CN102127047B - Method for separating 5-hydroxyl isoflavonoids monomeric compound in blackberry lily - Google Patents
Method for separating 5-hydroxyl isoflavonoids monomeric compound in blackberry lily Download PDFInfo
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- CN102127047B CN102127047B CN2011100036527A CN201110003652A CN102127047B CN 102127047 B CN102127047 B CN 102127047B CN 2011100036527 A CN2011100036527 A CN 2011100036527A CN 201110003652 A CN201110003652 A CN 201110003652A CN 102127047 B CN102127047 B CN 102127047B
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Abstract
The invention relates to the field of natural medicaments, in particular to the field for separating and purifying 5-hydroxyl isoflavonoids monomeric compound in irides blackberry lily. The method is characterized in that the method is used for separating and purifying high-purity monomeric compound iso-irigenin, irigenin and 5, 7, 4'-trihydroxy-6, 3', 5'-trimethoxy isoflavone from the blackberry lily by a complexing high-speed counter current color spectrum, and the complexing agent is copper nitrate. The separation method has the advantages of being simple, fast, high in separation efficiency, and good in product purity.
Description
Technical field
The present invention relates to natural medicine field; Be specifically related to the separation and purification field of NOVASOY 400 in the irides blackberry lily; Promptly utilize complexing the high speed adverse current chromatogram different irigenin of the monomeric compound of separating and purifying high-purity, irigenin and 5,7 from blackberry lily, 4 '-trihydroxy--6; 3 ', 5 '-method of trimethoxy NOVASOY 400.
Background technology
Blackberry lily is the dry rhizome of Iridaceae (Iridaceae) plant blackberry lily Belamcanda chinensis (L.) DC., have clearing heat and detoxicating, the effect of dissolving phlegm relieve sore throat.Contain polytype natural product in the blackberry lily, wherein isoflavonoid is its main active substances, has significant anti-inflammatory, biological activity and pharmacological action such as antibiotic, antiviral, anticancer.Different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-the trimethoxy NOVASOY 400 is the main active ingredient in the blackberry lily.They are isomerss, and separating difficulty is bigger.At present, both at home and abroad these three target compounds of traditional method separation and purification such as conventional column chromatography and recrystallization that adopt more.These method complex operations, waste time and energy, and stationary phase there is the non-reversibility adsorption to sample, causes sample loss serious.In addition,, can't it effectively be separated usually, need repeatedly repeat preparation and carry out purifying because target compound character is very approaching.
(High-speed counter current chromatography HSCCC) is a kind of newer liquid liquid distribution chromatography technology to high speed adverse current chromatogram.It need not any solid support or carrier, has therefore overcome the non-reversibility adsorption of traditional separation method to sample, and the theoretical recovery is 100%.Using traditional HSCCC to the different irigenin in the blackberry lily, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-when the trimethoxy NOVASOY 400 separated, different irigenin can't be separated with irigenin.
Summary of the invention
The invention discloses the method for NOVASOY 400 in a kind of complexing high-speed countercurrent chromatography separation and purification blackberry lily, this technology has efficiently, fast, can under the condition that does not change hardware, significantly improve the advantage of separating effect.
Main active ingredient in the blackberry lily is the very proximate 5-hydroxy-isoflavone of a structure compounds, and traditional separation and purification chromatographic technique can't make them obtain to separate preferably at short notice.The present invention adopts the complexing high-speed countercurrent chromatography to separate monomeric compound in the blackberry lily, has obtained good separating resulting.
The contriver attempts to utilize traditional HSCCC that the monomer of NOVASOY 400 in the blackberry lily is separated, but because target compound character is very approaching, is difficult to separate; The contriver attempts to improve separating effect through in stationary phase, adding complexing agent, discovers that different complexing agents is isolated different irigenin, irigenin and 5 to NOVASOY 400 in the blackberry lily; 7; 4 '-trihydroxy--6,3 ', 5 '-effect of trimethoxy NOVASOY 400 is different.
