CN105367531A - Method for separating two homoisoflavonoids in fibrous roots of ophiopogon japonicusby adopting recycling high-speed counter-current chromatography - Google Patents
Method for separating two homoisoflavonoids in fibrous roots of ophiopogon japonicusby adopting recycling high-speed counter-current chromatography Download PDFInfo
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- CN105367531A CN105367531A CN201510857198.XA CN201510857198A CN105367531A CN 105367531 A CN105367531 A CN 105367531A CN 201510857198 A CN201510857198 A CN 201510857198A CN 105367531 A CN105367531 A CN 105367531A
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- dwarf lilyturf
- methylophiopogonanone
- methyl
- flavanone
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- 244000248557 Ophiopogon japonicus Species 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000010262 high-speed countercurrent chromatography Methods 0.000 title claims abstract description 12
- 238000004064 recycling Methods 0.000 title claims abstract description 6
- 229930195210 Ophiopogon Natural products 0.000 title claims description 4
- 239000000203 mixture Substances 0.000 claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 14
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- 239000002994 raw material Substances 0.000 claims abstract description 4
- XXDBCABHQMLKHQ-UHFFFAOYSA-N 2-methyl-2-phenyl-3h-chromen-4-one Chemical compound C1C(=O)C2=CC=CC=C2OC1(C)C1=CC=CC=C1 XXDBCABHQMLKHQ-UHFFFAOYSA-N 0.000 claims description 55
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 230000004087 circulation Effects 0.000 claims description 20
- 230000002411 adverse Effects 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 230000005526 G1 to G0 transition Effects 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 241001448424 Ophiopogon Species 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 239000000835 fiber Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 4
- UFMAZRUMVFVHLY-CYBMUJFWSA-N (3r)-5,7-dihydroxy-3-[(4-methoxyphenyl)methyl]-6,8-dimethyl-2,3-dihydrochromen-4-one Chemical compound C1=CC(OC)=CC=C1C[C@H]1C(=O)C2=C(O)C(C)=C(O)C(C)=C2OC1 UFMAZRUMVFVHLY-CYBMUJFWSA-N 0.000 abstract 8
- BXTNNJIQILYHJB-GFCCVEGCSA-N Methylophiopogonanone A Chemical compound C1=C2OCOC2=CC(C[C@@H]2COC3=C(C)C(O)=C(C(=C3C2=O)O)C)=C1 BXTNNJIQILYHJB-GFCCVEGCSA-N 0.000 abstract 8
- 239000003480 eluent Substances 0.000 abstract 3
- 229940079593 drug Drugs 0.000 abstract 1
- 238000002481 ethanol extraction Methods 0.000 abstract 1
- 238000003810 ethyl acetate extraction Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 239000000047 product Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- WMJVWGZVCINNNW-UHFFFAOYSA-N acetonitrile ethyl acetate hexane methanol hydrate Chemical compound O.OC.CC#N.CCCCCC.CCOC(C)=O WMJVWGZVCINNNW-UHFFFAOYSA-N 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000002790 anti-mutagenic effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000017574 dry cough Diseases 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- 239000002024 ethyl acetate extract Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 229930013032 isoflavonoid Natural products 0.000 description 1
- 150000003817 isoflavonoid derivatives Chemical class 0.000 description 1
- 235000012891 isoflavonoids Nutrition 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- HZVOZRGWRWCICA-UHFFFAOYSA-N methanediyl Chemical compound [CH2] HZVOZRGWRWCICA-UHFFFAOYSA-N 0.000 description 1
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- 238000005192 partition Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
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- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
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- 150000003384 small molecules Chemical class 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000035922 thirst Effects 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
- C07D311/38—2,3-Dihydro derivatives, e.g. isoflavanones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a method for separating two homoisoflavonoids in fibrous roots of ophiopogon japonicus by adopting recycling high-speed counter-current chromatography (HSCCC), and belongs to the technical field of natural medicines. The method comprises the following steps of conducting ethanol extraction, ethyl acetate extraction and silica-gel column chromatography on fibrous roots of ophiopogon japonicus serving as a raw material to obtain a mixture which only contains methylophiopogonanone A and methylophiopogonanone B; separating and purifying methylophiopogonanone A and methylophiopogonanone B in the mixture by using a high-speed counter-current instrument; and detecting eluents with HPLC, mixing the eluents of methylophiopogonanone A having the purity of more than 98 percent and mixing eluents of methylophiopogonanone B having the purity of more than 98 percent, and recovering the solvent by using a rotary evaporator to obtain monomeric compounds. The method can be used for efficiently and quickly separating the mixture of methylophiopogonanone A and methylophiopogonanone B in fibrous roots of ophiopogon japonicus, and has the advantages of saving a mobile phase, reducing the sample recovering burden, saving time and the like in comparison with conventional multiple HSCCC preparation.
