CN101012257A - Extraction and separation method for rhubarb willow leaf flavone component - Google Patents

Extraction and separation method for rhubarb willow leaf flavone component Download PDF

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CN101012257A
CN101012257A CN 200710067244 CN200710067244A CN101012257A CN 101012257 A CN101012257 A CN 101012257A CN 200710067244 CN200710067244 CN 200710067244 CN 200710067244 A CN200710067244 A CN 200710067244A CN 101012257 A CN101012257 A CN 101012257A
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compound
water
extraction
ethanol
rhubarb
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许传莲
赵钰岚
邹文涛
许根俊
赵辅昆
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Zhejiang Sci Tech University ZSTU
Zhejiang University of Science and Technology ZUST
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Abstract

The invention discloses an extracting separating method of flavone from rheum officinale Cynanchum, which is characterized by the following: affirming extracting condition through alcohol; setting the extracting quantity of ether and water stature butanol; comparing the relationship of flavone structure with silica gel thin-layer chromatography, gluglucosan gel thin-layer chromatography and polyamide thin-layer chromatography; affirming the approximate flavone compound to high-effective chromatographic separating purifying structure.

Description

The extraction and separation method of rhubarb willow leaf flavone component
Technical field
The invention belongs to the natural product extraction separation method, relate generally to a kind of extraction and separation method of rhubarb willow leaf flavone component, utilize polymeric amide to extract and separate Changbai Mountain peculiar sallow rhubarb willow leaf flavone compound in conjunction with efficient liquid-phase chromatography method.
Background technology
Sallow (Salix) is the Salicaceae xylophyta, and this genus is of a great variety, widely distributed, and the whole world has kind more than 500 approximately, and only the sallow that distributes in China just has kind more than 210.Willow is among the people long as medicinal history.LI Shi-Zhen is put down in writing in Compendium of Material Medica: willow is the herbal classic low-grades, and its property bitter cold, nontoxic can be treated the yellow subcutaneous ulcer in geomantic omen, sore, carbuncle and painful swelling, and phlegm heat is drenched disease, disease diseases such as arthritis with fixed pain caused by dampness [3]Willow as medicinal mainly be with the root of weeping willow (Salix babylonica L.) and fibrous root (willow root), branch (withy), root bast (Root-bark of Weeping Willow), leaf (willow leaf), flower (willow), reach the seed be used as medicine [4] such as (waddings) of tool hair.Rheum officinale willow (Salix raddeana laksh) mainly is distributed in China the Northeast, particularly Changbaishan area.Local among the people with its treatment stomatocace and dermatitis etc., effect is obvious.Do not see that about the research of its chemical ingredients and pharmacologically active report is arranged.
Research data shows that sallow generally contains flavonoid compound.Flavonoid compound in the sallow of having found is sorted out, mainly contained flavonoid, flavonols, flavanone alcohols, biflavone and flavanol compound etc.Flavonoid compound has many-sided biological activity, as immunoregulation effect, hypotensive effect, and antithrombotic, arteriosclerosis effect, anti-inflammatory action, antibiotic, antivirus action etc., the pharmaceutical use of sallow and the exploitation of resource have very big potentiality.
Chinese scholars is carried out chemical research to leaf, branch or the root skin of tens of kinds of willows, and the extraction and separation method that is adopted in chemical constitution study has: organic solvent lixiviate, system's solvent extration, classical column chromatography (as polyamide column chromatography, silica gel column chromatography, dextrane gel column chromatography), high performance liquid chromatography (HPLC), ply of paper are analysed, thin layer chromatography etc.; Shortcomings such as these classical extraction and separation method ubiquity fractional doses are few, separation efficiency is low.
This research is raw material with Changbai Mountain peculiar sallow rheum officinale willow, set up a kind of extraction and separation method of quick, a large amount of, high efficiency flavonoid compound, for further investigation rhubarb willow leaf flavone class chemical ingredients, pharmacologically active and find that new medicine resource is significant; Simultaneously, high performance liquid chromatography is set up in this research, for the rhubarb willow leaf flavone compounds provides quantitative analysis method fast and convenient, the method favorable reproducibility.
Summary of the invention
The objective of the invention is on the basis of traditional chromocor compound extraction and separation method, common polyamide column chromatography to be combined with half preparative high performance liquid chromatography, a kind of extraction and separation method of rhubarb willow leaf flavone component is provided.
