CN102924418A - Method for separating and purifying effective ingredients from fine felt hairy honeysuckle - Google Patents
Method for separating and purifying effective ingredients from fine felt hairy honeysuckle Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying 5-hydroxy-7, 3', 4'-triethoxy flavone, beta-sitosterol or/and betal-daucosterol from fine felt hairy honeysuckle. According to the method for separating and purifying 5-hydroxy-7, 3', 4'-triethoxy flavone, beta-sitosterol or/and betal-daucosterol from fine felt hairy honeysuckle, disclosed by the invention, the purity of each obtained compound is high, the new use value of fine felt hairy honeysuckle is developed, and the definition of pharmacodynamic material of fine felt hairy honeysuckle is favorable.
Description
Technical field
The present invention relates to the separation purification method of effective constituent in a kind of L. similis Hemsl, relate generally to 5-hydroxyl-7,3 ', 4 '-separation purification method of trimethoxy flavone, β-sitosterol, β-daucosterol.
Background technology
L. similis Hemsl Lonicera similes Hemsl is the main flow kind of river pan honeysuckle flower, it is wide to distribute, the resource standing stock are large, according to investigations, the Japanese Honeysuckle that Chengdu lotus pond Chinese medicinal materials specialized market is sold, the Japanese Honeysuckle that shop of Chinese medicines and medical institutions use in the Sichuan Province mostly is southern river pan honeysuckle flower (L. similis Hemsl), and sells to all parts of the country.Nanjiang County, Sichuan has built 22 Japanese Honeysuckle specialty Demonstration Villages at present, and whole county Japanese Honeysuckle total cultivated area reaches 260,000 mu, and L. similis Hemsl has accounted for more than 95%, existing 10.2 ten thousand mu of operations, produce 7650000 kilograms of dried honeysuckle flowers per year, realize 200,000,000 yuan of the gross output values, the producing region increases income 560 yuan per capita.The Japanese Honeysuckle industry has become the Main Economic mainstay industry of Nanjiang County.
In recent years, studies show that much the L. similis Hemsl chlorogenic acid content is high, it is suitable that pharmacological action and pharmacopeia are recorded kind.But river honeysuckle flower Dominant variety L. similis Hemsl is not selected in Chinese Pharmacopoeia yet at present, and especially " Chinese pharmacopoeia version in 2010 strengthened discriminating and assay to Chinese medicinal materials specificity composition, and L. similis Hemsl still lacks relevant fundamental research.
5-hydroxyl-7,3 ', 4 '-trimethoxy flavone to staphylococcus aureus etc. have anti-microbial effect (Xing Junbo is etc., In Flower Buds of Lonicera Japonica Thunb chemical constitution study [J]. Chinese Journal of New Drugs, 2002,11 (11): 856-859).β-sitosterol has the effect that reduces serum cholesterol, and it is atherosis to can be used for II type hyperlipidaemia and prevention of arterial; Simultaneously, this compound can also change into medicinal Vitamin D3 500,000 I.U/GM raw material and multiple hormone.β-daucosterol is one of neurilemmal main component, also has simultaneously oxidation resistant pharmacologically active.
At present, yet there are no separation and purification 5-hydroxyl-7,3 from L. similis Hemsl ', 4 '-relevant report of the compounds such as trimethoxy flavone.
Summary of the invention
The object of the present invention is to provide a kind of 5-hydroxyl-7,3 ', 4 '-separation purification method of trimethoxy flavone; Another object of the present invention is to provide the separation purification method of β-sitosterol, β-daucosterol.
The invention provides 5-hydroxyl-7,3 ', 4 '-separation purification method of trimethoxy flavone, it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95%v/v ethanol position successively;
(3) get gained ethanol position, reclaim solvent after, upper silica gel column chromatography is used hexanaphthene-ethyl acetate=20: 1 ~ 0: 1 successively, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, and the elutriant of per 0.16 column volume is 1 stream part, collects elutriant, the thin layer monitoring is got 39 ~ 59 parts, behind the recovery solvent, redissolve with methyl alcohol, go up again gel column, use methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallization, namely get 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone.
Wherein, in the step (1), the concrete operations of described extraction using alcohol are: be the extraction using alcohol of 90%V/V, 80%V/V, 70%V/V successively with concentration; Preferably, the concrete grammar of extraction using alcohol is: use respectively the alcohol reflux of 90%V/V, 80%V/V, 70%V/V, and the extraction using alcohol of each concentration 1-2 time, each 1-3 hour, material ratio was 1: 6-10.
