CN112630327B - Quick detection method for active ingredients of cistanche - Google Patents
Quick detection method for active ingredients of cistanche Download PDFInfo
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- CN112630327B CN112630327B CN202011394924.6A CN202011394924A CN112630327B CN 112630327 B CN112630327 B CN 112630327B CN 202011394924 A CN202011394924 A CN 202011394924A CN 112630327 B CN112630327 B CN 112630327B
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- 238000001514 detection method Methods 0.000 title claims abstract description 42
- 239000004480 active ingredient Substances 0.000 title claims abstract description 34
- 241000005787 Cistanche Species 0.000 title claims abstract description 20
- 230000014759 maintenance of location Effects 0.000 claims abstract description 36
- QFRYQWYZSQDFOS-UHFFFAOYSA-N verbascoside Natural products CC1OC(COC2C(O)C(COC3OC(C(O)C(O)C3O)C(=O)O)OC(Oc4cc(O)cc5OC(=CC(=O)c45)c6ccc(O)c(O)c6)C2O)C(O)C(O)C1O QFRYQWYZSQDFOS-UHFFFAOYSA-N 0.000 claims abstract description 26
- NJYVDFDTLLZVMG-UHFFFAOYSA-N echinacoside Natural products CC1OC(OC2C(O)C(OCCc3ccc(O)c(O)c3)OC(COC4OC(CO)C(O)C(O)C4O)C2OC(=O)C=Cc5cc(O)cc(O)c5)C(O)C(O)C1O NJYVDFDTLLZVMG-UHFFFAOYSA-N 0.000 claims abstract description 17
- FSBUXLDOLNLABB-ISAKITKMSA-N echinacoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)\C=C\C=2C=C(O)C(O)=CC=2)[C@@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O FSBUXLDOLNLABB-ISAKITKMSA-N 0.000 claims abstract description 17
- IDVRYIVYDAOHSS-UHFFFAOYSA-N 4-O-[(E)-caffeoyl]-3-O-(alpha-L-rhamnopyranosyl)-D-glucopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1C(OC(=O)C=CC=2C=C(O)C(O)=CC=2)C(CO)OC(O)C1O IDVRYIVYDAOHSS-UHFFFAOYSA-N 0.000 claims abstract description 16
- KCDXLWBGPYIKRW-UHFFFAOYSA-N Cistanoside H Natural products CC1OC(OC2C(O)C(CO)OC(OCCc3ccc(O)c(O)c3)C2OC(=O)C)C(O)C(O)C1O KCDXLWBGPYIKRW-UHFFFAOYSA-N 0.000 claims abstract description 16
- IDVRYIVYDAOHSS-CRADLMAESA-N [(2r,3r,4r,5r,6r)-5,6-dihydroxy-2-(hydroxymethyl)-4-[(2r,3s,4s,5s,6r)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-3-yl] (e)-3-(3,4-dihydroxyphenyl)prop-2-enoate Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](C)O[C@@H]1O[C@H]1[C@H](OC(=O)\C=C\C=2C=C(O)C(O)=CC=2)[C@@H](CO)O[C@@H](O)[C@@H]1O IDVRYIVYDAOHSS-CRADLMAESA-N 0.000 claims abstract description 16
- KARNWFDIYRKBOE-UHFFFAOYSA-N cistanoside F Natural products CC1OC(OC(C(O)C=O)C(OC(=O)C=Cc2ccc(O)c(O)c2)C(O)CO)C(O)C(O)C1O KARNWFDIYRKBOE-UHFFFAOYSA-N 0.000 claims abstract description 16
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 claims abstract description 15
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 claims abstract description 15
- MQDZZCJYWRZONM-UHFFFAOYSA-N Cistanoside A Natural products COc1ccc(CCOC2OC(COC3OC(CO)C(O)C(O)C3O)C(OC(=O)C=Cc4ccc(O)c(O)c4)C(OC5OC(C)C(O)C(O)C5O)C2O)cc1O MQDZZCJYWRZONM-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229930185474 acteoside Natural products 0.000 claims abstract description 13
- FBSKJMQYURKNSU-ZLSOWSIRSA-N acteoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)\C=C\C=2C=C(O)C(O)=CC=2)[C@@H](CO)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O FBSKJMQYURKNSU-ZLSOWSIRSA-N 0.000 claims abstract description 13
- FBSKJMQYURKNSU-UKQWSTALSA-N acteoside I Natural products C[C@@H]1O[C@H](O[C@@H]2[C@@H](O)[C@H](OCCc3ccc(O)c(O)c3)O[C@H](CO)[C@H]2OC(=O)C=Cc4ccc(O)c(O)c4)[C@H](O)[C@H](O)[C@H]1O FBSKJMQYURKNSU-UKQWSTALSA-N 0.000 claims abstract description 13
- 239000012086 standard solution Substances 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- FNMHEHXNBNCPCI-QEOJJFGVSA-N Isoacteoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@H](OCCC=2C=C(O)C(O)=CC=2)O[C@H](COC(=O)\C=C\C=2C=C(O)C(O)=CC=2)[C@H]1O FNMHEHXNBNCPCI-QEOJJFGVSA-N 0.000 claims abstract description 10
