC(=O)C1=CC(O)=C(O)C(O)=C1 RNKMOGIPOMVCHO-SJMVAQJGSA-N 0.000 claims abstract description 21
- NTNQTAISNHFKRA-CKYFFXLPSA-N (3r,4r,5r)-3,4-dihydroxy-5-(3,4,5-trihydroxybenzoyl)oxycyclohexene-1-carboxylic acid Chemical compound O[C@@H]1[C@H](O)C=C(C(O)=O)C[C@H]1OC(=O)C1=CC(O)=C(O)C(O)=C1 NTNQTAISNHFKRA-CKYFFXLPSA-N 0.000 claims abstract description 18
- 239000001263 FEMA 3042 Substances 0.000 claims abstract description 18
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims abstract description 18
- 235000015523 tannic acid Nutrition 0.000 claims abstract description 18
- 229920002258 tannic acid Polymers 0.000 claims abstract description 18
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 claims abstract description 18
- 229940033123 tannic acid Drugs 0.000 claims abstract description 18
- ZELMDXUEWHBWPN-UHFFFAOYSA-N 3,4,8,9,10-pentahydroxybenzo[c]chromen-6-one Chemical compound OC1=C(O)C(O)=C2C3=CC=C(O)C(O)=C3OC(=O)C2=C1 ZELMDXUEWHBWPN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000001514 detection method Methods 0.000 claims abstract description 14
- 238000000605 extraction Methods 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 81
- 239000012488 sample solution Substances 0.000 claims description 52
- 239000000243 solution Substances 0.000 claims description 42
- 239000013558 reference substance Substances 0.000 claims description 33
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 24
- 238000012360 testing method Methods 0.000 claims description 23
- 238000001228 spectrum Methods 0.000 claims description 16
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 14
- 239000000523 sample Substances 0.000 claims description 14
- -1 myrobalanin Chemical compound 0.000 claims description 11
- 230000014759 maintenance of location Effects 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 9
- 238000010276 construction Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 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 7
- 235000019253 formic acid Nutrition 0.000 claims description 7
- 238000002137 ultrasound extraction Methods 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 238000000691 measurement method Methods 0.000 claims description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
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- 241000001522 Terminalia chebula Species 0.000 description 117
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- 150000001875 compounds Chemical class 0.000 description 19
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- 239000003643 water by type Substances 0.000 description 9
- 239000000463 material Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
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- 206010068319 Oropharyngeal pain Diseases 0.000 description 2
- 201000007100 Pharyngitis Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 229920001461 hydrolysable tannin Polymers 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
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- 239000011550 stock solution Substances 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 229930186301 urolithin Natural products 0.000 description 2
- 240000006891 Artemisia vulgaris Species 0.000 description 1
- 235000003261 Artemisia vulgaris Nutrition 0.000 description 1
- 241000221032 Combretaceae Species 0.000 description 1
- 206010013952 Dysphonia Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000012671 Gastrointestinal haemorrhages Diseases 0.000 description 1
- 208000010473 Hoarseness Diseases 0.000 description 1
- 102220477148 Putative uncharacterized protein PP632_S24D_mutation Human genes 0.000 description 1
- 206010038084 Rectocele Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 208000013116 chronic cough Diseases 0.000 description 1
- 208000019902 chronic diarrheal disease Diseases 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000001360 collision-induced dissociation Methods 0.000 description 1
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- 230000003203 everyday effect Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 125000001736 nosyl group Chemical group S(=O)(=O)(C1=CC=C([N+](=O)[O-])C=C1)* 0.000 description 1
- 238000003909 pattern recognition Methods 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- JXOHGGNKMLTUBP-JKUQZMGJSA-N shikimic acid Natural products O[C@@H]1CC(C(O)=O)=C[C@H](O)[C@@H]1O JXOHGGNKMLTUBP-JKUQZMGJSA-N 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 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
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- 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)
Abstract
The invention relates to the field of traditional Chinese medicine analysis, in particular to a method for measuring the content of multiple components in myrobalan and constructing a characteristic fingerprint. The content determination method provided by the invention can be used for simultaneously determining the myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid in the myrobalan, has high sensitivity and good precision, repeatability and stability, can be used for detecting the content of the tannins in the extracting solution of each step in the tannins component extraction process, and can be used for carrying out related detection in the research and quality evaluation of the tannins components of the myrobalan, and can be used for constructing the characteristic fingerprint of the myrobalan. The method can also be used for measuring the content of tannins in fructus Chebulae containing similar tannins and constructing characteristic fingerprint.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine analysis, in particular to a method for measuring the content of multiple components in myrobalan and constructing a characteristic fingerprint.
Background
The Chinese medicine myrobalan is a dry mature fruit of the plant myrobalan Terminalia chebula Retz of the family Combretaceae or the plant myrobalan Terminalia chebula Retz, var. Tomentosa Kurt, has the effects of astringing intestines to check diarrhea, astringing lung to check cough, reducing fire to relieve sore throat, and is mainly used for treating chronic diarrhea, chronic dysentery, hematochezia rectocele, lung deficiency asthma and cough, chronic cough and sore throat with hoarseness.