Wherein, in the step (2), water, 80%/V ethanol, 95%V/V ethanol elution are collected 95%v/v ethanol position successively; Described macroporous adsorbent resin is D101 type macroporous adsorbent resin.
Wherein, in the step (2), upper column liquid concentration is counted 0.02-0.2g/ml with crude drug, than upper column quantity less than 2.8g medicinal material/g dried resin.
Further, in the step (2), upper column liquid concentration is counted 0.10g/ml with crude drug, than upper column quantity 2.74g medicinal material/g dried resin.
Wherein, in the step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volumes; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes.
Wherein, in the step (3), described gel column is SephadexLH-20 type gel column; Recrystallization solvent is methyl alcohol.
The present invention also provides the separation purification method of β-sitosterol, and it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95% ethanol position successively;
(3) get gained ethanol position, after reclaiming solvent, upper silica gel column chromatography, usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, the elutriant of per 0.16 column volume is 1 stream part, collects elutriant, the thin layer monitoring, get 17 ~ 22 parts, after reclaiming solvent, recrystallization namely gets β-sitosterol;
In the step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes.
The present invention also provides the separation purification method of β-daucosterol, and it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95% ethanol position successively;
(3) get gained ethanol position, after reclaiming solvent, upper silica gel column chromatography, usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, the elutriant of per 0.16 column volume is 1 stream part, collects elutriant, the thin layer monitoring, get 120 ~ 124 parts, after reclaiming solvent, recrystallization namely gets β-daucosterol;
In the step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 1 1.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volumes; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes; Hexanaphthene-ethyl acetate=3:1,4.05 times of column volumes; Hexanaphthene-ethyl acetate=2:1,1.67 times of column volumes; Hexanaphthene-ethyl acetate=1:1,2.12 times of column volumes; 1.62 times of column volumes of ethyl acetate.
The present invention also provide from L. similis Hemsl, separate simultaneously 5-hydroxyl-7,3 ', 4 '-method of trimethoxy flavone, β-sitosterol and β-daucosterol, it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95%v/v ethanol position successively;
(3) get gained ethanol position, after reclaiming solvent, upper silica gel column chromatography, with hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, and the elutriant of per 0.16 column volume is 1 stream part, collect elutriant, the thin layer monitoring, condition of gradient elution is as follows: hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 1 1.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volumes; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes; Hexanaphthene-ethyl acetate=3:1,4.05 times of column volumes; Hexanaphthene-ethyl acetate=2:1,1.67 times of column volumes; Hexanaphthene-ethyl acetate=1:1,2.12 times of column volumes; 1.62 times of column volumes of ethyl acetate;
(4) get 17 ~ 22 parts of stream parts, behind the recovery solvent, recrystallization namely gets β-daucosterol;
Get 39 ~ 59 parts, reclaim solvent after, redissolve with methyl alcohol, go up again gel column, use methanol-eluted fractions, the collection elutriant, the recovery solvent, recrystallization, namely get 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone;
Get 120 ~ 124 parts, behind the recovery solvent, recrystallization namely gets β-daucosterol.
Beneficial effect of the present invention is as follows:
(1) the invention provides separation and purification 5-hydroxyl-7 from L. similis Hemsl, 3 ', 4 '-separation purification method of trimethoxy flavone, β-daucosterol, β-sitosterol, each compound purity that obtains is higher, for the L. similis Hemsl medicinal material has searched out new use value; Wherein, 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone, β-daucosterol are to separate first to obtain from L. similis Hemsl.
(2) the 5-hydroxyl-7 that from L. similis Hemsl, separates simultaneously provided by the invention, 3 ', 4 '-method of trimethoxy flavone, β-daucosterol, β-sitosterol, can be under same courses of action, separate successively obtaining above-mentioned three kinds of compounds, compare with the independent separation method of compound, the present invention more saves reagent dosage, improve the utilization ratio of crude drug, greatly saved the separation and purification cost of compound.
(3) 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone has anti-microbial effect to staphylococcus aureus etc., match with antibiotic, the antiviral effect of L. similis Hemsl, may be the effective constituent that its function of reflection cures mainly, the present invention is to the effective separation of this compound, and also the effective substance for clear and definite L. similis Hemsl provides possibility.