- FNMHEHXNBNCPCI-RYEKTNFUSA-N isoacteoside Natural products C[C@@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](COC(=O)C=Cc3ccc(O)c(O)c3)O[C@@H](OCCc4ccc(O)c(O)c4)[C@@H]2O)[C@H](O)[C@H](O)[C@H]1O FNMHEHXNBNCPCI-RYEKTNFUSA-N 0.000 claims abstract description 10
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 8
- 239000013558 reference substance Substances 0.000 claims description 55
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 241000336291 Cistanche deserticola Species 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- YSCJAYPKBYRXEZ-HZPINHDXSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4ar,6ar,6bs,8as,12as,14ar,14br)-4,4,6a,6b,11,11,14b-heptamethyl-8a-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxycarbonyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-3-hydroxy-4-[(2s,3r,4s, Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C(O)=O)[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YSCJAYPKBYRXEZ-HZPINHDXSA-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
- 241000336316 Cistanche tubulosa Species 0.000 claims description 4
- ILRCGYURZSFMEG-RKQHYHRCSA-N Salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RKQHYHRCSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- QSVJYFLQYMVBDR-UHFFFAOYSA-N Ergosterin Natural products C1C(O)CCC2(C)C3=CCC4(C)C(C(C)C=CC(C)C(C)C)CCC4C3=CC=C21 QSVJYFLQYMVBDR-UHFFFAOYSA-N 0.000 claims description 2
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 claims description 2
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 claims description 2
- DNVPQKQSNYMLRS-APGDWVJJSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)/C=C/[C@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-APGDWVJJSA-N 0.000 claims description 2
- 241000336315 Cistanche salsa Species 0.000 claims 2
- 230000035945 sensitivity Effects 0.000 abstract description 3
- KZLDMAIXPXOZCX-UHFFFAOYSA-N Tubuloside A Natural products OC1C(O)C(O)C(C)OC1OC1C(OC(C)=O)C(OCCC=2C=C(O)C(O)=CC=2)OC(COC2C(C(O)C(O)C(CO)O2)O)C1OC(=O)C=CC1=CC=C(O)C(O)=C1 KZLDMAIXPXOZCX-UHFFFAOYSA-N 0.000 description 4
- JXRBUKCEZLRNOI-BRJAJVCCSA-N [(2r,3r,4s,5r,6r)-5-acetyloxy-6-[2-(3,4-dihydroxyphenyl)ethoxy]-2-[[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]-4-[[(2s,3r,4r,5r,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]methoxy]oxan-3-yl] (e)-3-(3,4-dihydroxyphenyl)prop-2-enoate Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1CO[C@@H]1[C@@H](OC(C)=O)[C@H](OCCC=2C=C(O)C(O)=CC=2)O[C@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 JXRBUKCEZLRNOI-BRJAJVCCSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001632052 Haloxylon ammodendron Species 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 241000308150 Orobanchaceae Species 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 230000002124 endocrine Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229930182489 iridoid glycoside Natural products 0.000 description 1
- 150000008145 iridoid glycosides Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- KDSWDGKIENPKLB-QJDQKFITSA-N verbascoside Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](OC(=O)CCC=2C=C(O)C(O)=CC=2)[C@@H](CO)O[C@@H](OCCC=2C=C(O)C(O)=CC=2)[C@@H]1O KDSWDGKIENPKLB-QJDQKFITSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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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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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- 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
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- 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