The technical solution adopted for the present invention to solve the technical problems.The extraction and separation method of this rhubarb willow leaf flavone component, the extraction and separation process key step is as follows:
(1.1) sample extraction:
After drying, rhubarb willow leaf is ground into meal, microwave extraction after the adding alcohol immersion, and extracting temperature is middle high fire, united extraction liquid after filtering, concentrating under reduced pressure becomes concentrated extract; Use isopyknic extracted with diethyl ether after the water dissolution with 3 times of amount volumes, combining water layer discards ether layer; Water layer is again with the water-saturated n-butanol extraction of 3/4 volume, and is complete until the water-saturated n-butanol extraction; N-butanol extraction phase decompression and solvent recovery, water-soluble macroporous adsorbent resin, the ethanol elution gone up of medicinal extract with 30%;
(1.2) separation of flavonoid compound
Macroporous adsorbent resin 30% ethanol eluate is concentrated and Silon mixing drying, dry method is splined on polyamide column, use volume percent 30% and 40% ethanol gradient elution respectively, the stream part with 30% and 40% is left standstill after concentrating, and has produced light yellow cotton-shaped and particulate state precipitation; These two kinds of precipitations are used ethyl alcohol recrystallization respectively, get compound 1 and compound 2; 30% and 40% ethanol supernatant liquor is lyophilized into powder after reclaiming solvent, utilize the further separation and purification of preparative high performance liquid chromatography, on high performance liquid chromatography, be moving phase with the first alcohol and water, preparative column is Zorbax SB C18, flow velocity is 4.0ml/min, sample introduction concentration is 10mg/ml, sample size is 500 μ l-1000 μ l, when methyl alcohol: when water is 40: 60 (v/v), monomeric compound 3, compound 4 and compound 9 have been obtained, when methyl alcohol: when water is 38: 62 (v/v), obtained monomeric compound 5, compound 6, compound 7 and compound 8.
The explanation of the extraction process of rhubarb willow leaf flavone compounds of the present invention:
The alcoholic acid extraction conditions: taking by weighing sample 5g, is solvent with 20%, 40%, 60% ethanol respectively.A. add the 60ml solvent in 80 ℃ of heating in water bath refluxing extraction 1 hour.B. add 60ml solvent ultrasonic extraction 3 times, each 20 minutes.C. add in the solvent 60ml microwave high temperature extraction 3 times, each 8 minutes.According to the determination of total flavonoids result, determine ethanol microwave extraction, each 8 minutes with 20%.
Extracted with diethyl ether consumption: inquired into of the influence of ether amount to oil-soluble impurities in the ethanol extraction, the result shows that the volume of ether consumption and water is suitable, extracts 3 times, substantially oil-soluble impurities in the ethanol extraction is removed, the ether layer color is colourless substantially when extracting for the third time.
Water-saturated n-butanol extraction consumption detects n-butanol extraction efficient with HPLC and thin layer chromatography (TLC), find down irradiation (254-365nm) of ultraviolet lamp behind the amalgamation liquid thin-layer developing of 3 extractions, near Rf value 0.20,0.40,0.51,0.60,0.70, glassy yellow fluorescence is arranged.Extract the 4th n-butanol layer thin-layer chromatography and only see the burgundy spot near the solvent front, corresponding retention time does not detect in the HPLC spectrogram.Water-saturated n-butanol extraction 4 times are described, flavones ingredient extracts substantially fully.The TLC condition: thin layer plate is the high-efficient silica gel plate, and developping agent is an ethyl acetate: butanone: formic acid: water (50: 30: 10: 10).HPLC condition: chromatographic column: Zorbax SB C18 (4.6 * 150mm, 5 μ m); Moving phase: methyl alcohol: water=40: 60; Flow velocity: 1.0ml/min; Detect wavelength: 254nm; Column temperature: 25 ℃, sample size 20 μ l.
Determining of separation method
Flavonoid compound structure and silica gel thin-layer chromatography (TLC) behavior: benzene has been compared in this research: pyridine: formic acid (36: 9: 5), toluene: ethyl formate: several developping agents of formic acid (5: 4: 1), the result fails separately, most spot hangovers also concentrate near the start line, the polarity of this explanation developping agent is too small, change the benzene in the developping agent, toluene into ethyl acetate and add an amount of water enhancing polarity, with ethyl acetate: butanone: formic acid: water (50: 30: 10: 10) launch, the chromatography behavior makes moderate progress, but flavonoid glycoside and aglycon are at the R of silica gel thin-layer fBe worth very approachingly, show that silica gel column chromatography can not separate flavonoid glycoside and aglycon are fine.
Flavonoid compound structure and polymeric amide thin-layer chromatography (TLC) behavior: ethanol has been compared in this research: water (3: 2); Water: ethanol: methyl ethyl diketone (4: 2: 1) and water: ethanol: butanone: methyl ethyl diketone (13: 3: 3: 1) several developping agents, the compound separation effect is better, the disaccharide flavonoid glycoside is walked in the solvent front, the Rf value is between 0.69-0.73, monose flavonoid glycoside Rf value is between 0.55-0.62, and aglycon Rf value is between 0.30-0.55.