Description of drawings
Fig. 1 control substance of Rutin solution and need testing solution UV-VIS scintigram
Fig. 2 control substance of Rutin solution canonical plotting
The performance analysis of the dissimilar macroporous resin Static Adsorption of Fig. 3
Fig. 4 sample solution concentration is investigated interpretation of result
Fig. 5 leakage plot figure
Fig. 6 L. similis Hemsl extracts schema
Fig. 7 L. similis Hemsl total flavone part chemical ingredients separation process figure
Fig. 8 β-sitosterol (hydrogen spectrogram of β-sitosterol)
Fig. 9 β-sitosterol (carbon spectrogram of β-sitosterol)
Figure 105-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone) hydrogen spectrogram
Figure 11 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone) carbon spectrogram
Figure 12 β-daucosterol (hydrogen spectrogram of β-Daucosterin)
Figure 13 β-daucosterol (carbon spectrogram of β-Daucosterin)
The HPLC collection of illustrative plates of Figure 14 L. similis Hemsl
Embodiment
Instrument
Rotatory evaporator RE-5203(Shanghai Yarong Biochemical Instrument Plant)
The multiplex vacuum pump SHB-of circulating water type III (Zhengzhou Greatwall Scientific Industrial ﹠ Trading Co., Ltd.)
ZF-90 type dark box type ultraviolet projectoscope (Shanghai Gu Cun electric light instrument plant).
Reagent and reagent
Ethanol, methyl alcohol, chloroform, methylene dichloride, acetone, sherwood oil, ethyl acetate, propyl carbinol, aluminum chloride (more than be analytical pure, the Long Huagongshijichang of Chengdu section produces).Column chromatography silica gel (Haiyang Chemical Plant, Qingdao produces, 100～200 orders, 200～300 orders), tlc silica gel G(Haiyang Chemical Plant, Qingdao produce).
Crude drug source and evaluation
Test used L. similis Hemsl medicinal material and purchase in Nanjiang County, Sichuan Province, identify through professor Ma Yuying of Chengdu University of Traditional Chinese Medicine, be caprifoliaceae plant L. similis Hemsl Lonicera similis Hemsl.
(1) take by weighing the dry flower 1.9Kg of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70%, material ratio are 1: 8, filter, and decompression recycling ethanol gets medicinal extract.After measured, contain total flavones 3.39% in this extract.
(2) get above-mentioned medicinal extract, add water preparation upper prop liquid, upper column liquid concentration is counted 0.10g/ml with crude drug, upper macroporous adsorptive resins, successively water, concentration be 10%, 20%, 40%, 60%, 80%, the ethanol elution of 95%V/V, collect 95% ethanol position;
(3) get gained ethanol position, behind the recovery solvent, upper silica gel column chromatography (200-300 order, 350g, column volume 925ml), usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, and every stream part 150ml collects elutriant, the thin layer monitoring, merge same stream part, get 39 ~ 59 parts, behind the recovery solvent, redissolve with methyl alcohol, go up again the SephadexLH-20 gel column, use methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallization, namely get 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (35mg), the rate of transform is 0.17%.
The condition of gradient elution is:
Hexane-ethyl acetate=20: 1 (997.5ml), hexanaphthene-ethyl acetate=10: 1 (990ml), hexanaphthene-ethyl acetate=8: 1 (3050ml), hexanaphthene-ethyl acetate=6: 1 (970ml), hexanaphthene-ethyl acetate=5: 1 (1901ml), hexanaphthene-ethyl acetate=4:1 (1855ml), hexanaphthene-ethyl acetate=3:1 (3755ml), hexanaphthene-ethyl acetate=2:1 (1550ml), hexanaphthene-ethyl acetate=1:1 (1960ml), ethyl acetate (1500ml), ethyl acetate-methyl alcohol=5:1 (2035ml), ethyl acetate-methyl alcohol=3:1 (1430ml), ethyl acetate-methyl alcohol=2:1 (1335ml), ethyl acetate-methyl alcohol=1:1 (1500ml), methyl alcohol (1500ml).
Compound identification:
The gained compound is yellow powder, mp167 ~ 169 ℃.Develop the color spray AlCl under the TLC ultraviolet lamp
3Yellow fluorescence is arranged, and hydrochloric acid-magnesium powder reaction is positive, and ferric chloride reaction is positive.Tentatively be judged as thus flavonoid or flavonols compound.