The fructus Chebulae contains a large amount of bioactive components including polyphenols, flavonoids, polysaccharides, terpenes, etc., wherein the most content is tannins, mainly hydrolysable tannins such as gallic acid, punicalagin, corilagin, ellagic acid, etc. Analysis and content measurement of chemical substances in myrobalan and research of characteristic fingerprints are helpful for quality evaluation of medicinal materials, discovery of active ingredients and research of action mechanisms. However, the existing analysis and detection method can only quantitatively detect one or a few tannin compounds in the myrobalan, and is insufficient for more comprehensively reflecting the quality fluctuation condition of the tannin components.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for measuring the content of multiple components in myrobalan and constructing a characteristic fingerprint and application thereof. The content determination method provided by the invention can be used for simultaneously and accurately detecting more than ten tannins in the myrobalan, has high sensitivity and good precision, repeatability and stability, and can be used for constructing the characteristic fingerprint of the myrobalan and the characteristic fingerprint of the fructus chebulae which has common components with the myrobalan.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a content determination method of multiple components in fructus Chebulae comprises extracting fructus Chebulae with methanol water solution, and determining content of the components to be detected by ultra high performance liquid chromatography (UPLC), wherein the components to be detected comprise myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalanin, urea M5, myrobalanin tannic acid, myrobalan Lin Rousuan and ellagic acid; the chromatographic conditions of the ultra performance liquid chromatography include:
chromatographic column: pentabromophenyl high performance liquid chromatography column;
mobile phase: mobile phase A is 0.095% -0.105% v/v formic acid aqueous solution, mobile phase B is methanol, gradient elution is carried out, and the gradient elution is carried out according to the following procedures:
the flow rate is: 0.28 to 0.32ml/min;
the detection wavelength is as follows: 250-290 nm;
the chromatographic conditions can detect various tannin components in the myrobalan, including myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid, and the chromatographic conditions have high sensitivity and good precision, repeatability and stability, and can be used for quality evaluation of the myrobalan, content detection of the tannin components in the myrobalan extract or extract, and construction of a characteristic fingerprint of the myrobalan. The content determination method can also be used for determining the content of the tannin compounds in the fructus chebulae and the fructus chebulae containing similar tannin compounds and constructing a characteristic fingerprint.
With reference to the first aspect, the volume percentage concentration of the methanol aqueous solution is 40% -100%. Further preferred is methanol, i.e. 100% v/v methanol.
Preferably, the extraction mode is ultrasonic extraction, the extraction temperature is 30-50 ℃, and the extraction time is 10-30 min.
With reference to the first aspect, the pentabromophenyl high performance liquid chromatography column was Cosmosil PBr Packed Column (2.1X100 mm,2.6 μm). The chromatographic column not only can separate large polar compounds under the reverse phase condition, but also can be applied under the condition of 100% water, and is suitable for separating polar compounds; and has obvious advantages for cyclic compounds or amine compounds. For the components to be detected in the myrobalan, the chromatographic column has a good separation effect.
In combination with the first aspect, mobile phase A is preferably 0.10% v/v aqueous formic acid.
With reference to the first aspect, the flow rate is 0.3ml/min.
With reference to the first aspect, the chromatographic conditions further include a column temperature of 30 to 60 ℃; preferably 30 ℃.
With reference to the first aspect, the mass-volume ratio of the myrobalan to the aqueous methanol solution is 1:50-500 (g: ml). Preferably, 1:250 to 500 (g: ml) is used.
With reference to the first aspect, the content determination method includes the following steps:
step a, extracting myrobalan with a methanol aqueous solution to prepare a sample solution;
b, preparing a reference substance solution of the component to be detected;
and c, measuring the reference substance solution and the test sample solution by using the ultra-high performance liquid chromatography, and calculating the content of the index component to be measured in the test sample by using a standard curve method.
In a second aspect, the invention also provides a method for constructing the characteristic fingerprint of the myrobalan, which comprises the following steps:
s1, preparing a reference substance solution of index components; the index component comprises myrobalan hypoacid, gallic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid;
s2, extracting different batches of myrobalan with methanol respectively to obtain sample solutions of different batches of myrobalan, measuring the reference substance solution and the sample solution by using an ultra-high performance liquid chromatography in the content measuring method, recording fingerprint patterns, introducing all the fingerprint patterns into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, taking a batch of myrobalan patterns as reference patterns, selecting time width of 0.1min, performing peak matching by using a Mark peak by using a median method, performing pattern recognition on the reference patterns to obtain fingerprint patterns and reference fingerprint patterns, selecting peaks with strong absorption signals and good separation degree and stability as characteristic peaks, and calculating the relative retention time and relative peak area of each characteristic peak.
The characteristic fingerprint constructed by the characteristic fingerprint construction method of the myrobalan provided by the invention can obtain the spectrum information of the 12 tannins compounds, can be used for rapidly evaluating the tannins compounds in the myrobalan, and provides data support for the quality research of the myrobalan.