The column chromatography behavior of flavonoid compound structure and dextrane gel Sephadex LH-20: this research is elutriant with methyl alcohol, flow rate control is at 1ml/min, every 10ml is a stream part, carry out thin-layer chromatography (TLC) and follow the tracks of detection, the yellow ring of the appearance between stream part 33-60 is same compound, and the Rf value is near 0.75.A stream part 78-95 is same compound, and the Rf value is near 0.79.A stream part 104-123 is same compound, and the Rf value is near 0.85.This explanation separates these three kinds of compounds that obtain through glycan gel Sephadex LH-20 and is Flavone aglycone, does not obtain flavonoid glycoside.
The chromatographic behavior of flavonoid compound structure and preparative high performance liquid chromatography: this institute wants the flavones ingredient of separation and purification to belong to the bigger compound of polarity, adopt the reverse-phase chromatography of octadecyl bonding phase, mixed solvent system with methyl alcohol and water has compared methyl alcohol: water (40: 60; 38: 62; 36: 64) etc. moving phase, they are applicable to polyamide column chromatography different concentration ethanol wash-out stream part respectively.When sample size be 10mg/ml in 200 μ l, sample introduction concentration, when flow velocity is 4.0ml/min, each peak in the sample separation separates good, substantially reach baseline separation, going out the peak in 50min finishes, reduce separation costs in order to save disengaging time, strengthen sample size gradually, reach baseline separation according to the bigger main peak of HPLC figure peak area and get final product.Sample size is controlled at 500-1000 μ l.Be divided into from obtaining 7 flavonoid compounds.
The effect that the present invention is useful is: the present invention investigates extracting method and separation method respectively, has determined alcoholic acid extraction conditions, extracted with diethyl ether consumption, water-saturated n-butanol extraction consumption; The relation that has compared rhubarb willow leaf flavone compounds structure and silica gel thin-layer chromatography, dextrane gel thin-layer chromatography, the behavior of polymeric amide thin-layer chromatography, and analyzed the chromatographic behavior relation of flavonoid compound structure and preparative high performance liquid chromatography, determined that classical column chromatography is in conjunction with chromocor compound that the modern liquid chromatography technology-preparative high performance liquid chromatography separation and purification structure is close.This extraction and separation process can be applicable to suitability for industrialized production.Separating used column packing is Silon, and extraction, eluting solvent are medical alcohol, and material is recyclable to be utilized again, nontoxic, pollution-free.Quick, easy, the method favorable reproducibility, workable of high performance liquid chromatography that this research is set up, be applicable to the quantitative analysis of flavonoid compound in the rhubarb willow leaf, for quality and products thereof the quality of control rhubarb willow leaf provides feasible detection means and theoretical foundation.
Description of drawings:
Fig. 1 is the schematic flow sheet of invention;
Embodiment:
Below in conjunction with embodiment the present invention is further described, embodiment will help to understand the present invention better, but the present invention is not limited only to following embodiment.
Extraction and separation process sees that flow process is shown in Figure 1:
1. sample extraction
Rhubarb willow leaf (adopting July near a river regional) dries back 4.8Kg, be ground into meal (80 mesh sieve), added 20% alcohol immersion 2 hours, microwave extraction then, extracting temperature is middle high fire, each 8 minutes, extract altogether 3 times, filter back united extraction liquid, concentrating under reduced pressure becomes concentrated extract 1.68g, and productive rate is 35%.With isopyknic extracted with diethyl ether 3 times, combining water layer (be divided into two-layer, take off layer) discards ether layer with 3 times of water gagings dissolving back; Water layer is used 3/4 volume again, and (water layer: water-saturated n-butanol propyl carbinol=3: 4 (v/v)) extracts 3 times, according to thin-layer chromatography (TLC) and HPLC water layer is detected, and determines the water-saturated n-butanol extraction fully.N-butanol extraction phase decompression and solvent recovery, the water-soluble macroporous resin D101 (0.3-1.29mm 〉=9%) that goes up of medicinal extract, use 10% respectively, 30%, 50%, 70% ethanol elution, each component polyamide layer and high-efficient silica gel plate thin-layer chromatography, 5 (v/v)) and acetate ethanol developping agent is respectively water: ethanol: butanone: methyl ethyl diketone (65: 15: 15:: butanone: formic acid: water (50: 30: 10: 10 (v/v)), ultraviolet lamp (254-365nm) is observed down and is shown glassy yellow fluorescence and sapphirine fluorescence, deepen with the smoked spot colors of ammonia, be brown, yellow-green colour, aureus does not wait.Show that according to the thin-layer chromatography detected result most of composition concentrates on macroporous adsorbent resin 30% ethanol elution phase, is flavones ingredient, and the chromatography effect on polyamide layer is better, can separate, therefore, next step selects polyamide column chromatography separating flavone compounds.
