CN1974590B - Process of extracting luteolin-O-beta-D-glucoside from elsholtzia bodinieri - Google Patents
Process of extracting luteolin-O-beta-D-glucoside from elsholtzia bodinieri Download PDFInfo
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- CN1974590B CN1974590B CN200610128294A CN200610128294A CN1974590B CN 1974590 B CN1974590 B CN 1974590B CN 200610128294 A CN200610128294 A CN 200610128294A CN 200610128294 A CN200610128294 A CN 200610128294A CN 1974590 B CN1974590 B CN 1974590B
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Abstract
The present invention relates to extraction of medicine from plant material, and is especially process of extracting luteolin-7-O-beta-D-glucoside from Elsholtzia bodinieri. The process includes the following steps: extracting Elsholtzia bodinieri with solvent, dissolving the extracted concentrated solution or extractum in water, extracting and defatting with weak polar or medium polar organic extractant, extracting the defatted water phase with water immiscible low carbon alcohol extractant, concentrating and drying the extracted phase, and re-crystallizing to obtain luteolin-7-O-beta-D-glucoside. Or, the defatted water phase is adsorbed with macroporous resin and eluted with eluent, and the eluted liquid is concentrated, dried and re-crystallized to obtain luteolin-7-O-beta-D-glucoside. The extracting process is simple, low in power consumption, environment friendly and high in extracting rate, and the product is used in developing medicine for various diseases.
Description
Technical field
The present invention relates to a kind of method, relate in particular to a kind of method of from East Perillae, extracting luteolin-7-O-β-D-glucoside from the plant extract medicine.
Background technology
East Perillae (Elsholtzia bodinieri Vaniot.) is that Labiatae (Labiatae) elscholtiza belongs to (Elsholtzia) plant.Its herb all can be used as medicine, and is used for the treatment of cold, headache bodily pain, toothache caused by fire of deficiency type, havies loose bowels, acute conjunctivitis, hepatitis, throat pain, maldigestion, closes disease such as urine; Tender leaf can be done tea-drinking usefulness, and clearing heat and detoxicating effect is arranged.In the chemical ingredients Separation Research of East Perillae being carried out system, find, luteolin-(luteolin-7-O-β-D-glucoside) content is higher in East Perillae for 7-O-β-D-glucoside, luteolin-7-O-β-D-glucoside has cough-relieving, eliminates the phlegm, antiinflammation, and the effect that reduces rabbit experimental atherosclerosis and cholesterol arranged, rat skin lower injection, the effect of the capillary permeability of enhancing is arranged, but from East Perillae, do not extract the bibliographical information of luteolin-7-O-β-D-glucoside at present.
Summary of the invention
The purpose of this invention is to provide a kind of method of from East Perillae, extracting luteolin-7-O-β-D-glucoside.
The present invention realizes above-mentioned purpose by the following technical solutions: the method for extracting luteolin-7-O-β-D-glucoside from East Perillae, at first East Perillae is utilized solvent extraction, the extraction solvent is an ethanol, methyl alcohol, the solvent that acetone and water are formed with different proportioning, East Perillae is 1 with the weight ratio of extracting solvent: 8-20, after concentrating, extracting solution gets concentrated solution or medicinal extract, concentrated solution or medicinal extract is water-soluble, organic extractant extraction degreasing with low-pole or middle polarity, water after the degreasing is used and the immiscible low-carbon alcohol kind of extractants extraction of water again, alcohols extraction phase concentrate drying gets luteolin-7-O-β-D-glucoside behind the recrystallization.
The extraction agent of low-pole or middle polarity is sherwood oil, hexanaphthene, normal hexane, chloroform or ethyl acetate, with the immiscible low-carbon alcohol kind of extractants of water be C
4～C
10Alcohols.
With the immiscible low-carbon alcohol kind of extractants of water be propyl carbinol, 1-amylalcohol, cyclopentanol, hexalin or isooctyl alcohol.
The another kind of method of from East Perillae, extracting luteolin-7-O-β-D-glucoside, at first East Perillae is utilized solvent extraction, the extraction solvent is an ethanol, methyl alcohol, the solvent that acetone and water are formed with different proportioning, East Perillae is 1 with the weight ratio of extracting solvent: 8-20, after concentrating, extracting solution gets concentrated solution or medicinal extract, concentrated solution or medicinal extract are dissolved in water, organic solvent extracting extracting degreasing with low-pole or middle polarity, water after the degreasing is crossed macroporous adsorbent resin, after washing the solubility impurity that anhydrates earlier with water, use the eluent wash-out again, the elutriant concentrate drying gets luteolin-7-O-β-D-glucoside behind the recrystallization.