ESI-MS?m/z:329[M+H]
+。
1H-NMR(C
5D
5N,400MHz)δ:7.07(1H,s,H-3),6.60(1H,s,H-6),6.73(1H,s,H-8),7.57(1H,s,H-2′),7.06(1H,d,J=8.6Hz,H-5'),7.66(1H,d,J=8.6Hz,H-6′),13.60(1H,s,5-OH),3.82(3H,s,-OCH
3),3.79(3H,s,-OCH
3),3.72(3H,s,-OCH
3)。
13C-NMR(C
5D
5N,100MHz)δ:164.4(C-2,s),104.9(C-3,d),182.9(C-4,s),162.7(C-5,s),98.8(C-6,d),166.0(C-7,s),93.1(C-8,d),158.2(C-9,s),106.0(C-10,s),123.2(C-1',s),110.0(C-2',d),149.6(C-3',s),153.2(C-4',s),112.20(C-5',d),120.7(C-6',d),56.5(-OCH
3),56.4(-OCH
3),56.2(-OCH
3)。Above data and document are basically identical, thus be defined as 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone.
Compound VII 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone)
HPLC detects:
Chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m)
Moving phase: acetonitrile-0.4% phosphoric acid (22:78)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Sample size: 10 μ l
Detect wavelength: 269nm
Under above selected condition, 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone and sample in other component chromatographic peaks can substantially separate, 5-hydroxyl-7,3 ', 4 '-resolution that trimethoxy flavone is adjacent chromatographic peak is greater than 1.5, and peak shape is symmetrical, sharp-pointed, and theoretical plate number is 6000.The performance of considering different instruments, chromatographic column may there are differences, thus decide number of theoretical plate with 5-hydroxyl-7,3 ', 4 '-the trimethoxy flavone meter should be not less than 4000.
Detect through HPLC, the 5-hydroxyl-7,3 that separation obtains among the embodiment 1 ', 4 '-trimethoxy flavone, purity is 99.5%; In the L. similis Hemsl medicinal material, 5-hydroxyl-7,3 ', 4 '-content of trimethoxy flavone is 1.08%.
The separation purification method of embodiment 2 β-sitosterols
(1) take by weighing the dry flower 475g of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70%, material ratio are 1: 8, filter, and decompression recycling ethanol gets medicinal extract 95g, (being that 1g medicinal extract is equivalent to 5g crude drug amount).After measured, contain total flavones 3.39% in this extract.
(2) get above-mentioned medicinal extract, add water preparation upper prop liquid, upper column liquid concentration is counted 0.10g/ml with crude drug, upper macroporous adsorptive resins, successively water, concentration be 10%, 20%, 40%, 60%, 80%, the ethanol elution of 95%V/V, collect 95% ethanol position;
(3) get gained ethanol position, behind the recovery solvent, upper silica gel column chromatography (200-300 order, 350g), usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, every stream part 150ml, collect elutriant, the thin layer monitoring merges same stream part, get 17 ~ 22 parts, after reclaiming solvent, recrystallization namely gets β-sitosterol (158mg).
Compound identification:
The gained compound is the white plates crystallization, mp137.6～138.0 ℃.The Liebermann-Burchard reaction is positive.
1δ 5.25 is rare hydrogen proton signal in the H-NMR spectrum, and δ 3.26 is for connecting proton signal on the oxygen carbon.δ: 0.62,0.93,0.86,0.88,0.77,0.75 is methyl proton signal, and pointing out this compound is steroid compound.
13Have 29 carbon signals in the C-NMR spectrum, 140.83,120.97 two typical rare carbon signals wherein, 71.01 for connecting carbon signal, determines therefore to contain that oxygen carbon connects is a hydroxyl, and remaining carbon signal all is in high field.
13C-NMR(100MHz,DMSO-d
6)δ:37.11（C-1）,31.58（C-2）,71.01（C-3）,42.05（C-4）,140.83（C-5）,120.97（C-6）,30.87（C-7）,31.84（C-8）,50.31（C-9）,35.97（C-10）,20.74（C-11）,39.72（C-12）,41.59（C-13）,56.75（C-14）,56.24（C-15）,27.88（C-16）,56.05（C-17）,10.85（C-18）,18.71（C-19）,36.24（C-20）,17.87（C-21）,33.68（C-22）,25.80（C-23）,45.87（C-24）,28.99（C-25）,18.39（C-26）,17.97（C-27）,22.73（C-28）,10.85（C-29）。With it
13The data of β-sitosterol contrast in C-NMR data and the document, basically identical, simultaneously consistent with β-sitosterol reference substance thin-layer chromatography Rf value and colour developing behavior, and mixed melting point do not descend, so compound 1 is accredited as β-sitosterol.