Description
Technical field
The invention belongs to natural medicine technical field, about the separation purification method of compound in Rootlet Ophiopogonis, be specifically related to utilize circulation high speed adverse current chromatogram from fibrous root of Radix Ophiopogonis extract, isolate the method for methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B.
Background technology
The Chinese medicine tuber of dwarf lilyturf (OphiopogonisRadix) is Liliaceae Ophiopogon perennial evergreen herbaceous plant tuber of dwarf lilyturf
ophiopogonjaponicus(L.f) dried root of Ker-Gawl. is one of traditional conventional Chinese medicine; There is nourishing Yin and promoting production of body fluid, effect that moistening lung clears away heart-fire; Cure mainly dryness of the lung dry cough, deficiency of Yin consumptive disease coughed, thirsty, the interior heat of larynx numbness pharyngalgia, Tianjin wound is quenched one's thirst, vexed insomnia, the dry constipation of intestines etc.This product main product, in Zhejiang Province and Sichuan Province, is called as the Zhejiang tuber of dwarf lilyturf and river tuber of dwarf lilyturf respectively.Research shows, homoisoflavone is the small molecule active composition that in the tuber of dwarf lilyturf, a class is important, and its parent nucleus skeleton carbon number is 16, many methylene radical between the B ring and C ring than common isoflavonoid; This compounds has antitumor, anti-inflammatory, suppression phosphorylation, estrogen antagonist, antimutagenic, antibechic, antimycotic, many-sided biological activity and the effect such as inducing vasodilation and hepatocyte protection.
Methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B(structural formula are shown in Fig. 1) be homoisoflavone constituents main in the tuber of dwarf lilyturf, but due to structural similitude, in the solution such as water, methyl alcohol, acetonitrile, solubleness is low.The method separating effect such as domestic and international adopted silica gel column chromatography, gel chromatography and preparative liquid chromatography is all undesirable at present, and it usually cannot be made within a short period of time to be effectively separated, and need carry out repeatedly purifying and repeat preparation, complex operation, wastes time and energy.And adopting Tubers of Ophiopogon japonicus medicinal material to be prepared, cost is high, also can consume the herb resource tuber of dwarf lilyturf in a large number.Rootlet Ophiopogonis is the waste in the tradition medicinal part block root course of processing tuber of dwarf lilyturf, usually used as animal-feed or directly abandon.
A kind of new chromatographic separating and purifying technology based on liquid-liquid partition mechanism of high speed adverse current chromatogram (HSCCC).It need not any solid-state upholder or carrier, but utilizes two phase solvent system in the spiral tube of high speed rotating, set up a kind of special one-way fluid dynamic equilibrium.When wherein one as stationary phase, another is as moving phase, can retain a large amount of stationary phase in the process of continuous wash-out.Owing to not needing solid support, the separation of material realizes according to the difference of its partition ratio in two-phase, thus avoids the sample loss, inactivation, sex change etc. that cause because of irreversible adsorption, is particularly suitable for the separation of natural bioactive ingredients.But the HSCCC of experiment discovery single routine is separated methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture effect is also undesirable.
Summary of the invention
For prior art Problems existing, the object of the invention is to design provides a kind of technical scheme adopting circulation high speed adverse current chromatogram to be separated the method for two kinds of homoisoflavones from Rootlet Ophiopogonis, and the method has efficiently, fast, under the condition not changing hardware, significantly can improve the advantage of separating effect.
Contriver finds the chemical constitution study tuber of dwarf lilyturf, and in Rootlet Ophiopogonis, methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B component content are significantly higher than content in block root, and Rootlet Ophiopogonis thus can be selected to be that raw material carries out separation and purification.
Contriver attempts to utilize a HSCCC to be separated with methyl flavanone tuber of dwarf lilyturf B methyl flavanone tuber of dwarf lilyturf A in Rootlet Ophiopogonis, but due to target compound character very close, separating effect is undesirable.Contriver found through experiments circulation HSCCC can significantly improve separating effect; Different moving phase all can be applied, and just the number of times of separating effect and circulation is different.Circulation high speed adverse current chromatogram hardware condition is consistent with conventional high speed adverse current chromatogram, just spiral tube outlet is connected with the entrance of moving phase pump after sample introduction certain hour.Therefore two components be not separated completely after flash liberation in main frame can enter high-speed counter-current chromatograph and be separated, so repeatedly.After two peaks on detector are separated completely, cut off the connection of spiral tube outlet and pump intake, and locate to collect two kinds of target products that effluent can obtain having satisfactory purity at the former.