In a third aspect, the invention further provides a myrobalan characteristic fingerprint, wherein the myrobalan characteristic fingerprint comprises 12 characteristic fingerprint peaks: sequentially comprises myrobalan hypoacid, gallic acid, 5-galloylshikimic acid, punicalagin A, punicalagin B, corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, myrobalan tannic acid, myrobalan Lin Rousuan, ellagic acid and 4-O- (3 ', 4' -Di-O-galloyl-alpha-L-rhamnopyra nosyl) elagic acid.
In combination with the third aspect, the peak of corilagin is taken as a reference peak (H6), and the relative retention time of each common peak and the reference peak is within a range of ±5% of a prescribed value; the specified value is: the peak H1 (gallic acid) is 0.2220-0.2453, the peak H2 (gallic acid) is 0.4239-0.4685, the peak H3 (5-gallic acid acyl shikimic acid) is 0.6435-0.7112, the peak H4 (punicalagin A) is 0.7405-0.8185, the peak H5 (punicalagin B) is 0.8456-0.9346, the peak H6 (corilagin) is 1.000, the peak H7 (1, 3, 6-tri-O-galloyl-beta-D-glucose) is 1.056-1.167, the peak H8 (myrobalamine) is 1.167-1.290, the peak H9 (myrobalamine) is 1.306-1.444, the peak H10 (myrobalamine Lin Rousuan) is 1.501-1.659, the peak H11 (ellagic acid) is 1.532-1.693, and the peak H728 (4-O- (3' -Di-O-galloyl-alpha-L-glucose) is 1.910.
In a fourth aspect, the invention also provides application of the content determination method in content determination of fructus chebulae immaturus tannins components and construction of characteristic fingerprints.
The content determination method of the myrobalan provided by the invention can determine the content of the fructus chebulae tannins, and construct the characteristic fingerprint of fructus chebulae, so as to quickly reflect the quality condition of fructus chebulae.
The invention has the beneficial effects that:
the method for measuring the content of the multi-component in the myrobalan can simultaneously measure 12 tannin compounds in the myrobalan, and is verified by methodology that the method for measuring the content has high sensitivity and good precision, repeatability and stability, and meets the requirement of methodology. The invention also establishes a characteristic fingerprint spectrum construction method of the myrobalan and obtains 12 characteristic fingerprint peaks of the myrobalan. According to the invention, the corilagin is selected as a reference peak, and the relative retention time of the common peak of the characteristic fingerprint spectrum of the myrobalan is determined. The content determination method provided by the invention can be used for determining the content of the tannin compounds in the fructus chebulae and fructus chebulae containing similar tannin compounds and constructing characteristic fingerprint patterns, and can realize quality evaluation and sample detection of the fructus chebulae and fructus chebulae.
Drawings
FIG. 1 is a liquid chromatogram of a sample solution of different materials in example 3;
FIG. 2 is a TIC mass spectrum of the sample solution of different materials in example 3;
FIG. 3 is a characteristic fingerprint spectrum and a control fingerprint spectrum of 35 batches of myrobalan samples in example 4;
FIG. 4 is a characteristic fingerprint of 28 batches of fructus Canarii albi samples and a control fingerprint of example 5;
FIG. 5 is a liquid chromatogram of the control solutions of examples 1, 6, 7, and 8;
FIG. 6 is a liquid chromatogram obtained using different columns in comparative example 1.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The fructus Chebulae contains a large amount of bioactive components, wherein the most content is tannins, mainly contains hydrolysable tannins such as gallic acid, punicalagin, corilagin, ellagic acid, etc. The existing analysis and detection method can only quantitatively detect one or a few tannin compounds in the myrobalan, and is insufficient for more comprehensively reflecting the quality fluctuation condition of the tannin components. Aiming at the problem, the embodiment of the invention provides a content determination method for multiple components in myrobalan and a characteristic fingerprint construction method for the myrobalan, and provides a characteristic fingerprint construction method for fructus chebulae based on the content determination method.
The invention is further illustrated in the following examples.
The main instrumentation used in the following examples:
ultra-high performance liquid chromatograph (ACQUITY)H class plus, waters company, usa); agilent Infinitiy II 1260 series 6550Q-TOF high resolution mass spectrometer (Agilent Co., U.S.); one ten-thousandth balance (ME 204, METTLER company, switzerland); one ten thousandth balance (New Classic MS, METTER, switzerland); intelligent ultrasonic cleaner (DL-720B, shanghai communications instruments limited); a high-speed cryocentrifuge (5430R, eppendorf, germany);
the reagents used in the following examples were:
methanol (chromatographic purity, sigma-Aldrich trade limited); absolute ethanol (analytically pure, division of Kangkode technologies, tianjin); formic acid (chromatographic purity, shanghai Ala Biochemical technologies Co., ltd.); purified water (drohent food and beverage limited, guangzhou); dimethyl sulfoxide (analytically pure, tianjin, meta. Chemical reagent plant).
Reagents used in the following examples:
myrobalan (Beijing Tongrentang Tianjin Hexi big pharmacy Co., ltd.); gallic acid (J05 GB 153704), punicalagin (J03 HB 186918), corilagin (A02 GB 143893), myrobalan tannic acid (M29 GB 143373), myrobalan Lin Rousuan (J21 HB 174660), 1,3, 6-tri-O-galloyl-beta-D-glucose (J11 GB 154490), ellagic acid (S24D 11G 135548), all purchased from Shanghai source leaf Biotechnology Co., ltd; the myrobalan hypoacid, 4-galloylshikimic acid, 5-galloylshikimic acid, myrobalan tannin and urolithin M5 are all self-made by the laboratory (purity is more than 98%).