The extraction agent of low-pole or middle polarity is sherwood oil, hexanaphthene, normal hexane, chloroform or ethyl acetate.
Macroporous adsorbent resin is the macroporous adsorbent resin of low-pole, nonpolar or middle polarity, and eluent is the alcohol-water system eluent.
Selecting low-pole, water content for use is that 60-70%, specific surface area are 480-520m
2/ g, mean pore size are 130-
Macroporous adsorbent resin.
Method of the present invention can be extracted luteolin-7-O-β-pure product of D-glucoside from East Perillae, extraction process is simple, easy to prepare, energy consumption is little, it mainly is solvent with the alcohol-water system, non-environmental-pollution, solvent can be recycled, the extraction yield height, crude product extracts can obtain the luteolin-7-O-β-D-glucoside of content at 60-80%, obtain the luteolin-7-O-β-D-glucoside of content at 95-99% behind the purifying, luteolin-7-O-β-D-glucoside can be used for research and development treatment cardiovascular and cerebrovascular diseases, bronchitis, asthma, the medicine of diseases such as lung cancer.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of reference substance;
Fig. 2 is the high-efficient liquid phase chromatogram of sample of the present invention.
Embodiment
Embodiment 1, East Perillae picks up from the Hong Hezhou of Yunnan Province, Elsholtzia bodinieri Vaniot herbs 500g is pulverized, alcohol-water with 4000ml is made extraction agent, refluxing extraction, wherein the alcoholic acid volume percent is 70%, after extracting solution is evaporated to no ethanol, the East Perillae extract concentrated solution, use and concentrated solution equal volume of ethyl acetate three times, after water concentrating under reduced pressure behind the ethyl acetate extraction is removed residual ethyl acetate, lyophilize gets luteolin-7-O-β-thick product of D-glucoside, HPLC, it is 80% that UV measures content, and crude product is carried out crystallization and recrystallization, then get luteolin-7-O-β-pure product of D-glucoside, HPLC, it is 96% that UV measures content.
Embodiment 2, Elsholtzia bodinieri Vaniot herbs 500g is pulverized, alcohol-water with 7000ml is made extraction agent, refluxing extraction, wherein the alcoholic acid volume percent is 90%, after extracting solution is evaporated to no ethanol, gets the East Perillae concentrated extract, East Perillae medicinal extract is made aqeous suspension, with with suspension equal-volume chloroform extraction three times, the water behind the chloroform extraction is again with the extraction of 1-amylalcohol, behind the 1-amylalcohol extraction phase concentrating under reduced pressure, vacuum-drying, then get luteolin-7-O-β-D-glucoside crude product, HPLC, UV measures content and is about 61%, and crude product is carried out crystallization and recrystallization, then get luteolin-7-O-β-pure product of D-glucoside, HPLC, it is 98% that UV measures content.
Embodiment 3, Elsholtzia bodinieri Vaniot herbs 500g is pulverized, acetone-water with 10000ml is made extraction agent, refluxing extraction, wherein the volume percent of acetone is 60%, after extracting solution is evaporated to no acetone, get the East Perillae extract concentrated solution, use and twice degreasing of concentrated solution equal-volume petroleum ether extraction, water is crossed macroporous adsorbent resin, wash with water to effluent liquid colourless, with 95% ethanol is the eluent wash-out, behind the elutriant concentrating under reduced pressure, and lyophilize again, get luteolin-7-O-β-D-glucoside elaboration, HPLC, UV measure content and are about 90%; Crude product and elaboration are carried out crystallization and recrystallization, then get luteolin-7-O-β-pure product of D-glucoside, it is 99% that HPLC, UV measure content.Wherein, macroporous adsorbent resin is selected low-pole for use, granularity mm (0.3-1.25) 〉=90%, and water content 70%, average specific surface area is 520m
2/ g, mean pore size
Macroporous adsorbent resin.
Luteolin-7-O-β-D-glucoside product is identified:
One, proterties: content is 99%, buff powder, mp 251-253 ℃; HCl-Zn reacting positive, TLC are met ethanol solution of sulfuric acid heating displaing yellow, and the thin layer hydrolysis detects glucose and luteolin.