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Abstract
The invention discloses a rapid detection method of active ingredients of cistanche, which comprises the following steps: respectively preparing standard solutions of acteoside, isoacteoside, echinacoside, angioside A, 2' -acteoside, cistanoside A, cistanoside F and salidroside; then carrying out liquid chromatography detection on the sample to be detected and 8 standard solutions at the wavelength of 330nm or 275 nm; and comparing the retention time of the chromatographic peak of the sample to be detected with the reference retention time of the standard solution to determine whether the sample to be detected contains the 8 cistanche active ingredients. The method can simultaneously determine 8 cistanche active ingredients in the sample to be detected, and can accurately quantify the content of each active ingredient in the sample to be detected according to the standard curve of the standard substance and the chromatographic peak area of the sample to be detected, thereby greatly improving the detection efficiency, reducing the detection cost, and having the advantages of simple and rapid detection method, high accuracy and good sensitivity.
Description
Technical Field
The invention belongs to the technical field of cistanche detection, and particularly relates to a rapid detection method for cistanche active ingredients.
Background
Cistanchis Herba Herba, also called Jinsun bamboo shoot, herba Germinae, cistanchis Herba, herba Cistanchis, is dry fleshy stem with scaly leaf of Cistanchis Herba Y.C.Ma or Cistanchis Herba Cistanchis Tubulosa (Schrenk) Wight of Orobanchaceae, and is mainly produced in inner Mongolia, gansu, xinjiang, qinghai, etc. The plant is parasitic plant parasitic on the root of haloxylon ammodendron in desert, absorbs nutrient and water from haloxylon ammodendron host, has the reputation of desert ginseng, has high medicinal value and is a traditional famous and precious Chinese medicinal material. It is warm in nature, sweet and salty in taste, and is an essential herb for invigorating kidney, strengthening yang, loosening bowel to relieve constipation, and also has the effects of resisting fatigue, resisting aging, regulating endocrine, enhancing immunity, enhancing memory, etc. At present, a great deal of studies on chemical components of cistanche have been carried out by a great number of scholars at home and abroad, and currently, the chemical components of cistanche which are separated and identified mainly comprise phenylethanoid glycosides, iridoid glycosides, lignins, amino acids, saccharides and the like. Wherein the phenethyl alcohol total glycosides are main active ingredients in Cistanchis herba, and have multiple functions of tonifying yang, resisting oxidation, resisting aging, enhancing memory, etc. Patent CN106053626B provides a method for rapidly determining active ingredients of echinacoside and verbascoside in cistanche, which mainly utilizes ultra-high performance liquid chromatography-mass spectrometry to determine and analyze the contents of the two active ingredients, however, the method can only detect the two active ingredients at the same time, and the detection efficiency is low.
Disclosure of Invention
Aiming at the defect of low detection efficiency in the prior art when the active ingredients of the cistanche deserticola are detected, the invention provides a rapid detection method for the active ingredients of the cistanche deserticola, which can simultaneously and rapidly detect 8 effective active ingredients of the cistanche deserticola and obviously improve the detection efficiency, and meanwhile, the detection method is simple and rapid, has high accuracy, good sensitivity and low cost.