The contriver adopts different complexing agent to add in the stationary phase above-mentioned three kinds of monomeric compounds in the blackberry lily: different irigenin (I), irigenin (II) and 5,7,4 '-trihydroxy--6,3 ', 5 '-trimethoxy NOVASOY 400 (III) separates, and the result sees table 1.
Table 1I, II, partition ratio and the separating size of III in different complexing agents
aComplexing agent concentration is 0.1mol/L, and solvent systems is a sherwood oil: ETHYLE ACETATE: methyl alcohol: water=3: 5: 3: 5
b"-" do not dissolve in being illustrated in down mutually.
Suitable complexing agent should possess following condition: 1. in descending mutually, have enough solubleness; 2. the partition ratio of treating isolated compound is between 0.5-2; 3. the separating size of compound is more than 1.5.
Visible by table 1: copper sulfate solubleness in descending mutually is too small, therefore can not use.When using zinc chloride as complexing agent, the partition ratio of different irigenin and irigenin is less than 0.5, and their separating size is less than 0.5, and therefore, separating effect is bad.When using aluminum chloride as complexing agent, the separating size of different irigenin and irigenin can't reach 1.5 equally.Evidence has only cupric nitrate could satisfy above three conditions simultaneously as complexing agent, reaches excellent separating effect.Formed technical scheme of the present invention on this basis.
The present invention adopts complexing the high-speed countercurrent chromatography different irigenin of separation and purification, irigenin and 5,7 from the blackberry lily NOVASOY 400,4 '-trihydroxy--6; 3 ', 5 '-the trimethoxy NOVASOY 400, method comprises: with sherwood oil: ETHYLE ACETATE: methyl alcohol: water=2.8-3.2: 4.8-5.2: 2.8-3.2: 4.8-5.2 is by corresponding volume ratio preparation two-phase solvent; On be moving phase mutually, down to be added to the complexing agent cupric nitrate be stationary phase, is dissolved in the blackberry lily NOVASOY 400 mutually; Pump into stationary phase from tail end toward high-speed counter-current chromatograph; Treat to rotate main frame after pipeline fully is full of stationary phase, pump into moving phase simultaneously, the receiving target composition.
Wherein pump into the preferred 10mL/min of flow velocity of stationary phase.
The preferred 0.05mol/L-0.15mol/L of the concentration of cupric nitrate in the solvent systems of complexing high speed adverse current chromatogram.
The preferred 800-900rpm of engine speed.
Moving phase pumps into the preferred 0.8-2.0mL/min of speed.
The blackberry lily NOVASOY 400 preferably with blackberry lily through extraction using alcohol, CH
2Cl
2Extraction obtains behind the ODS post excessively.Preferred preparation method comprises: the dry rhizome of getting blackberry lily is pulverized ethanol water or the methanol aqueous solution heating and refluxing extraction of back with 70%-100%.Behind filtration and the merging filtrate, concentrating under reduced pressure becomes medicinal extract.Use dichloromethane extraction then.Dichloromethane extraction medicinal extract is crossed anti-phase, with the methanol-water wash-out, gets the blackberry lily NOVASOY 400 after the elutriant merging concentrates.
Use the complexing high speed adverse current chromatogram with different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-separation and purification of trimethoxy NOVASOY 400.
Complexing high speed adverse current chromatogram two phase solvent system is a sherwood oil: ETHYLE ACETATE: methyl alcohol: water=2.8-3.2: 4.8-5.2: 2.8-3.2: 4.8-5.2; On be moving phase mutually, down to be added to the complexing agent cupric nitrate be stationary phase, is dissolved in the blackberry lily NOVASOY 400 mutually; Speed with 10mL/min from tail end toward high-speed counter-current chromatograph pumps into stationary phase; Treat to rotate main frame after pipeline fully is full of stationary phase, pump into moving phase simultaneously, according to color atlas receiving target composition.