2. the separation of flavonoid compound
Macroporous adsorbent resin 30% ethanol eluate is concentrated and Silon mixing drying (the 100mL concentrated solution adds the 45g Silon), dry method is splined on polyamide column, use 10% respectively, 20%, 30%, 40%, 50%, 60%, 70% (volume percent) ethanol gradient elution, high-efficient silica gel plate thin-layer chromatography is followed the tracks of and is detected, stream part with 30% and 40% is left standstill after concentrating, and has produced light yellow cotton-shaped and particulate state precipitation, and precipitation is insoluble in ethanol, be partially soluble in methyl alcohol, dissolve in the mixed solution of first alcohol and water.These two kinds of precipitations are used ethyl alcohol recrystallization (ethanol: water=9: 1 (v/v), room temperature) respectively, get compound 1 (130.2mg) and 2 (78.5mg).30% and 40% ethanol supernatant liquor is lyophilized into powder after reclaiming solvent, utilizes the further separation and purification of preparative high performance liquid chromatography, on high performance liquid chromatography with methyl alcohol: water (40: 60; 38: 62; 36: 64) be moving phase, preparative column is Zorbax SB C18 (9.4 * 250mm, 5 μ), flow velocity is 4.0ml/min, sample introduction concentration is 10mg/ml, and sample size is 500 μ l-1000 μ l, has obtained monomeric compound 3 (12.5mg), 4 (14.8mg), 5 (97mg), 6 (120.6mg), 7 (124.5mg), 8 (24.2mg), 9 (100mg).
Above-mentioned 9 compounds are accredited as following compound through chemical reaction, physicochemical constant mensuration and wave spectrums such as UV spectrum, infrared spectra, mass spectrum and nucleus magnetic resonance:
Compound 1 buff powder, m.p.243-245 ℃ ℃, hydrochloric acid-magnesium powder reaction is positive, and the Molish reaction is positive.UVλmax(MeOH)nm：254，348。IR(KBr)cm -1：3520，3480，1650，1605，1590，1495，1435，830。HRFAB-MS M-1 m/z:593.1528 (C 27H 30O 15Calculated value: 539.1511), EIMS m/z:594. 1HNMR(600MHz-DMSO-d 6)δ：3.87(3H，s，C-4’-OMe)，7.62(1H，d，J＝8.4Hz，H-6’)；7.47(1H，s，H-2’)；7.14(1H，d，J＝6.0Hz，H-5’)；6.84(1H，d，J＝6.0Hz，H-3)；6.82(1H，d，J＝2.0Hz，H-8)，6.47(1H，d，J＝9.6Hz，H-6)；5.46(1H，d，J＝3.6Hz，HO-glu)，5.23(1H，d，J＝3.0Hz，HO-glu)，5.18(1H，d，J＝3.6Hz，HO-glu)，5.09(1H，d，J＝7.2Hz，glu-1-H)；4.97(1H，d，J＝4.5Hz，HO-ara)，4.95(1H，d，J＝5.0Hz，HO-ara)，4.77(1H，s，HO-ara)，4.56(1H，s，HO-ara)，4.52(1H，s，HO-ara)，4.17(1H，d，J＝6.0Hz，ara-1-H)，12.97，9.45(each?1H，s，OH)。The aglycon part 13The CNMR spectral data conforms to document.Compound 1 is diosmetin 7-O-α-L-arabopyranose (1 → 6) β-D glucopyranoside.
Compound 2 buff powders, m.p.243-245 ℃, hydrochloric acid-magnesium powder reaction is positive, and the Molish reaction is positive.UVλmax(MeOH)nm：254，348。IR(KBr)cm -1：3520，3480，1650，1605，1590，1495，1435，830。HRFAB-MS M -1M/z:593.1528 (calculated value: 539.1511), EIMS m/z:594. 1H-NMR(500MHz-DMSO-d 6)δ：3.88(3H，s，C-4’-OMe)，7.60(1H，dd，J＝2.0Hz，8.5Hz，H-6’)；7.48(1H，d，J＝2.5Hz，H-2’)；7.16(1H，d，J＝8.5Hz，H-5’)；6.83(1H，s，H-3)；6.82(1H，d，J＝2.0Hz，H-8)，6.50(1H，d，J＝2.0Hz，H-6)；5.44(1H，d，J＝4.5Hz，HO-glu)，5.22(1H，d，J＝4.5Hz，HO-glu)，5.18(1H，d，J＝5.0Hz，HO-glu)，5.06(1H，d，J＝7.5Hz，glu-1-H)；4.97(1H，d，J＝4.5Hz，HO-xyl)，4.95(1H，d，J＝5.0Hz，HO-xyl)，4.88(1H，d，J＝4.5Hz，HO-xyl)，4.18(1H，d，J＝7.5Hz，xyl-H-1)，12.94，9.44(each?1H，s，OH)。The Flavone aglycone carbon atom 13The CNMR spectral data conforms to document.Compound 2 is diosmetin 7-O-β-D-xylopyranose (1 → 6) β-D glucopyranoside.