In order to achieve the purpose, the invention adopts the technical scheme that:
a rapid detection method for active ingredients of cistanche deserticola, which comprises the following steps:
step 1, preparing an ergosterin reference substance, an isoergosterin reference substance, an echinacoside reference substance, an angioside A reference substance, a 2' -acetylverbascoside reference substance, a cistanoside A reference substance, a cistanoside F reference substance and a salidroside reference substance into standard solutions respectively;
and 2, performing liquid chromatography detection on the sample to be detected and the 8 standard solutions obtained in the step 1: taking a formic acid solution as a mobile phase A, taking acetonitrile as a mobile phase B for gradient elution, and combining a diode array detector to perform chromatographic detection at the wavelength of 330nm or 275 nm;
and 3, comparing the retention time of the chromatographic peak of the 8 standard solutions in the step 1 with the reference retention time to determine whether the sample to be detected contains acteoside, isoacteoside, echinacoside, tubular flower glycoside A, 2' -acetyl verbascoside, cistanoside A, cistanoside F and salidroside.
Further, the retention time of the acteoside control at the wavelength of 330nm is 18.532min, the retention time of the isoacteoside control at the wavelength of 330nm is 19.719min, the retention time of the echinacoside control at the wavelength of 330nm is 13.880min, the retention time of the tubuloside A control at the wavelength of 330nm is 17.751min, the retention time of the 2' -acteoside control at the wavelength of 330nm is 21.525min, the retention time of the cistanoside A control at the wavelength of 330nm is 15.971min, the retention time of the cistanoside F control at the wavelength of 330nm is 9.135min, and the retention time of the rhodioloside control at the wavelength of 275nm is 9.492min.
Further, 8 reference substances are respectively dissolved by a methanol-water mixed solution with the volume ratio of 1.
Further, the procedure of gradient elution in step 2 is as follows: the sample size is 10 μ L, wherein at 0min and 10min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%;
at 22min and 25min, the volume fraction of the mobile phase A is 60%, and the volume fraction of the mobile phase B is 40%;
at 28min and 30min, the volume fraction of mobile phase A was 30% and the volume fraction of mobile phase B was 70%.
Further, the gradient elution procedure further comprises: the flow rate of sample injection is 0.4mL/min, and the column temperature is 30 ℃.
Further, the sample to be tested is at least one of cistanche, cistanche tubulosa, a substance containing cistanche or a substance containing cistanche tubulosa.
Further, adding the cistanche powder into a methanol-water mixed solution with the volume ratio of 1.
Further, the method also comprises the quantitative detection of the active ingredients in the sample to be detected, which specifically comprises the following steps: preparing 8 reference substances into different concentration gradients, drawing a standard curve according to the concentration of the reference substances and the chromatographic peak area during liquid chromatography detection, and quantitatively determining the content of the active ingredients in the sample to be detected according to the standard curve and the chromatographic peak area of the sample to be detected.
Compared with the prior art, the invention has the beneficial effects that: the invention effectively measures the cistanche active ingredients in the sample to be measured by combining the high performance liquid chromatograph with the diode array detector, wherein the diode array detector can simultaneously detect multi-channel wavelengths, can simultaneously measure 8 cistanche active ingredients in the sample to be measured according to the retention time difference of different substances under different wavelengths and by taking the standard solution as the reference, and can accurately quantify the content of each active ingredient in the sample to be measured according to the standard curve of the standard substance and the chromatographic peak area of the sample to be measured, thereby greatly improving the detection efficiency, reducing the detection cost, and the detection method is simple, rapid, high in accuracy and good in sensitivity.