Reagents used in the following examples: the information of the producing places and sources of the myrobalan and the fructus chebulae are shown in table 1 (HZ represents the myrobalan and XQG represents fructus chebulae).
TABLE 1 Chebula and fructus Canarii albi medicinal material origin and source information
The myrobalan and fructus chebulae powder adopted in the following examples are all powder obtained by pulverizing the above medicinal materials and sieving with No. 3 sieve.
Example 1
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of sample solution
Taking 0.2g of myrobalan powder, precisely weighing, placing into a 50ml volumetric flask, adding a proper amount of methanol, carrying out ultrasonic extraction at 30 ℃ for 20min, standing at room temperature, using methanol to fix the volume to scale, shaking uniformly, taking a proper amount of solution for centrifugation at 13700rpm for 10min, and taking supernatant as a sample solution;
step b, preparation of reference substance solution
Taking 12 kinds of reference substances, precisely weighing, dissolving the reference substances of myrobalan hypoacid and gallic acid with water, dissolving the reference substances of 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose with methanol, dissolving the reference substances of punicalagin (punicalagin A & B), chebulin, urolithin M5, chebulin tannic acid, chebula Lin Rousuan and ellagic acid with dimethyl sulfoxide, and preparing into reference substance stock solutions with the concentration of 10.014mg/ml, 7.028mg/ml, 1.022mg/ml, 1.662mg/ml, 1.092mg/ml, 0.854mg/ml, 4.048mg/ml, 3.846mg/ml, 1.190mg/ml, 1.530mg/ml, 1.864mg/ml and 0.5844mg/ml respectively.
Precisely measuring appropriate amount of chebulimic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, punicalagin (punicalagin A & B), chebulin, urolithin M5, chebulin tannic acid, chebula Lin Rousuan and ellagic acid reference substance stock solution, placing into a 10ml volumetric flask, adding dimethyl sulfoxide to volume to scale, shaking, and obtaining mixed reference substance solution. The mixed reference solution contains 500.7 mugwort acid/ml, 210.84 mug/ml gallic acid, 51.1 mug/ml 4-galloylshikimic acid, 66.48 mug/ml 5-galloylshikimic acid, 242.88 mug/ml punicalagin (punicalagin A & B), 65.52 mug/ml corilagin, 68.32 mug/ml 1,3, 6-tri-O-galloyl-beta-D-glucose, 230.76 mug/ml chebulin, 35.7 mug/ml urolithin M, 183.6 mug/ml chebulin tannic acid, lin Rousuan 186.4.4 mug/ml chebulin, 175.32 mug/ml ellagic acid; and (5) gradually diluting to obtain a series of reference substance solutions with different concentrations.
Step c, using Agilent Infinitiy II 1260 ultra-high performance liquid chromatograph to connect 6550Q-TOF high resolution mass spectrometer in series to measure the mixed reference substance solution and the sample solution, wherein the chromatographic conditions are as follows:
chromatographic column: cosmosil PBr Packed Column (2.1X100 mm,2.6 μm);
column temperature: 30 ℃;
sample injection amount: 2. Mu.L;
flow rate: 0.3ml/min;
mobile phase a was 0.10% v/v aqueous formic acid and mobile phase B was methanol, and linear gradient elution was performed with the following elution procedure:
the detection wavelength is 270nm;
the mass spectrum conditions are as follows: data are collected in the negative ion mode, and the collection mode is Auto MS/MS. The ion source parameters were set as follows: the gas temperature is 200 ℃; the flow rate of the drying gas is 12L/min; the atomization pressure was 40psi; the sheath gas temperature is 350 ℃; the nozzle voltage is-1.5 kV; the capillary voltage is-4.0 kV; the Fragmentor is 390V; mass to charge ratio (m/z) range MS scanned by TOF analyzer 1 100-1500 MS 2 50-1500. Automatic selection at MS by collision induced dissociation 1 Ions at the first 3 th position of the intensity in the spectrum trigger MS/MS fragmentation with collision energy of 30V. Data acquisition was implemented on Agilent MassHunter Workstation Data Acquisition software.
Test example 1
The test examples of the present invention provide a methodological investigation of the multicomponent content determination method of example 1, wherein:
(1) Linear relationship and limit of detection, limit of quantification study
A series of mixed reference solutions were prepared as in step b of example 1, and peak areas were measured by ultra high performance liquid chromatography under the same chromatographic conditions as in step c of example 1. Sampling for 2 times in parallel, drawing a regression equation by taking the concentration of each compound reference substance as an abscissa x (mug/ml) and the peak area of the corresponding reference substance as an ordinate y; and (3) measuring the detection limit and the quantitative limit of myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid by taking the signal to noise ratio S/N=3 as the detection Limit (LOD) and the S/N=10 as the quantitative Limit (LOQ). The results are shown in Table 2.