Two, spectrogram: EI-MS m/z (%):
286 (aglycon) (100), 270 (5), 258 (16), 257 (5), 212 (38), 162 (3), be shown flavonoid compound, push away to such an extent that molecular formula is C
21H
20O
11, consistent with the mass spectrum molecular ion peak; Compound
13In C-NMR, the DEPT spectrum, in δ C 60.6-105 scope, typical glucose feature carbon absorption signal occurs, be shown flavonoid glycoside.
1H-NMR (400Hz, DMSO-d
6) among the δ, 12.98 (1H, s), 10.00 (1H, s) and 9.41 (1H, three peaks s) belong to respectively and are 5,4 ' and the hydroxyl signal of 3 ' position, 7.43 (1H, dd, J=2.0 8.4Hz) is the proton signal of 6 ' position, 7.41 (1H, d is J=2.0Hz) with 6.89 (1H, d, J=8.0Hz) then be 2 ' and the peak of 5 ' position, 6.78 (1H, d, J=2.0Hz) and 6.43 (1H, d J=2.0Hz) are 8 and 6 proton signals respectively, 6.75 (1H, s) then be 3 proton signals, 5.08 (1H, d, J=6.8Hz) be sugared anomeric proton signal, be β-type.Comprehensive all spectroscopic datas, through and the document contrast, determine this compound be 5,3 ', 4 '-trihydroxyflavone-7-O-β-D-glucoside, i.e. luteolin-7-O-β-D-glucoside.
Three, assay: select the Agilent high performance liquid chromatograph for use, day island proper Tianjin UV-250 of company spectrophotometer; Chromatographic column: Agilent ODS (5 μ m, 21mm * 150mm), moving phase: methyl alcohol-1.5% glacial acetic acid aqueous solution (60: 40v/v); Flow velocity: 0.3mLmin
-1Detect wavelength: 360nm; Column temperature: 30 ℃; Sample size: 5 μ L, selecting methyl alcohol for use is chromatographically pure, other reagent is analytical pure.Luteolin-7-O-β-D-glucoside reference substance self-control, structure are by UV, and IR, spectrum methods such as NMR identify that purity is measured greater than 99% through the HPLC area normalization method, can make quantitative usefulness, and the high-efficient liquid phase chromatogram of reference substance as shown in Figure 1.
(1), the investigation of typical curve
1, the investigation of high performance liquid phase typical curve
Get reference substance storing solution 2.5mL, with 10 times of methyl alcohol dilutions, accurately draw mark liquid 3.0,4.0,5.0,6.0,7.0 μ L sample introduction is measured by above-mentioned chromatographic condition, is X-coordinate with sample size (X), with peak area (Y) is ordinate zou, gets typical curve to be: the Y=305.7167+7864.7871Xr=0.9994 linearity range is 0.3～0.7 μ g.
2, the investigation of spectrophotometry typical curve
Draw reference substance storing solution 1.0,1.5,2.0,2.5 successively with valinche respectively, 3.0 3.5mL is diluted to 25mL with methyl alcohol in colorimetric cylinder, mixing, measuring absorbancy at 360nm wavelength place, is X-coordinate with strength of solution (X), is ordinate zou with absorbancy (Y), gets typical curve and is:
Y=-0.00155+44.5428X r=0.9999 linearity range is 0.004～0.014gL
-1
(2), precision and circulation ratio
1, the precision of high-efficient liquid phase technique and circulation ratio
Get same sample solution continuous sample introduction 5 times, peak area RSD=0.045%.Accurately take by weighing 5 parts in same sample, prepare sample solution, the sample high-efficient liquid phase chromatogram of measuring as shown in Figure 2, the RSD=2.1% of its peak area with aforesaid method.
2, precision of spectrophotometry and circulation ratio
Get same sample solution METHOD FOR CONTINUOUS DETERMINATION 5 times, get the RSD=0.045% of absorbancy.Accurately take by weighing 5 parts in same sample, prepare sample solution, measure in accordance with the law, get the RSD=2.1% of absorbancy with aforesaid method.