Compound 3 yellow powders, m.p.167-170 ℃, Molish reaction and hydrochloric acid-magnesium powder reaction all is positive UV λ max (MeOH) nm:258,266,290sh, 348, IR (KBr) cm-1:3480,3348,1650,1605,1590,1495,1435,830.EI-MS?m/z：580.2(C26H28015)。1HNMR(300MHz-DMSO-d6)δ12.6(1H，bs，5-OH)，5.09(1H，d，J＝8.7Hz，1”-H)，4.16(1H，d，J＝7.2Hz，1”’-H)，7.74(1H，d，J＝6.0Hz，H-5)，7.46(1H，d，J＝8.4Hz，H-2’，H-6’)，6.90(1H，d，J＝7.8Hz，H-5’)，6.77(1H，d，J＝13.0Hz，H-8)，6.46(1H，d，J＝6.6Hz，H-6)。13CNMR (DMSO-d6) δ 161.2 (C-2), 104.0 (C-3), 181.9 (C-4), 162.9 (C-5),, 99.5 (C-6), 164.6 (C-7), 94.7 (C-8), 156.9 (C-9), 105.3 (C-10), 121.1 (C-1 '), 113.5 (C-2 '), 145.9 (C-3 '), 150.3 (C-4 '), 116.0 (C-5 '), (119.2 C-6 '), carbon atom and aglycon part on the sugar 13The CNMR spectral data is consistent with bibliographical information, and compound 3 is luteolin 7-O-α-L-arabopyranose (1 → 6) β-D-pyrans heteroside.
Compound 4 yellow powders, mp.172-174 ℃, hydrochloric acid-magnesium powder reaction is positive.UVλmax(MeOH)nm：258，266，290sh，348，IR(KBr)cm -1：3480，3348，1650，1605，1590，1495，1435，830。EI-MS?m/z：580.2(C 2NH 28O 15)。 1HNMR (DMSO-d 6) δ 12.6 (s, 5-OH), 5.09 (1H, d, J=5.2Hz, 1 "-H); 3.94 (1H, d, J=9.0Hz, 1 -H, 7.74 (1H; s, H-5), 7.46 (1H, d, J=8.4Hz; H-2 ', H-6 '), 6.90 (1H, d, J=7.8Hz; H-5 '), 6.77 (1H, d, J=13.0Hz, H-8); 6.46 (1H, d, J=6.6Hz, H-6), 13CNMR (DMSO-d 6) spectrum δ: 161.2 (C-2), 104.0 (C-3), 181.9 (C-4), 162.9 (C-5), 99.5 (C-6), 164.6 (C-7), 94.7 (C-8), 156.9 (C-9), 105.3 (C-10), 121.1 (C-1 '), 113.5 (C-2 '), 145.9 (C-3 '), 150.3 (C-4 '), (116.0 C-5 '), 119.2 (C-6 '), carbon atom and aglycon part on the sugar 13The CNMR spectral data is consistent with bibliographical information.Compound 4 is inferred as luteolin 7-O-β-D-xylopyranose (1 → 6) β-D-glucopyranoside.
Compound 5 yellow powders, m.p.163-165 ℃, hydrochloric acid magnesium powder reacting positive, Molish reacting positive.UVλmax(MeOH)nm：265，290sh，325sh，358；.IR(KBr)?cm -1：3300，1655，1608.EI-MSm/z：594(C 27H 30O 15). 1HNMR(DMSO-d 6)δ：5.07(1H，d，J＝5.2Hz，glu-H-1)
3.88 (1H, d, J=9.6Hz, rha-H-1), 4.37 (3H, s, rha-H-5), 5.48 (1H, d, J=7.0Hz, OH-), 6.21 (1H, d, J=2.0Hz, H-6), 6.42 (1H, d, J=2.0Hz, H-8), 7.12 (1H, d, J=8.7, H-5 '), 8.05 (1H, d, J=9.0Hz, H-6 '), 7.99 (1H, d, J=9.3Hz, H-2 '), 10.85 (s, 7-OH), 12.61 (s, 5-OH). 13CNMR (DMSO-d6) composes δ: 156.50 (C-2), 133.13 (C-3), 177.39 (C-4), 161.18 (C-5), 98.79 (C-6), 164.53 (C-7), 93.79 (C-8), 156.97 (C-9), 103.84 (C-10), 122.88 (C-1 '), carbon atom and aglycon part on the sugar 13The CNMR spectral data is consistent with bibliographical information, infers that compound 5 is a kaempferol 3-O-rutinoside.