As is clear from Table 2, the components have good linear relationship in the respective concentration ranges (r 2 Not less than 0.999), the concentration range of the detection limit is 0.0998-0.9779 mug/ml, and the concentration range of the quantitative limit is 0.2559-1.956 mug/ml, which shows that the sensitivity of the method for measuring the content of the multi-component in the myrobalan provided by the invention is higher.
(2) Precision test
(a) Within-day precision test
Precisely sucking the same sample solution, repeating sample injection for 6 times, and measuring peak area by ultra-high performance liquid chromatography under the same chromatographic conditions as in step c in example 1. RSD values were calculated as peak areas, respectively, and the results are shown in table 3.
Table 3 within day precision test results (n=6)
As can be seen from Table 3, the daily precision RSD of the method for measuring the content of the multiple components in the myrobalan provided in example 1 is less than 1.0%.
(b) Daytime precision test
Continuously sucking the same sample solution precisely every day for 3 days, repeating sample injection for 6 times, and measuring peak area by ultra-high performance liquid chromatography under the same chromatographic conditions as in step c in example 1. The results are shown in Table 4.
Table 4 daytime precision test results (n=3)
As can be seen from Table 4, the method for measuring the content of multiple components in myrobalan provided in example 1 has a daytime precision RSD of less than 2.7% and a good measuring method precision.
(3) Repeatability test
A test solution of 6 parts of the powder of chebula fruit was prepared in parallel according to the method of step a in example 1, the above test solution was measured by ultra-high performance liquid chromatography, and the content of each compound was calculated, and the chromatographic conditions were the same as in step c in example 1. The contents of chebulimic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, chebulin, urolithin M5, chebulin tannic acid, chebula Lin Rousuan, ellagic acid were calculated by standard curve and RSD values were calculated. The results are shown in Table 5.
As can be seen from Table 5, in this test, myrobalan hypoacid 41.48mg/g, gallic acid 12.65mg/g, 4-galloylshikimic acid 2.530mg/g, 5-galloylshikimic acid 3.760mg/g, punicalagin (punicalagin A & B) 9.692mg/g, corilagin 3.809mg/g, 1,3, 6-tri-O-galloyl-beta-D-glucose 3.467mg/g, chebulin 17.56mg/g, urolithin M5 2.707mg/g, chebulin 9.985mg/g, chebula Lin Rousuan 7.878mg/g, ellagic acid 28.76mg/g, and RSD of each compound was less than 3.0%, indicating that the detection method was excellent in reproducibility.
(4) Stability test
Preserving the sample solution at 10 ℃, precisely sucking the same sample solution at 0, 2, 4, 6, 8, 10 and 12 hours, and measuring the sample solution by ultra-high performance liquid chromatography under the same chromatographic conditions as in the step c in the example 1. RSD values were calculated from peak areas of chebulimic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, chebulin, urolithin M5, chebulin tannic acid, chebulin Lin Rousuan, ellagic acid. The results are shown in Table 6.
As is clear from Table 6, the stability test RSD of the liquid chromatography condition of the method for measuring the content of multiple components in myrobalan provided in example 1 is less than 1.2%, which indicates that the test solution has good stability in 12 hours at normal temperature.
(5) Sample recovery test
About 0.1g of myrobalan powder is precisely weighed, and 16.6ml of mixed reference solution (300.4 mug/ml of myrobalan hypo-acid, 84.34 mug/ml of gallic acid, 18.40 mug/ml of 4-galloylshikimic acid, 29.92 mug/ml of 5-galloylshikimic acid, 72.86 mug/ml of punicalagin (punicalagin A & B), 32.76 mug/ml of corilagin, 20.50 mug/ml of 1,3, 6-tri-O-galloyl-beta-D-glucose, 115.4 mug/ml of myrobalanin, 5.85 mug/ml of urolithin M, 64.26 mug/ml of myrobalan tannic acid, lin Rousuan 55.92.92 mug/ml of myrobalan and 175.3 mug/ml of ellagic acid) is added.
6 parts of myrobalan sample solution is prepared, each sample is measured for 2 times in parallel, the sample solution is measured by adopting an ultra-high performance liquid chromatography, and the chromatographic conditions are the same as in the step c in the example 1. And (3) examining the accuracy of the method according to the sample adding recovery rate and the RSD value calculated by the sample adding recovery rate formula.
The sample recovery rate formula is as follows:
the results are shown in Table 7.
As shown in Table 7, the average recovery rate of 97.41-113.4% under the liquid chromatography conditions of the multi-component content measuring method in myrobalan provided in example 1, RSD values are less than 6.5%, which indicates that the recovery rate of each compound is basically in accordance with the requirement, and the method is accurate and feasible.
Tables 2 to 7 show that the method for measuring the content of the multiple components in the myrobalan provided by the embodiment 1 basically meets the research requirements of methodology and can be used for quantitative analysis of the myrobalan.
Example 2
In this example, the content of tannins in different batches of myrobalan was measured by the content measurement method in example 1. The procedure is as in example 1 and the results are shown in Table 8.
Example 3
In this example, the content of tannins in the fructus Canarii albi was measured by the method of example 1.