The present invention is directed to all very close situation of osajin constituent structure, polarity in the blackberry lily; On traditional high speed adverse current chromatogram, improve; Creationary utilization complexing high-speed countercurrent chromatography separates ordinary method can't isolating different irigenin, irigenin and 5,7,4 '-trihydroxy--6; 3 ', 5 '-the trimethoxy NOVASOY 400.This method is simply quick, has the advantage of separation efficiency height, good product purity.
Description of drawings
Fig. 1 be the blackberry lily crude extract through the isolating color atlas of complexing high speed adverse current chromatogram (wherein flow point " I " is different irigenin, and flow point " II " is an irigenin, flow point " III " is 5,7,4 '-trihydroxy--6,3 ', 5 '-the trimethoxy NOVASOY 400)
Fig. 2 be the blackberry lily crude extract high-efficient liquid phase chromatogram (wherein 1 for different irigenin, and 2 is irigenin, 3 be 5,7,4 '-trihydroxy--6,3 ', 5 '-the trimethoxy NOVASOY 400)
Fig. 3 is the high-efficient liquid phase chromatogram of different irigenin
Fig. 4 is the high-efficient liquid phase chromatogram of irigenin
Fig. 5 is 5,7,4 '-trihydroxy--6,3 ', 5 '-high-efficient liquid phase chromatogram of trimethoxy NOVASOY 400
Embodiment
Embodiment 1
1, preparation blackberry lily crude extract
Get the dry rhizome 4kg of blackberry lily, pulverize the back with the extraction using alcohol of 3L 95% three times, 2h at every turn.Behind filtration and the merging filtrate, on 55 ℃ of Rotary Evaporators, reclaim solvent.Behind the gained medicinal extract dilute with water respectively with sherwood oil, methylene dichloride, ethyl acetate extraction.Dichloromethane extraction medicinal extract is crossed the ODS column chromatography, and with 40%MeOH, 50%MeOH is wash-out 500mL and 800ml respectively.The 50%MeOH elutriant merges according to HPLC inspection knowledge, finally obtains crude extract 1.1g.
2, the complexing high speed adverse current chromatogram separates preparation
Use complexing high speed adverse current chromatogram (Shenzhen is with the biochemical ltd in field) with different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-separation and purification of trimethoxy NOVASOY 400.
The high speed adverse current chromatogram two phase solvent system is a sherwood oil: ETHYLE ACETATE: methyl alcohol: water=3: 5: 3: 5.In separating funnel, placed 12 hours behind the shake well, make it fill a part layering.Separate mutually two then, the cupric nitrate that in descending mutually, adds 0.1mol/L is as stationary phase, and is last as moving phase.Speed with 10ml/min in the high-speed counter-current chromatograph pumps into stationary phase, treats to rotate main frame after pipeline fully is full of stationary phase, pumps into moving phase simultaneously.The high speed adverse current chromatogram column volume is 300ml, and the stationary phase retention rate is 78.3%, and flow velocity is 1ml/min, and rotating speed is 850rpm, and the detection wavelength is 254nm.Get the extract obtained powder 100mg of step 1 and be dissolved in the 10ml moving phase,, receive flow point according to detector by the sampling valve sample introduction; Wherein flow point " I " is different irigenin, and flow point " II " is an irigenin, and flow point " III " is 5; 7; 4 '-trihydroxy--6,3 ', 5 '-the trimethoxy NOVASOY 400.See Fig. 1.
3, the purity check at high-speed counter-current peak and structure determination
The HPLC analysis condition is following:
Chromatographic column: Ultimate TM XB-C
18(250mm * 4.6mm, 5 μ m); Column temperature: 30 ℃; Moving phase: water (0.1% trifluoroacetic acid)-methyl alcohol; Gradient: 0-15min, 40%-50% methyl alcohol; 15-35min, 50%-70% methyl alcohol; 35-45min, 57%-65% methyl alcohol; Detect wavelength: 254nm; Flow velocity: 1mL/min.