Compound 193-195 ℃ of .UVmax of 6 yellow powder m.p (MeOH) nm (1og): 252,310sh, 358.IR (KBr) cm-1:3759,3363,1653,1604.EI-MS:m/z 448 (C 21H 20O 11). 1HNMR (300MHz-DMSO-d 6) δ: 5.44 (1H, d, J=7.2Hz, H-1 "), 6.11 (1H, d, J=1.8Hz, H-8); 6.33 (1H, d, J=7.2Hz, H-6), 6.88 (1H, d, J=9.0Hz, H-3 ' H-5 '); 8.03 (1H, d, J=1.8Hz, H-2 ', H-6 ') 12.48 (s, 5-OH) 13CNMR (DMSO-d 6): 156.56 (C-2), 133.03 (C-3), 177.05 (C-4), 159.96 (C-5), 99.26 (C-6), 161.11 (C-7), 93.93 (C-8), 156.78 (C-9), 103.13 (C-10), 101.34 (C-1 "), 100.85 (C-1 ), 74.20 (C-2 "), 77.50 (C-3 "), 70.57 (C4 "), 76.39 (C-5 "); 66.87 (C-6 "), 70.32 (C-2 ), 69.86 (C-3 ), (71.79 C-4 ), 68.23 (C-5 ), 17.70 (C-6 ).Carbon atom and aglycon part on the sugar 13The spectral data of CNMR and bibliographical information are identical substantially, infer compound 6 and are kaempferol 3-O-β-D glucopyranoside.
Compound 7 yellow powder m.p.174-176 ℃ .UVmax (MeOH) nm:258,266,290sh, 365.IR (KBr) cm -1: 3759,3363,1653,1604,1507,1370,1250.EI-MS m/z:464 (C 21H 20O 12). 1HNMR (300MHz-DMSO-d6) δ: 12.62 (bs, 5-OH), 5.47 (1H, d, J=7.2Hz, glu-1-H), 7.59 (1H, d, J=1.8Hz, H-2 ', H-6 '), 6.85 (1H, d, J=9.0Hz, H-5 '), 6.37 (1H, s, H-6), 6.18 (1H, d, J=1.8Hz, H-8). 13CNMR (DMSO-d 6): δ: 156.27 (C-2), 133.30 (C-3), 177.40 (C-4), 161.20 (C-5), 98.59 (C-6), 164.04 (C-7), 93.43 (C-8), 156.13 (C-9), 103.94 (C-10), 100.85 (C-1 "), 74.05 (C-2 "), 76.47 (C-3 "); 69.91 (C-4 "), 77.50 (C-5 "), 60.94 (C-6 "), carbon atom and aglycon part on the sugar 13The spectral data of CNMR is consistent with bibliographical information, infers compound 7 and is Quercetin 3-O-β-D glucopyranoside.
Compound 8 yellow powder m.p.233-235 ℃ .UVmax (MeOH) nm:254,270 (s), 300ah, 361.IR (KBr) cm -1: 3400,1650,1600,1500,1380,1200. EI-MS m/z:464 (C 21H 20O 12). 1HNMR (300MHz-DMSO-d 6) δ: 12.62 (bs, 5-OH), 10.86 (7-OH), 7.66 (1H, dd, J=9.0Hz, 2.1Hz, 6 '-H), 7.52 (1H, d, J=2.1Hz, 2 '-H), 6.88 (1H, d, J=9.0Hz, 5 '-H), 6.40 (1H, d, J=1.8Hz, 8-H), 6.19 (1H, d, J=1.8Hz, 6-H), 5.37 (1H, d, J=8.8Hz, gal-1-H). 13CNMR (DMSO-d6): 156.11 (C-2), 133.36 (C-3), 177.28 (C-4), 161.04 (C-5), 98.60 (C-6), 163.91 (C-7), 93.42 (C-8), 156.13 (C-9), 103.86 (C-10), 121.88 (C-1 '), 115.86 (C-2 '), 144.66 (c-3 '), 148.29 (C-4 '), 115.08 (C-5 '), 120.98 (C-6 '), 101.73 (C-1 "); 71.19 (gal-C-2), 73.14 (gal-C-3), 67.78 (gal-C-4); 75.78 (gal-C-5), carbon atom and aglycon part on 60.11 (gal-C-6) sugar 13The spectral data of CNMR is consistent with bibliographical information, infers compound 8 and is Quercetin 3-O-β-D-galactopyranoside.