Step a, preparation of a sample solution: the difference from example 1 is that the myrobalan powder is replaced with the fructus chebulae powder;
step b, preparation of a reference substance solution: as in example 1;
step c, similar to example 1, results are shown in Table 9.
The liquid chromatogram of each medicinal material sample solution is shown in figure 1, the TIC mass spectrum is shown in figure 2, and the qualitative detection result is shown in table 10.
Example 4
The embodiment of the invention provides a method for constructing a myrobalan characteristic fingerprint, which comprises the following steps:
step 1, preparing a sample solution of myrobalan powder and a mixed reference substance solution according to the preparation methods of the sample solution and the reference substance solution in the embodiment 1;
step 2, the same as step c in embodiment 1, record the characteristic fingerprint.
The data is imported into 2012 edition of traditional Chinese medicine chromatographic fingerprint similarity evaluation system for processing, the spectrum of the myrobalan HZ35 is taken as a reference spectrum, and 12 main characteristic peaks with strong absorption signals and good separation degree and stability are selected and marked as common peaks, as shown in figure 3. And calculating the similarity between the myrobalan fingerprint and the control fingerprint, and finding that the similarity between the sample of 35 batches of myrobalan and the control fingerprint is more than 0.66, so that the difference between the batches of myrobalan is larger. According to the determination result of the relative retention time of each compound in the fingerprint of 35 batches of myrobalan medicinal materials, 12 common chromatographic peaks with larger peak area and better peak shape and separation degree are selected as characteristic fingerprint peaks. Standard control fingerprint is generated, H6 peak is used as reference peak, and relative retention time and relative peak area of 12 common chromatographic peaks are shown in tables 11 and 12. By comparison with a control chromatogram, each characteristic fingerprint peak is identified as myrobalan hypo-acid (H1), gallic acid (H2), 5-galloyl shikimic acid (H3), punicalagin A (H4), punicalagin B (H5), corilagin (H6), 1,3, 6-tri-O-galloyl-beta-D-glucose (H7), myrobalan tannin (H8), myrobalan tannic acid (H9), myrobalan Lin Rousuan (H10), ellagic acid (H11), 4-O- (3 ', 4' -Di-O-galloyl-alpha-L-rhamnopyrranosyl) elgilac acid (H12).
Example 5
The embodiment of the invention provides a construction method of a characteristic fingerprint of fructus chebulae immaturus, which comprises the following steps:
step 1, preparing a test solution of fructus chebulae immaturus powder and a mixed reference solution according to the preparation methods of the test solution and the reference solution in the embodiment 1.
Step 2, the same as step c in embodiment 1, record the characteristic fingerprint.
The data is imported into 2012 edition traditional Chinese medicine chromatographic fingerprint similarity evaluation system for processing, the spectrum of fructus chebulae immaturus XQG24 is taken as a reference spectrum, and 11 main characteristic peaks with strong absorption signals and good separation degree and stability are selected and calibrated as common peaks, as shown in figure 4. And (3) calculating the similarity between the UPLC spectrum of each sample and the control fingerprint spectrum, and finding that the similarity between 28 batches of fructus chebulae immaturus samples and the control fingerprint spectrum is more than 0.86, and the similarity is good. According to the measurement results of the relative retention time of each compound in 28 batches of fructus chebulae immaturus fingerprint, 11 common chromatographic peaks with larger peak area and better peak shape and separation degree are selected as characteristic fingerprint peaks. Standard control fingerprint was generated, and the relative retention time and relative peak area of 11 common chromatographic peaks were shown in tables 13 and 14 using the X5 peak (corilagin) as a reference peak. Comparing with the control chromatogram, the characteristic fingerprint peaks are respectively myrobalan hypoacid (X1), gallic acid (X2), punicalagin A (X3), punicalagin B (X4), corilagin (X5), 1,3, 6-tri-O-galloyl-beta-D-glucose (X6), myrobalan tannin (X7), urolithin M5 (X8), myrobalan tannic acid (X9), myrobalan Lin Rousuan (X10) and ellagic acid (X11).
Taking the peak of the corilagin as a reference peak (X5), wherein the relative retention time of each common peak and the reference peak is within a range of +/-5% of a specified value; the specified value is: the peak X1 (gallic acid) is 0.2232-0.2466, the peak X2 (gallic acid) is 0.4239-0.4686, the peak X3 (punicalagin A) is 0.7401-0.818, the peak X4 (punicalagin B) is 0.8442-0.933, the peak X5 (corilagin) is 1.000, the peak X6 (1, 3, 6-tri-O-galloyl-beta-D-glucose) is 1.057-1.168, the peak X7 (myrobalanin) is 1.167-1.290, the peak X8 (urolithin M5) is 1.201-1.327, the peak X9 (myrobalan acid) is 1.307-1.444, the peak H10 (myrobalan Lin Rousuan) is 1.503-1.662, and the peak X11 (ellagic acid) is 1.533-1.694.
Example 6
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: as in example 1;
step b, preparation of a reference substance solution: as in example 1;
step c, the above mixed control solution and the test sample solution were measured by using an ultra-high performance liquid chromatograph of Waters ACQUITY UPLC H-Class PLUS, and the chromatographic conditions were substantially the same as those of example 1 except that the column temperature was 40 ℃.