The present invention realizes especially by following methods:
A kind of described method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis, is characterized in that comprising following processing step:
1) take Rootlet Ophiopogonis as raw material, obtain only containing methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture through extraction using alcohol, extraction into ethyl acetate and silica gel column chromatography;
2) the methyl flavanone tuber of dwarf lilyturf A that obtains of step 1) and methyl flavanone tuber of dwarf lilyturf B mixture application high-speed counter-current instrument carry out separation and purification, high-speed counter-current instrument condition for methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture be dissolved in upper mutually in, in high-speed counter-current chromatograph, lower phase is pumped into the speed of 10 ~ 20mL/min, be full of completely after stationary phase until pipeline and rotate main frame with 600 ~ 1000rpm, pump into moving phase with the speed of 1 ~ 3mL/min simultaneously, after running certain hour, spiral tube outlet is connected with the entrance of moving phase pump, according to HSCCC separation case, circulate after 3 ~ 8 times, cut off the connection of outlet and entrance, effluent is collected from exit,
3) step 2) the effluent liquid HPLC that obtains detects, and the collection liquid respectively methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B purity being greater than 98% merges, and uses Rotary Evaporators recycling design, obtains monomeric compound.
A kind of described method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis, is characterized in that in described step 1), Rootlet Ophiopogonis is Zhejiang Rootlet Ophiopogonis and Fibre Ophiopogon.
A kind of described method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis, it is characterized in that silica gel column chromatography condition in described step 1): with sherwood oil and ethyl acetate volume ratio for 100:0,50:1,20:1,10:1,5:1 wash-out respectively, elutriant is known according to HPLC inspection and is merged.
A kind of described method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis, it is characterized in that described step 2) high speed adverse current chromatogram two phase solvent system normal hexane: ethyl acetate: ethanol: acetonitrile: water volume ratio is 2:1 ~ 3:1 ~ 4:0 ~ 1.5:0.5 ~ 2, or normal hexane: ethyl acetate: methyl alcohol: acetonitrile: water volume ratio is 2:1 ~ 3:1 ~ 4:0 ~ 1.5:0.5 ~ 2, upper is stationary phase mutually, and lower is moving phase mutually.
Methyl flavanone tuber of dwarf lilyturf A in Rootlet Ophiopogonis can efficiently, rapidly effectively be separated with B mixture by the present invention, and the present invention and routine are compared with repeatedly prepared by HSCCC, have saving moving phase, reduces sample and reclaims advantages such as bearing, save time.
Accompanying drawing explanation
Fig. 1 is methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B structural formula;
Fig. 2 is the HPLC spectrogram of the blend sample of methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B;
Fig. 3 is the circulation HSCCC spectrogram of methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B;
Fig. 4 is the HPLC color atlas of methyl flavanone tuber of dwarf lilyturf A;
Fig. 5 is the HPLC color atlas of methyl flavanone tuber of dwarf lilyturf B.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1
(1) methyl flavanone tuber of dwarf lilyturf A and the preparation of methyl flavanone tuber of dwarf lilyturf B blend sample
Get the dry fibrous root 6kg of the Zhejiang tuber of dwarf lilyturf, pulverize the EtOH Sonicate assisted extraction 2 times of rear 30L80%, each 1h, filter and after merging filtrate, reclaim Extraction solvent with on Rotary Evaporators.With extraction into ethyl acetate after gained medicinal extract dilute with water.Ethyl acetate extract (46.97g) crosses silica gel column chromatography, with sherwood oil: ethyl acetate (100:0,50:1,20:1,10:1,5:1) respectively wash-out 2000mL elutriant knows merging according to HPLC inspection, and HPLC analysis condition is as follows: chromatographic column is ThermoC
18(250mm × 4.6mm, 5 μm), column temperature is 30 DEG C, and moving phase adopts water-acetonitrile (35:65); Determined wavelength is 285nm; Flow velocity is set to 1mL/min.Finally obtain only detecting color atlas as shown in Figure 2 containing methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture 1.50g, HPLC.
(2) circulation high speed adverse current chromatogram is separated preparation
Application TBE-300A type high-speed counter-current instrument (Shanghai Tongtian Biotechnology Co., Ltd.) will obtain methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture separation purifying above.