Compound 9 yellow powder m.p 186-188 ℃ of .UV λ max (MeOH) nm:258,268sh, 300sh, 359.IR (KBr) cm -1: 3400 (OH), 1678 (C=O), 1620,1618 (Ar) .EI-MS m/z:610 (C 27H 30O 16). 1HNMR (300MHz-DMSO-d6) δ: 3.72 (1H, d, J=10.0Hz, H-1 ), 4.39 (3H, s, H-5 ), 5.48 (1H, d, J=7.0Hz, H-1 "), 6.21 (1H, d; J=2.0Hz, H-6), 6.42 (1H, d, J=2.0Hz, H-8); 7.57 (1H, d, J=8.6,2.0Hz, H-5 '), 7.59 (1H; dd, J=8.6,2.0Hz, H-6 "), 7.60 (1H, d, J=3.0Hz, H-2 '), 10.85 (s, 7-OH), 12.61 (s, 5-OH). 13CNMR (DMSO-d6): 156.45 (C-2), 133.32 (C-3), 177.48 (C-4), 161.25 (C-5), 98.66 (C-6), 164.11 (C-7), 93.58 (C-8), 156.65 (C-9), 104.0 (C-10), 121.67 (C-1 '), 115.25 (C-2 '), 144.84 (C-3 '), 148.44 (C-4 '), 116.26 (C-5 '), 121.64 (C-6 '), 101.75 (C-1 "); 74.10 (C-2 "), 76.52 (C-3 "), 70.58 (C-4 "), 75.92 (5 "); 69.95 (C-6 "), (100.78 C-1 ), 70.02 (C-2 ), 70.42 (C-3 ), (71.86 C-4 ), (68.28 C-5 ), 17.78 (C-6 ), carbon atom and aglycon part on the sugar 13The spectral data of CNMR is consistent with bibliographical information, infers compound 9 and is Quercetin 3-O-rutinoside.
3. utilize the content of high effective liquid chromatography for measuring rhubarb willow leaf flavone compound
Chromatographic condition
Chromatographic column: Zorbax SB C 18(4.6 * 150mm, 5 μ m); Moving phase: methyl alcohol: water=40: 60; Flow velocity: 1.0ml/min;
Detect wavelength: the 254nm column temperature: 25 ℃, sample size 20 μ l.
Test sample preparation and mensuration
Precision takes by weighing rhubarb willow leaf (, May, July and October) and (Lusuihe River October) each 3.000g near a river, adds 20% alcohol immersion 12h, and the excusing from death ripple extracts 30min, the centrifugal 10min of 5000r/min.The accurate 5ml supernatant liquor of drawing, in furnace pot, 80 ℃ of evaporates to dryness of water-bath., in the 5ml volumetric flask, shake up with moving phase dissolving and constant volume, cross 0.25 μ m millipore filtration.Standby.
Rate of recovery experiment:
The accurate brilliant liquid of rhubarb willow leaf sample of drawing known content, accurate diosmetin 7-O-α-L-arabopyranose (1 → 6) β-D glucopyranoside reference substance that adds is measured average recovery rate 97.5%, RSD%=4.2 altogether 5 times.
The precision experiment:
Withinday precision: the accurate reference substance solution 20 μ l that draw, repeat sample introduction six times, the RSD of diosmetin 7-O-α-L-arabopyranose (1 → 6)-β-D glucopyranoside is 0.55%.
Day to day precision: same date is not drawn standardized solution 10 μ l, repeats sample introduction 6 times, and the RSD of diosmetin 7-O-α-L-arabopyranose (1 → 6)-β-D glucopyranoside is 1.53%.
The circulation ratio experiment:
Get with a collection of rhubarb willow leaf, extract by sample preparation methods at same date not, measure by above-mentioned chromatographic condition sample introduction, RSD is 2.01% (n=5) as a result.

Claims (2)

1, a kind of extraction and separation method of rhubarb willow leaf flavone component is characterized in that: described extraction and separation process key step is as follows:
(1.1) sample extraction:
After drying, rhubarb willow leaf is ground into meal, microwave extraction after the adding alcohol immersion, and extracting temperature is middle high fire, united extraction liquid after filtering, concentrating under reduced pressure becomes concentrated extract; Use isopyknic extracted with diethyl ether after the water dissolution with 3 times of amount volumes, combining water layer discards ether layer; Water layer is again with the water-saturated n-butanol extraction of 3/4 volume, and is complete until the water-saturated n-butanol extraction; N-butanol extraction phase decompression and solvent recovery, water-soluble macroporous adsorbent resin, the ethanol elution gone up of medicinal extract with 30%;
(1.2) separation of flavonoid compound
Macroporous adsorbent resin 30% ethanol eluate is concentrated and Silon mixing drying, dry method is splined on polyamide column, use volume percent 30% and 40% ethanol gradient elution respectively, the stream part with 30% and 40% is left standstill after concentrating, and has produced light yellow cotton-shaped and particulate state precipitation; These two kinds of precipitations are used ethyl alcohol recrystallization respectively, get compound 1 and compound 2; 30% and 40% ethanol supernatant liquor is lyophilized into powder after reclaiming solvent, utilize the further separation and purification of preparative high performance liquid chromatography, on high performance liquid chromatography, be moving phase with the first alcohol and water, preparative column is Zorbax SB C18, flow velocity is 4.0ml/min, sample introduction concentration is 10mg/ml, sample size is 500 μ l-1000 μ l, when methyl alcohol: when water is 40: 60 (v/v), monomeric compound 3, compound 4 and compound 9 have been obtained, when methyl alcohol: when water is 38: 62 (v/v), obtained monomeric compound 5, compound 6, compound 7 and compound 8.