Example 7
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: as in example 1;
step b, preparation of a reference substance solution: as in example 1;
step c, the above mixed control solution and the test sample solution were measured by using an ultra-high performance liquid chromatograph of Waters ACQUITY UPLC H-Class PLUS, and the chromatographic conditions were substantially the same as in example 1 except that the column temperature was 50 ℃.
Example 8
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: as in example 1;
step b, preparation of a reference substance solution: as in example 1;
step c, the above mixed control solution and the test sample solution were measured by using an ultra-high performance liquid chromatograph of Waters ACQUITY UPLC H-Class PLUS, and the chromatographic conditions were substantially the same as those of example 1 except that the column temperature was 60 ℃.
As shown in FIG. 5, the liquid chromatograms of the sample solutions in examples 1, 6, 7 and 8 can separate the peaks of 13 tannins compounds under the column temperature condition of 30-60 ℃, wherein the peak type separation degree of 13 compounds under the column temperature condition of 30 ℃ is better.
Example 9
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: taking 0.2g of myrobalan powder, precisely weighing, placing into a 25ml volumetric flask, adding a proper amount of methanol, carrying out ultrasonic extraction at 30 ℃ for 20min, standing at room temperature, using methanol to fix the volume to scale, shaking uniformly, taking a proper amount of solution for centrifugation at 13700rpm for 10min, and taking supernatant as a sample solution; sample solutions were prepared in the same manner with 60%, 80% and 100% v/v as extraction solvents.
Step b, preparation of a reference substance solution: as in example 1.
And c, measuring the mixed reference substance solution and the test sample solution by using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, wherein chromatographic conditions are the same as in example 1.
The peak areas of the respective components in the liquid chromatograms obtained for the respective sample solutions are shown in table 15.
TABLE 15 peak areas of tannins content of sample solutions obtained in different extraction solvents
As is clear from Table 15, the peak area of the tannins component was the highest and the extraction effect was the best when methanol was used as the extraction solvent.
Example 10
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: precisely weighing 1, 0.2 and 0.1g of myrobalan powder, respectively placing into 50ml, 25ml, 50ml and 50ml volumetric flasks, respectively precisely adding a proper amount of methanol, carrying out ultrasonic extraction at 30 ℃ for 20min, standing at room temperature, fixing volume to scale with methanol, shaking uniformly to obtain solutions with feed liquid ratios of 1:50, 1:125, 1:250 and 1:500, respectively taking a proper amount of solutions to be centrifuged at 13700rpm for 10min, and taking supernatant as a sample solution.
Step b, preparation of a reference substance solution: as in example 1.
And c, measuring the mixed reference substance solution and the test sample solution by using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, wherein chromatographic conditions are the same as in example 1.
The peak areas of the respective components in the liquid chromatograms obtained for the respective sample solutions are shown in table 16.
TABLE 16 peak areas of tannins in sample solutions obtained at different feed to liquid ratios
As can be seen from Table 16, when the feed-liquid ratio is 1:250 and 1:500, the peak areas of the tannins are both high, the extraction effect is excellent, and the preferred feed-liquid ratio is 1:250..
Example 11
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: taking 0.2g of myrobalan powder, precisely weighing, placing into a 50ml volumetric flask, adding a proper amount of methanol, respectively performing ultrasonic extraction at 30 ℃ for 10, 20 and 30min, standing at room temperature, fixing the volume to a scale with methanol, shaking uniformly, respectively taking a proper amount of supernatant as a sample solution after centrifugation at 13700rpm for 10 min.
Step b, preparation of a reference substance solution: as in example 1.
And c, measuring the mixed reference substance solution and the test sample solution by using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, wherein chromatographic conditions are the same as in example 1.
The peak areas of the respective components in the liquid chromatograms obtained for the respective sample solutions are shown in table 17.
TABLE 17 peak areas of tannins content of sample solutions obtained at different sonication times
As can be seen from Table 17, the peak areas of tannins are higher when the ultrasonic time is 20min and 30min, the extraction effect is better, and the ultrasonic time is selected to be 20min.
Example 12
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparation of a sample solution: taking 0.2g of myrobalan powder, precisely weighing, placing into a 50ml volumetric flask, adding a proper amount of methanol, respectively performing ultrasonic extraction at 30, 40 and 50 ℃ for 20min, standing at room temperature, fixing the volume to a scale with methanol, shaking uniformly, respectively taking a proper amount of supernatant as a sample solution after centrifugation at 13700rpm for 10 min.
Step b, preparation of a reference substance solution: as in example 1.
And c, measuring the mixed reference substance solution and the test sample solution by using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, wherein chromatographic conditions are the same as in example 1.
The peak areas of the respective components in the liquid chromatograms obtained for the respective sample solutions are shown in table 18.
TABLE 18 peak areas of tannins content of sample solutions obtained at different ultrasonic temperatures
As can be seen from Table 18, the peak area of tannins component was the highest at 30℃with the optimum extraction effect.