Drawings
FIG. 1 is a chromatogram of a control product of acteoside of example 1 of the present invention at a wavelength of 330 nm;
FIG. 2 is a chromatogram of an isoacteoside control at a wavelength of 330nm according to example 1 of the present invention;
FIG. 3 is a chromatogram of a reference echinacoside sample at a wavelength of 330nm according to example 1 of the present invention;
FIG. 4 is a chromatogram of a sinotubular glycoside A control at a wavelength of 330nm in example 1 of the present invention;
FIG. 5 is a chromatogram of a reference 2' -acteoside sample at 330nm in example 1 of the present invention;
FIG. 6 is a chromatogram of cistanoside A control at a wavelength of 330nm in example 1 of the present invention;
FIG. 7 is a chromatogram of cistanoside F control at a wavelength of 330nm in example 1 of the present invention;
FIG. 8 is a chromatogram of a salidroside control at a wavelength of 275nm according to example 1 of the present invention;
FIG. 9 is a chromatogram of a sample to be tested at a wavelength of 330nm in example 2 of the present invention;
FIG. 10 is a chromatogram of a sample to be tested at a wavelength of 275nm in example 2 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The sources of the instrument equipment and the material reagent adopted in the embodiment of the invention are as follows:
a high performance liquid chromatograph: equipped with a diode array detector from Agilent, USA;
acteoside reference substance, isoacteoside reference substance, echinacoside reference substance, tubuloside A reference substance, 2' -acteoside reference substance, cistanoside A reference substance, cistanoside F reference substance, and salidroside reference substance are all purchased from Dowangsi Biotech limited;
methanol (chromatographically pure), acetonitrile (chromatographically pure) and formic acid (chromatographically pure) were purchased from Thermo Fisher Scientific, inc.
EXAMPLE 1 determination of the Retention time of the Standard solution
The embodiment provides a detection method of a cistanche active ingredient standard solution, which comprises the following steps:
step 1, respectively weighing 20mg of acteoside reference substance, 20mg of isoacteoside reference substance, 20mg of echinacoside reference substance, 10mg of tubuloside A reference substance, 10mg of 2' -acteoside reference substance, 10mg of cistanoside A reference substance, 10mg of cistanoside F reference substance and 20mg of salidroside reference substance, dissolving the components in a 25mL measuring flask by using a methanol-water mixed solution with a volume ratio of 1;
and 2, performing liquid chromatography detection on the 8 standard solutions by using a high performance liquid chromatograph, wherein the detection method specifically comprises the following steps: using 0.1% formic acid solution as mobile phase A and acetonitrile as mobile phase B to perform gradient elution, wherein the procedure of the gradient elution is as follows: the column temperature is 30 ℃, and the sample injection amount is 10 mu L each time;
wherein, at the time of 0min and 10min, the volume fraction of the mobile phase A is 80%, the volume fraction of the mobile phase B is 20%, and the sample injection flow rate is 0.4mL/min;
at 22min and 25min, the volume fraction of the mobile phase A is 60%, the volume fraction of the mobile phase B is 40%, and the sample injection flow rate is 0.4mL/min;
at 28min and 30min, the volume fraction of the mobile phase A is 30%, the volume fraction of the mobile phase B is 70%, and the sample injection flow rate is 0.4mL/min;
and (3) carrying out chromatographic detection at the wavelength of 330nm or 275nm by combining with a diode array detector, and counting the retention time of each control, namely the time from the sample introduction of the control to the appearance of the peak of the chromatographic peak. The detection results of the 8 kinds of reference substances are shown in figures 1-8, wherein figure 1 is chromatogram of acteoside reference substance at 330 nm; FIG. 2 is a chromatogram of isoacteoside control at 330 nm; FIG. 3 is a chromatogram of echinacoside reference substance at 330 nm; FIG. 4 is a chromatogram of a tubuloside A control at a wavelength of 330 nm; FIG. 5 is a chromatogram of a 2' -acteoside control at a wavelength of 330 nm; FIG. 6 is a chromatogram of cistanoside A reference substance at wavelength of 330 nm; FIG. 7 is a chromatogram of cistanoside F reference substance at wavelength of 330 nm; FIG. 8 is a chromatogram of salidroside control at a wavelength of 275 nm. Chromatogram of standard solution of 8 active ingredients of cistanche deserticola is obtained according to chromatogram, retention time of each component is obtained, and the result is shown in table 1.