(3), the stability and the rate of recovery
1, the stability of high-efficient liquid phase technique and the rate of recovery
Get same sample solution, room temperature places 0,2,4,6, and 8h is sample introduction respectively, and recording peak area mean value is 3.936 * 10
3, RSD=2.2%.Get each 5mL mixing of reference substance storing solution and sample solution, totally five parts, measure the rate of recovery and see Table 1.
2, the spectrophotometry stability and the rate of recovery
Get each 5mL mixing of reference substance storing solution and sample solution, totally five parts, measure the rate of recovery and see Table 1.
(4), content results
1, high effective liquid chromatography for measuring
Sample thief storing solution 2.5mL is three parts respectively, and with 10 times of methyl alcohol dilutions, under selected chromatographic condition, sample introduction 5 μ L record samples contg and see Table 1.
2, determined by ultraviolet spectrophotometry
Draw three parts of sample storing solution 2.5mL respectively, be diluted to 25mL, record content and see Table 1 with methyl alcohol.
Table 1 HPLC and UV method measurement result are relatively
*The there was no significant difference as a result that P＞0.01, two kind of method is measured.
Claims (9)
1. from East Perillae, extract the method for luteolin-7-O-β-D-glucoside, East Perillae is utilized solvent extraction, it is characterized in that, concentrated solution or medicinal extract after extracting is water-soluble, organic extractant extraction degreasing with low-pole or middle polarity, water after the degreasing is used and the immiscible low-carbon alcohol extraction agent extraction of water again, and the extraction phase concentrate drying gets luteolin-7-O-β-D-glucoside behind the recrystallization.
2. the method for from East Perillae, extracting luteolin-7-O-β-D-glucoside as claimed in claim 1, it is characterized in that, the extraction agent of low-pole or middle polarity is sherwood oil, hexanaphthene, normal hexane, chloroform or ethyl acetate, with the immiscible low-carbon alcohol extraction agent of water be C
4～C
10Alcohol.
3. the method for the luteolin that extracts from East Perillae as claimed in claim 1 or 2-7-O-β-D-glucoside is characterized in that, with the immiscible low-carbon alcohol extraction agent of water be propyl carbinol, 1-amylalcohol, cyclopentanol, hexalin or isooctyl alcohol.
4. the method for the luteolin that from East Perillae, extracts as claimed in claim 3-7-O-β-D-glucoside, it is characterized in that, the extraction solvent solvent that to be ethanol, methyl alcohol or acetone formed with different proportioning with water, East Perillae is 1 with the weight ratio of extracting solvent: 8-20.
5. from East Perillae, extract the method for luteolin-7-O-β-D-glucoside, East Perillae is utilized solvent extraction, it is characterized in that, concentrated solution or medicinal extract after extracting are dissolved in water, with the organic extractant extraction degreasing of low-pole or middle polarity, the water after the degreasing is crossed macroporous adsorbent resin, the eluent wash-out, the elutriant concentrate drying gets luteolin-7-O-β-D-glucoside behind the recrystallization.
6. the method for extracting luteolin-7-O-β-D-glucoside from East Perillae as claimed in claim 5 is characterized in that the extraction agent of low-pole or middle polarity is sherwood oil, hexanaphthene, normal hexane, chloroform or ethyl acetate.
7. as the method for claim 5 or the 6 described luteolin that from East Perillae, extracts-7-O-β-D-glucosides, it is characterized in that, macroporous adsorbent resin is the macroporous adsorbent resin of low-pole, nonpolar or middle polarity, and eluent is the alcohol-water system eluent.
8. the method for the luteolin that extracts from East Perillae as claimed in claim 7-7-O-β-D-glucoside is characterized in that, selecting low-pole, water content for use is that 60-70%, specific surface area are 480-520m
2/ g, mean pore size are 130-140
Macroporous adsorbent resin.
9. the method for the luteolin that from East Perillae, extracts as claimed in claim 8-7-O-β-D-glucoside, it is characterized in that, the extraction solvent solvent that to be ethanol, methyl alcohol or acetone formed with different proportioning with water, East Perillae is 1 with the weight ratio of extracting solvent: 8-20.
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| CN105732762A (en) * | 2016-03-31 | 2016-07-06 | 王婧 | Method for extracting perilla oil and ursolic acid from perilla seeds |
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| 赵东保 等.凤尾茶化学成分研究.中药材28 2.2005,28(2),94-96. |
| 赵东保 等.凤尾茶化学成分研究.中药材28 2.2005,28(2),94-96. * |
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