Comparative example 13
The embodiment of the invention provides a method for measuring the content of multiple components in myrobalan, which comprises the following steps:
step a, preparing a solution to be tested: preparation of the control solution as in example 1;
step b, measuring the solution to be measured by using a Waters ACQUITY UPLC H-Class PLUS ultra-high performance liquid chromatograph, wherein the chromatographic conditions are as follows:
chromatographic column: ZORBAX SB-C18 (4.6X100 mm,1.8 μm), ACQUITY UPLC HSS T3 (2.1X100 mm,1.8 μm) or Cosmosil PBr Packed Column (2.1X100 mm,2.6 μm);
column temperature: 30 DEG C
Sample injection amount: 2. Mu.L;
flow rate: 0.3ml/min;
mobile phase a was 0.1% v/v aqueous formic acid and mobile phase B was methanol, and linear gradient elution was performed with the following elution procedure:
the chromatogram obtained when the column is ZORBAX SB-C18 is shown in FIG. 6, panel A; the chromatogram for column ACQUITY UPLC HSS T3 is shown in figure 6B; the chromatogram for column Cosmosil PBr Packed Column is shown in figure 6 as C.
As can be seen from fig. 6, the Cosmosil PBr Packed Column chromatographic column is more suitable for separating tannins (similar in polarity) in myrobalan and has better separation degree.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A method for measuring the content of multiple components in myrobalan is characterized in that a methanol aqueous solution is used for extracting the myrobalan, and an ultra-high performance liquid chromatography is used for measuring the content of the components to be measured, wherein the components to be measured comprise myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalanin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid; the chromatographic conditions of the ultra performance liquid chromatography include:
chromatographic column: pentabromophenyl high performance liquid chromatography column;
mobile phase: mobile phase A is 0.095% -0.105% v/v formic acid aqueous solution, mobile phase B is methanol, gradient elution is carried out, and the gradient elution is carried out according to the following procedures:
the flow rate is: 0.28 to 0.32ml/min;
the detection wavelength is as follows: 250-290 nm.
2. The content measurement method according to claim 1, wherein the volume percentage concentration of the aqueous methanol solution is 40% to 100%; and/or
The extraction mode is ultrasonic extraction, the extraction temperature is 30-50 ℃, and the extraction time is 10-30 min.
3. The method according to claim 1, wherein the pentabromophenyl high performance liquid chromatography column is Cosmosil PBr Packed Column, with a specification of 2.1X100 mm, 2.6. Mu.m.
4. The assay according to claim 1, wherein mobile phase a is 0.10% v/v aqueous formic acid; and/or.
The flow rate was 0.3ml/min.
5. The content measuring method according to claim 1, wherein the chromatographic condition further comprises a column temperature of 30 to 60 ℃.
6. The content measuring method according to any one of claims 1 to 5, comprising the steps of:
step a, extracting myrobalan with a methanol aqueous solution to prepare a sample solution;
b, preparing a reference substance solution of the component to be detected;
and c, measuring the reference substance solution and the test sample solution by using the ultra-high performance liquid chromatography, and calculating the content of the index component to be measured in the test sample by using a standard curve method.
7. The method for constructing the characteristic fingerprint spectrum of the myrobalan is characterized by comprising the following steps of:
s1, preparing a reference substance solution of index components; the index component comprises myrobalan hypoacid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin (punicalagin A & B), corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid;
s2, extracting different batches of myrobalan with methanol respectively to obtain test sample solutions of different batches of myrobalan, measuring the reference sample solution and the test sample solution by using an ultra-high performance liquid chromatography in the content measuring method, recording characteristic fingerprints, introducing all the characteristic fingerprints into traditional Chinese medicine chromatographic fingerprint similarity evaluation system software, taking a batch of myrobalan fingerprints as reference fingerprints, selecting peaks with strong absorption signals and good separation degree and stability as characteristic peaks, and calculating the relative retention time and the relative peak area of each characteristic peak.
8. The characteristic fingerprint spectrum of the myrobalan is characterized by comprising 12 characteristic fingerprint peaks: sequentially comprises myrobalan hypoacid, gallic acid, 5-galloylshikimic acid, punicalagin A, punicalagin B, corilagin, 1,3, 6-tri-O-galloyl-beta-D-glucose, myrobalan tannin, urolithin M5, myrobalan tannic acid, myrobalan Lin Rousuan and ellagic acid.
9. According to claimThe myrobalan characteristic fingerprint spectrum is characterized in that the peak of the corilagin is taken as a reference peak, and the relative retention time of each common peak and the reference peak is within a range of +/-5% of a specified value; the specified value is: the H1 peak is 0.2220 – 0.2453 The H2 peak is 0.4239 – 0.4685 The H3 peak is 0.6435 – 0.7112 The H4 peak is 0.7405 – 0.8185 The H5 peak is 0.8456 – 0.9346 The H6 peak was 1.000 and the H7 peak was 1.056 – 1.167 The H8 peak was 1.167 – 1.290 The H9 peak is 1.306 – 1.444 The H10 peak was 1.501 – 1.659 H11 peak 1.532 – 1.693 The H12 peak was 1.728 – 1.910。
10. The use of the content determination method according to any one of claims 1 to 6 in content determination of fructus chebulae immaturus tannins components and in construction of characteristic fingerprints.
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