TABLE 1 results of standard solution retention time measurements
The retention time of salidroside and cistanoside F are close, and the diode array detector can be used for simultaneously carrying out multichannel wavelength detection, so that the detection results under different wavelengths can be detected by one needle, wherein salidroside has strong absorption at 275nm, but the cistanoside F hardly absorbs under the same wavelength, and therefore, even if the retention time is close, the salidroside and the cistanoside F can be obviously distinguished according to the difference of the wavelengths.
EXAMPLE 2 detection of samples to be tested
In this embodiment, a rapid detection method for active ingredients in cistanche deserticola powder is provided, and it should be clear to those skilled in the art that any substance containing the above 8 cistanche deserticola active ingredients can be used as a sample to be detected.
Preparation of a sample to be tested: taking cistanche powder (purchased from Wuhan Kestan Biotechnology Co., ltd.) and screening the powder through a No. four sieve, precisely weighing 1g of the sieved cistanche powder, placing the powder into a 100mL brown measuring flask, then adding a methanol-water mixed solution with a volume ratio of 1. And taking the supernatant, and filtering the supernatant by using a 0.45-micron filter membrane to obtain the sample to be detected.
Performing liquid chromatography detection on the sample to be detected by using a high performance liquid chromatograph, wherein the detection method is the same as the step 2 in the embodiment 1, the retention time of the chromatographic peak of the 8 standard liquids detected in the embodiment 1 is taken as the reference retention time, and the retention time of the chromatographic peak of the sample to be detected is compared with the reference retention time to judge whether the sample to be detected contains the 8 substances, wherein the detection result with the wavelength of 330nm is shown in fig. 9, and the detection result with the wavelength of 275nm is shown in fig. 10.
Referring to FIG. 9, the sample to be tested showed distinct peaks at wavelength of 330nm at 13.806min and 18.407min, which correspond to the reference retention times of echinacoside and acteoside in example 1, i.e., the sample to be tested contained a large amount of echinacoside and acteoside in the active ingredient of Cistanchis herba. Meanwhile, according to the attached figure 10, the sample to be detected has no peak at a wavelength of 275nm in about 9.492min, which indicates that the sample to be detected does not contain salidroside.
The method can be used for simultaneously detecting whether the sample to be detected contains the 8 cistanche active ingredients or not, and further is used for detecting and controlling the product quality.
EXAMPLE 3 quantitative determination of active ingredients in samples to be tested
Respectively weighing acteoside reference substances, isoacteoside reference substances, echinacoside reference substances, tubulin A reference substances, 2' -acetylverbascoside reference substances, cistanoside A reference substances, cistanoside F reference substances and salidroside reference substances, dissolving the reference substances by using a methanol-water mixed solution with the volume ratio of 1 to prepare different concentration gradients, respectively carrying out liquid chromatography detection on the reference substances with different concentrations (the sample injection amount is 5 mu L), taking the concentration (unit: mg/mL) of the reference substances as a horizontal coordinate and the chromatographic peak area (mAU) during liquid chromatography detection as a vertical coordinate, and respectively drawing corresponding standard curves for 8 different reference substances, wherein the standard curves are shown in the following table:
calculating the chromatographic peak area according to the liquid chromatographic test result chart of the sample to be tested in fig. 9 and fig. 10, and substituting the chromatographic peak area into the standard curve to obtain the concentration of each active ingredient in the sample to be tested. The result shows that the sample to be detected has an obvious peak at the wavelength of 330nm at 13.806min, the peak area of the corresponding echinacoside is 946.70mAU, and the concentration of the echinacoside is 1.17mg/mL according to a standard curve; the peak appears obviously at 18.407min, corresponds to acteoside, the peak area is 343.20mAU, and the content is 0.92mg/mL according to a standard curve; a peak appears at 17.645min, the peak area of the corresponding tubular flower glycoside A is 30.23mAU, and the content of the tubular flower glycoside A is 0.027mg/mL according to a standard curve; has a peak at 9.108min, corresponding to cistanoside F, with a peak area of 18.10mAU, and a content of 0.099mg/mL according to the standard curve; the peak appears at 19.668min, corresponds to isoergosteride, has a peak area of 19.61mAU, and has a content of 0.032mg/mL according to a standard curve; has a peak at 15.385min corresponding to cistanoside A, and has a peak area of 14.55mAU, and a content of 0.085mg/mL according to the standard curve; it has a peak at 21.444min corresponding to 2' -acteoside, and its peak area is 4.49mAU, and its content is 0.75mg/mL according to standard curve. Furthermore, the sample to be detected has no peak at a wavelength of 275nm within about 9.492min, that is, the content of salidroside in the sample to be detected is 0mg/mL.