Peak I:ESI-MS:m/z 359 [M-H]
- 1H NMR (500MHz, DMSO-d
6) δ: 12.63 (1H, s, 5-OH), 8.46 (1H, s, H-2), and 6.73 (1H, d, J=2.0Hz, H-2 '), 6.68 (1H, d, J=2.0Hz, H-6 '), 6.33 (1H, s, H-6), 3.81 (3H, s, 8-OCH
3), 3.78 (3H, s, 4 '-OCH
3), 3.71 (3H, s, 5 '-OCH
3);
13C NMR (125MHz, DMSO-d
6) δ: 154.7 (C-2), 122.1 (C-3), 180.1 (C-4), 157.5 (C-5), 99.3 (C-6); 156.8 (C-7), 127.5 (C-8), 149.8 (C-9), 104.1 (C-10), 126.0 (C-1 '); (110.4 C-2 '), 150.3 (C-3 '), 136.4 (C-4 '), 152.9 (C-5 '); (104.6 C-6 '), 59.9 (8-OMe), 60.9 (4 '-OMe), 55.8 (5 '-OMe).
Peak II:ESI-MS:m/z 359 [M-H]
- 1H NMR (500MHz, DMSO-d
6) δ: 13.04 (1H, s, 5-OH), 10.82 (1H, s, 7-OH), 9.29 (1H, s, 3 '-OH), 8.38 (1H, s, H-2), and 6.72 (1H, d, J=1.8Hz, H-2 '), 6.67 (1H, d, J=1.8Hz, H-6 '), 6.52 (1H, s, H-8), 3.80 (3H, s, 5 '-OCH
3), 3.76 (3H, s, 4 '-OCH
3), 3.71 (3H, s, 6-OCH
3);
13C NMR (125MHz, DMSO-d
6) δ: 155.3 (C-2), 122.2 (C-3), 180.8 (C-4), 153.8 (C-5), 132.0 (C-6); 158.0 (C-7), 94.4 (C-8), 153.4 (C-9), 105.3 (C-10), 126.6 (C-1 '); (105.0 C-2 '), 150.7 (C-3 '), 136.9 (C-4 '), 153.1 (C-5 '); (110.8 C-6 '), 60.4 (6-OMe), 60.4 (4 '-OMe), 56.3 (5 '-OMe).
Peak III:ESI-MS:m/z 359 [M-H]
- 1H NMR (500MHz, DMSO-d
6) δ: 13.07 (1H, s, 5-OH), 8.39 (1H, s, H-2), 6.86 (2H, s, H-2 ', H-6 '), 6.50 (1H, s, H-8), 3.79 (6H, s, 3 '-OMe, 5 '-OMe), 3.76 (3H, s, 6-OMe);
13C NMR (125MHz, DMSO-d
6) δ: 154.5 (C-2), 122.0 (C-3), 180.5 (C-4), 153.3 (C-5), 131.6 (C-6); 158.0 (C-7), 94.0 (C-8), 152.8 (C-9), 104.8 (C-10), 120.8 (C-1 '); (106.9 C-2 '), 147.8 (C-3 '), 135.9 (C-4 '), 147.8 (C-5 '); (106.9 C-6 '), 60.0 (6-OMe), 56.2 (3 '-OMe, 5 '-OMe).
Color atlas is seen Fig. 2～Fig. 5.Wherein different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-purity of trimethoxy NOVASOY 400 is respectively 95.06%, 96.98% and 93.69%.
1, preparation blackberry lily crude extract
The preparation of blackberry lily crude extract is with embodiment 1.
2, the complexing high speed adverse current chromatogram separates preparation
Use complexing high speed adverse current chromatogram (Shenzhen is with the biochemical ltd in field) with different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-separation and purification of trimethoxy NOVASOY 400.