2, the extraction and separation method of rhubarb willow leaf flavone component according to claim 1 is characterized in that: described extraction and separation process key step is as follows:
(2.1) sample extraction:
Rhubarb willow leaf dries back 4.8Kg, is ground into meal with 80 mesh sieves, adds 20% alcohol immersion 2 hours, then microwave extraction, extracting temperature is middle high fire, each 8 minutes, extracts altogether 3 times, filter back united extraction liquid, concentrating under reduced pressure becomes concentrated extract 1.68g, and productive rate is 35%; With after the water dissolution of 3 times of amount volumes with isopyknic extracted with diethyl ether 3 times, combining water layer discards ether layer and takes off layer; Water layer extracts 3 times with the water-saturated n-butanol of 3/4 volume again, until the water-saturated n-butanol extraction fully; N-butanol extraction phase decompression and solvent recovery, water-soluble macroporous adsorbent resin, the ethanol elution gone up of medicinal extract with 30%;
(2.2) separation of flavonoid compound
Macroporous adsorbent resin 30% ethanol eluate is concentrated and Silon mixing drying, the 100mL concentrated solution adds the 45g Silon, dry method is splined on polyamide column, use 10% respectively, 20%, 30%, 40%, 50%, 60%, 70% (volume percent) ethanol gradient elution, the stream part with 30% and 40% is left standstill after concentrating, and has produced light yellow cotton-shaped and particulate state precipitation, these two kinds of precipitations are used ethyl alcohol recrystallization respectively, at ethanol: water=9: 1 (v/v), under the room temperature condition, compound 1 (130.2mg) and compound 2 (78.5mg); 30% and 40% ethanol supernatant liquor is lyophilized into powder after reclaiming solvent, utilize the further separation and purification of preparative high performance liquid chromatography, on high performance liquid chromatography, be moving phase with the first alcohol and water, preparative column is Zorbax SB C18, flow velocity is 4.0ml/min, sample introduction concentration is 10mg/ml, sample size is 500 μ l-1000 μ l, when methyl alcohol: when water is 40: 60 (v/v), obtained monomeric compound 3 (12.5mg), compound 4 (14.8mg) and compound 9 (100mg), when methyl alcohol: when water is 38: 62 (v/v), obtained monomeric compound 5 (97mg), compound 6 (120.6mg), compound 7 (124.5mg) and compound 8 (24.2mg).
CN 200710067244 2007-02-09 2007-02-09 Extraction and separation method for rhubarb willow leaf flavone component Pending CN101012257A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101810735A (en) * 2010-04-16 2010-08-25 安徽省瑞森生物科技有限责任公司 Method for extracting total flavone from ampelopsis grossedentata
CN103923138A (en) * 2014-05-08 2014-07-16 江西天施康中药股份有限公司 Preparation method and application of nicotiflorin
CN106243173A (en) * 2016-07-14 2016-12-21 塔里木大学 Extract and the method for isolated and purified hypolipidemic activity compound Quercetin 3 O β D galactoside from the fine horse Fructus Jujubae of South Sinkiang

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101810735A (en) * 2010-04-16 2010-08-25 安徽省瑞森生物科技有限责任公司 Method for extracting total flavone from ampelopsis grossedentata
CN103923138A (en) * 2014-05-08 2014-07-16 江西天施康中药股份有限公司 Preparation method and application of nicotiflorin
CN103923138B (en) * 2014-05-08 2016-03-23 江西天施康中药股份有限公司 A kind of preparation method of Nicotifiorin and application thereof
CN106243173A (en) * 2016-07-14 2016-12-21 塔里木大学 Extract and the method for isolated and purified hypolipidemic activity compound Quercetin 3 O β D galactoside from the fine horse Fructus Jujubae of South Sinkiang
CN106243173B (en) * 2016-07-14 2020-10-30 塔里木大学 Method for extracting, separating and purifying blood fat reducing active compound quercetin-3-O-beta-D-galactoside from southern Xinjiang jun jujubes

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