High speed adverse current chromatogram two phase solvent system normal hexane: ethyl acetate: ethanol: acetonitrile: water volume ratio 2:1.5:1:1:1, places 4 hours after shake well, makes its abundant layering in separating funnel.Then two-phase is separated, upper as stationary phase, lower to moving phase, ultrasonic degas 30min.Get methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture 100mg, be dissolved in 10mL moving phase, as sample to be separated after filtration.
Speed with 20mL/min in high-speed counter-current chromatograph pumps into stationary phase, and be fully full of after stationary phase until pipeline and rotate main frame with 900rpm, 2mL/min pumps into moving phase simultaneously, and determined wavelength is set to 285nm, treats that two-phase reaches balance, injects sample after baseline stability; During 60min, spiral tube outlet is connected with the entrance of moving phase pump, through 2 circulations; Cut off the connection of outlet and entrance during 360min, collect effluent from exit; Receive flow point according to detector, wherein first flowing out chromatographic peak is methyl flavanone tuber of dwarf lilyturf A, and rear outflow chromatographic peak is methyl flavanone tuber of dwarf lilyturf B.Circulation HSCCC separate colors spectrogram is shown in Fig. 3.
(3) sample purity analysis and structure determination
Adopt above-mentioned HPLC detection method, be stream part solution merging that 6.6min and 7.3min purity is greater than 98% by collecting the retention time obtained, HPLC detects color atlas and sees Figure 4 and 5.
Stream part after merging uses Rotary Evaporators recycling design, obtains methyl flavanone tuber of dwarf lilyturf A(1 respectively) 25mg, methyl flavanone tuber of dwarf lilyturf B(1) 60mg.
Methyl flavanone tuber of dwarf lilyturf A structure determination: HR-ESI-MS:m/z341.1031 [M-H]
-(C
19h
18o
6);
13cNMR (400MHz, δ, CDCl
3): 69.0(C-2), 46.9(C-3), 198.4(C-4), 159.8(C-5), 102.4(C-6), 160.8(C-7), 102.9(C-8), 157.9(C-9), 101.8(C-10), 32.7(C-11), 131.9(C-1 '), 109.6(C-2 '), 146.5(C-3 '), 148.0(C-4 '), 108.5(C-5 '), 122.3(C-6 '), 7.0(6-CH
3), 7.5(8-CH
3), 101.1(-O-CH
2-O-).
Methyl flavanone tuber of dwarf lilyturf B structure determination: HR-ESI-MS:m/z327.1238 [M-H]
-(C
19h
20o
5);
13cNMR (400MHz, δ, CDCl
3): 69.1(C-2), 47.0(C-3), 198.6(C-4), 159.8(C-5), 102.4(C-6), 160.4(C-7), 102.9(C-8), 157.9(C-9), 101.8(C-10), 32.1(C-11), 130.2(C-1 '), 130.3(C-2 '), 114.2(C-3 '), 158.6(C-4 '), 114.3(C-5 '), 130.3(C-6 '), 7.0(6-CH
3), 7.5(8-CH
3), 55.4(-O-CH
3).
Embodiment 2
(1) methyl flavanone tuber of dwarf lilyturf A and the preparation of methyl flavanone tuber of dwarf lilyturf B blend sample
With embodiment 1.
(2) circulation high speed adverse current chromatogram is separated preparation
Instrument is with embodiment 1.Two-phase solvent system n-hexane-ethyl acetate-methanol-acetonitrile-water volume ratio 2:2:3.5:1:1.5 is mixed, fully leaves standstill 4 hours after concussion, make its abundant layering.Get as stationary phase, lower to moving phase, respectively to two-phase solvent ultrasonic degas 30 minutes.By above-mentioned middle sample 90mg solvent in 20ml moving phase, filter sample solution is for subsequent use.The stationary phase prepared is pumped in high-speed counter-current chromatograph with the speed of 20mL/min, then opens high-speed counter-current chromatograph, regulate engine speed 800rpm, flow rate of mobile phase is 2ml/min, determined wavelength is set to 285nm, treats that two-phase reaches balance, injects sample after baseline stability; 50min, is connected spiral tube outlet with the entrance of moving phase pump, through 2 circulations; 310min, cuts off the connection of outlet and entrance, collects effluent from exit; Receive flow point according to detector, wherein first flowing out chromatographic peak is methyl flavanone tuber of dwarf lilyturf A, and rear outflow chromatographic peak is methyl flavanone tuber of dwarf lilyturf B.Analyze according to HPLC, purity is greater than 98% flow point solution and merges, use Rotary Evaporators recycling design, obtain methyl flavanone tuber of dwarf lilyturf A and the 40mg methyl flavanone tuber of dwarf lilyturf B of 18mg respectively.