The high speed adverse current chromatogram two phase solvent system is a sherwood oil: ETHYLE ACETATE: methyl alcohol: water=3.2: 5: 3.2: 4.8.In separating funnel, placed 12 hours behind the shake well, make it fill a part layering.Separate mutually two then, the cupric nitrate that in descending mutually, adds 0.075mol/L is as stationary phase, and is last as moving phase.Speed with 10ml/min in the high-speed counter-current chromatograph pumps into stationary phase, treats to rotate main frame after pipeline fully is full of stationary phase, pumps into moving phase simultaneously.The high speed adverse current chromatogram column volume is 300ml, and the stationary phase retention rate is 78.3%, and flow velocity is 1ml/min, and rotating speed is 900rpm, and the detection wavelength is 254nm.Get the extract obtained powder 100mg of step 1 and be dissolved in the 10mL moving phase,, receive flow point according to detector by the sampling valve sample introduction; Wherein flow point " I " is different irigenin, and flow point " II " is an irigenin, and flow point " III " is 5; 7; 4 '-trihydroxy--6,3 ', 5 '-the trimethoxy NOVASOY 400.
3, the purity check at high-speed counter-current peak and structure determination
Different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-purity check of trimethoxy NOVASOY 400 and structure identify with embodiment 1.Wherein different irigenin, irigenin and 5,7,4 '-trihydroxy--6,3 ', 5 '-purity of trimethoxy NOVASOY 400 is respectively 96.02%, 98.80% and 93.49%.
Claims (5)
1. one kind is adopted complexing the high-speed countercurrent chromatography different irigenin of separation and purification, irigenin and 5,7 from the blackberry lily NOVASOY 400,4 '-trihydroxy--6; 3 ', 5 '-method of trimethoxy NOVASOY 400, comprising: with sherwood oil: ETHYLE ACETATE: methyl alcohol: water=2.8-3.2: 4.8-5.2: 2.8-3.2: 4.8-5.2 is by corresponding volume ratio preparation two-phase solvent; On be moving phase mutually; Under to be added to the complexing agent cupric nitrate be stationary phase, be dissolved in the blackberry lily NOVASOY 400 mutually, pump into stationary phase from tail end toward high-speed counter-current chromatograph; Treat to rotate main frame after pipeline fully is full of stationary phase; Pump into moving phase simultaneously, the receiving target composition, wherein to pump into speed be 0.8-2.0mL/min to moving phase.
2. the process of claim 1 wherein that the flow velocity that pumps into stationary phase is 10mL/min.
3. the process of claim 1 wherein that the concentration of cupric nitrate is 0.05mol/L-0.15mol/L in the solvent systems of complexing high speed adverse current chromatogram.
4. the process of claim 1 wherein that engine speed is 800-900rpm.
5. the process of claim 1 wherein the blackberry lily NOVASOY 400 be with blackberry lily through extraction using alcohol, CH
2Cl
2Extraction obtains behind the ODS post excessively.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986556A (en) * | 2006-12-19 | 2007-06-27 | 中国人民解放军第二军医大学 | Blackberry lily isoflavone compound and its application in medical field |
| CN101301287A (en) * | 2008-06-12 | 2008-11-12 | 上海双科医药科技有限公司 | Use of composition of isoflavonoids from Belamcanda chinensis in preparing anti-hepatitis medicament |
| CN101797265A (en) * | 2010-04-09 | 2010-08-11 | 中国人民解放军第二军医大学 | Application of belamcanda chinensis total isoflavone or isoflavone compounds in preparing medicaments and food for preventing and treating women's disease |
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2011
- 2011-01-10 CN CN2011100036527A patent/CN102127047B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1986556A (en) * | 2006-12-19 | 2007-06-27 | 中国人民解放军第二军医大学 | Blackberry lily isoflavone compound and its application in medical field |
| CN101301287A (en) * | 2008-06-12 | 2008-11-12 | 上海双科医药科技有限公司 | Use of composition of isoflavonoids from Belamcanda chinensis in preparing anti-hepatitis medicament |
| CN101797265A (en) * | 2010-04-09 | 2010-08-11 | 中国人民解放军第二军医大学 | Application of belamcanda chinensis total isoflavone or isoflavone compounds in preparing medicaments and food for preventing and treating women's disease |
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