Furthermore, the content of each active component in the cistanche deserticola of three different batches (sample 1 batch number: 201902512; sample 2 batch number: 201903452, sample 3 batch number: 2019123453) is determined by the same method: the concentration of each active ingredient is obtained by measurement according to a standard curve, the specific mass of each active ingredient in the sample is calculated according to the volume, the percentage of the mass of each active ingredient in the total mass of the sample is calculated, and the measurement results are shown in the following table:
while the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims.
Claims (3)
1. A rapid detection method for active ingredients of cistanche deserticola is characterized by comprising the following steps:
step 1, preparing an ergosterin reference substance, an isoergosterin reference substance, an echinacoside reference substance, an angioside A reference substance, a 2' -acetylverbascoside reference substance, a cistanoside A reference substance, a cistanoside F reference substance and a salidroside reference substance into standard solutions respectively;
step 2, taking cistanche powder, adding a methanol-water mixed solution with the volume ratio of 1: taking a formic acid solution as a mobile phase A, taking acetonitrile as a mobile phase B for gradient elution, and combining a diode array detector to perform chromatographic detection at the wavelength of 330nm or 275 nm;
step 3, taking the retention time of the chromatographic peaks of the 8 standard liquids in the step 1 as reference retention time, and comparing the retention time of the chromatographic peaks of the sample to be detected with the reference retention time to determine whether the sample to be detected contains acteoside, isoacteoside, echinacoside, tubular flower glycoside A, 2' -acetyl verbascoside, cistanoside A, cistanoside F and salidroside;
the retention time of the acteoside control at the wavelength of 330nm is 18.532min, the retention time of the isoacteoside control at the wavelength of 330nm is 19.719min, the retention time of the echinacoside control at the wavelength of 330nm is 13.880min, the retention time of the angioside A control at the wavelength of 330nm is 17.751min, the retention time of the 2' -acteoside control at the wavelength of 330nm is 21.525min, the retention time of the cistanoside A control at the wavelength of 330nm is 15.971min, the retention time of the cistanoside F control at the wavelength of 330nm is 9.135min, and the retention time of the rhodioloside control at the wavelength of 275nm is 9.492min;
in the step 1, 8 reference substances are respectively dissolved by a methanol-water mixed solution with the volume ratio of 1;
the procedure for the gradient elution in step 2 was: the sample amount is 10 μ L each time, wherein at 0min and 10min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%;
at 22min and 25min, the volume fraction of mobile phase A is 60%, and the volume fraction of mobile phase B is 40%;
at 28min and 30min, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%;
the gradient elution procedure further comprises: the flow rate of sample injection is 0.4mL/min, and the column temperature is 30 ℃.
2. The method according to claim 1, wherein the sample to be tested is at least one of cistanche salsa, cistanche tubulosa, a substance containing cistanche salsa or a substance containing cistanche tubulosa.
3. The detection method according to claim 1, characterized in that it further comprises a quantitative detection of the active ingredients in the sample to be detected, in particular: preparing 8 reference substances into different concentration gradients, drawing a standard curve according to the concentration of the reference substances and the chromatographic peak area during liquid chromatography detection, and quantitatively determining the content of the active ingredients in the sample to be detected according to the standard curve and the chromatographic peak area of the sample to be detected.
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