Claims (4)
1. adopt circulation high speed adverse current chromatogram from Rootlet Ophiopogonis, be separated a method for two kinds of homoisoflavones, it is characterized in that comprising following processing step:
1) take Rootlet Ophiopogonis as raw material, obtain only containing methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture through extraction using alcohol, extraction into ethyl acetate and silica gel column chromatography;
2) the methyl flavanone tuber of dwarf lilyturf A that obtains of step 1) and methyl flavanone tuber of dwarf lilyturf B mixture application high-speed counter-current instrument carry out separation and purification, high-speed counter-current instrument condition for methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B mixture be dissolved in upper mutually in, in high-speed counter-current chromatograph, lower phase is pumped into the speed of 10 ~ 20mL/min, be full of completely after stationary phase until pipeline and rotate main frame with 600 ~ 1000rpm, pump into moving phase with the speed of 1 ~ 3mL/min simultaneously, after running certain hour, spiral tube outlet is connected with the entrance of moving phase pump, according to HSCCC separation case, circulate after 3 ~ 8 times, cut off the connection of outlet and entrance, effluent is collected from exit,
3) step 2) the effluent liquid HPLC that obtains detects, and the collection liquid respectively methyl flavanone tuber of dwarf lilyturf A and methyl flavanone tuber of dwarf lilyturf B purity being greater than 98% merges, and uses Rotary Evaporators recycling design, obtains monomeric compound.
2. a kind of method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis as claimed in claim 1, is characterized in that in described step 1), Rootlet Ophiopogonis is Zhejiang Rootlet Ophiopogonis and Fibre Ophiopogon.
3. a kind of method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis as claimed in claim 1, it is characterized in that silica gel column chromatography condition in described step 1): with sherwood oil and ethyl acetate volume ratio for 100:0,50:1,20:1,10:1,5:1 wash-out respectively, elutriant is known according to HPLC inspection and is merged.
4. a kind of method adopting circulation high speed adverse current chromatogram to be separated two kinds of homoisoflavones from Rootlet Ophiopogonis as claimed in claim 1, it is characterized in that described step 2) high speed adverse current chromatogram two phase solvent system normal hexane: ethyl acetate: ethanol: acetonitrile: water volume ratio is 2:1 ~ 3:1 ~ 4:0 ~ 1.5:0.5 ~ 2 or normal hexane: ethyl acetate: methyl alcohol: acetonitrile: water volume ratio is 2:1 ~ 3:1 ~ 4:0 ~ 1.5:0.5 ~ 2, upper is stationary phase mutually, and lower is moving phase mutually.
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| CN110426486A (en) * | 2019-08-01 | 2019-11-08 | 正大青春宝药业有限公司 | The discrimination method of Zhejiang Radix Ophiopogonis in Chinese materia medica preparation |
| CN114814034A (en) * | 2022-05-06 | 2022-07-29 | 浙江科技学院 | Liquid chromatography method for simultaneously detecting contents of saponin and flavone in Zhejiang ophiopogon root |
| CN116102529A (en) * | 2022-12-13 | 2023-05-12 | 浙江省林业科学研究院 | Method for separating methyl ophiopogon root flavanone A and methyl ophiopogon root flavanone B |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108129467A (en) * | 2017-11-30 | 2018-06-08 | 浙江科技学院 | A kind of HSCCC-DPPH is combined online activity analysis and the method for detaching Radix Ophiopogonis homoisoflavone |
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| CN114814034A (en) * | 2022-05-06 | 2022-07-29 | 浙江科技学院 | Liquid chromatography method for simultaneously detecting contents of saponin and flavone in Zhejiang ophiopogon root |
| CN114814034B (en) * | 2022-05-06 | 2024-02-13 | 浙江科技学院 | Liquid chromatography method for simultaneously detecting contents of saponin and flavone in ophiopogon japonicus |
| CN116102529A (en) * | 2022-12-13 | 2023-05-12 | 浙江省林业科学研究院 | Method for separating methyl ophiopogon root flavanone A and methyl ophiopogon root flavanone B |
| CN116102529B (en) * | 2022-12-13 | 2024-05-28 | 浙江省林业科学研究院 | Method for separating methyl ophiopogon root flavanone A and methyl ophiopogon root flavanone B |
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Effective date of registration: 20210122 Address after: 310023 No. 551, Xixi Road, Hangzhou, Zhejiang, Xihu District Patentee after: CHIATAI QINGCHUNBAO PHARMACEUTICAL Co.,Ltd. Address before: 310023 No. 318 stay Road, Xihu District, Zhejiang, Hangzhou Patentee before: ZHEJIANG University OF SCIENCE AND TECHNOLOGY |