Compound VI β-sitosterol (β-sitosterol)
The separation purification method of embodiment 3 β-daucosterol
(1) take by weighing the dry flower 475g of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70%, material ratio are 1: 8, filter, and decompression recycling ethanol gets medicinal extract 95g, (being that 1g medicinal extract is equivalent to 5g crude drug amount).After measured, contain total flavones 3.39% in this extract.
(2) get above-mentioned medicinal extract, add water preparation upper prop liquid, upper column liquid concentration is counted 0.10g/ml with crude drug, upper macroporous adsorptive resins, successively water, concentration be 10%, 20%, 40%, 60%, 80%, the ethanol elution of 95%V/V, collect 95% ethanol position;
(3) get gained ethanol position, behind the recovery solvent, upper silica gel column chromatography (200-300 order, 350g), usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, every stream part 150ml, collect elutriant, the thin layer monitoring merges same stream part, get 120 ~ 124 parts, after reclaiming solvent, recrystallization namely gets β-daucosterol (74.5mg).
Compound identification:
The gained compound is white powder, and mp289～290 ℃ are insoluble to sherwood oil, are slightly soluble in chloroform, are dissolved in methyl alcohol, and the Liebermann-Burchard reaction is positive, and supposition may be triterpene or steroid compound.
1H-NMR (400MHz, C
5D
6N): show even H signal δ: 4.42 (1H, t, J=3.0Hz, H-3) on the oxygen carbon of H signal δ on the double key carbon: 5.32 (1H, s, H-6) and.Simple substance occurs simultaneously at 4.57 places bimodal, and J=7.6Hz is sugared terminal hydrogen signal.And judge that by coupling Changshu this sugar is beta configuration.
13C-NMR (100MHz, C
5D
6N) δ: 39.37(C-1), 29.47(C-2), 78.56(C-3), 39.97(C-4), 140.92(C-5), 121.99(C-6), 32.22(C-7), 32.08(C-8), 50.36(C-9), 24.56(C-10), 20.05(C-11), 26.37(C-12), 42.52(C-13), 56.86(C-14), 23.42(C-15), 39.98(C-16), 56.26(C-17), 12.03(C-18), 21.33(C-19), 36.96(C-20), 19.25(C-21), 36.45(C-22), 37.51(C-23), 46.06(C-24), 30.29(C-25), 19.06(C-26), 20.05(C-27), 19.48(C-28), 12.21(C-29), 102.61(C-1 '), 75.39(C-2 '), 78.57(C-3 '), 71.69(C-4 '), 78.09(C-5 '), 62.84(C-6 '). δ 140.92,121.99 the existence of fignal center explanation ethylene linkage, δ 62.84～102.61 has 6 carbon, show to have a hexose fragment, wherein sugared end group carbon δ 102.61 with
1δ 4.57(1H among the H-NMR, d, J=7.6Hz) corresponding, show that hexose is beta configuration.Above spectral data and bibliographical information are basically identical, and be simultaneously consistent with β-daucosterol reference substance thin-layer chromatography Rf value and colour developing behavior, and mixed melting point does not descend.So the compound IX is accredited as β-daucosterol.
Compound IX β-daucosterol (β-Daucosterin)
Embodiment 2 macroporous resin purification technical studies
According to the literature, the main effective constituent of Japanese Honeysuckle class medicinal material is organic acid, flavonoid and saponins, and wherein organic acid is take chlorogenic acid as main, its extract separate, content assaying method is relatively ripe, but the quality that is used for estimating Japanese Honeysuckle is not had a specificity.And by L. similis Hemsl flavonoid, saponin component being carried out qualitative identification, the reaction of saponin component qualitative identification is not obvious, illustrates that saponin component content contained in the L. similis Hemsl is very little or do not contain specific examples of such components.The present invention separates the extraction that L. similis Hemsl carries out flavones ingredient by macroporous resin chromatogram, polymeric amide chromatogram, column chromatography chromatogram and gel column chromatography isochrome spectrometry, the preparation reference substance, and thin layer is differentiated and the research of content assaying method.
1.1 instrument, reagent, crude drug source
1.1.1 instrument
Rotary Evaporators: RE-5203(Shanghai Yarong Biochemical Instrument Plant)
The multiplex vacuum pump of circulating water type: SHB-III (Zhengzhou Greatwall Scientific Industrial ﹠ Trading Co., Ltd.)
Electronic balance: sky, TD6001(Tianjin and instrument plant)
Dark box type ultraviolet transilluminator: ZF-90(Shanghai Gu Cun electric light instrument plant)
Ultraviolet-visible spectrophotometer: sky, UV1100(Shanghai U.S. Science and Technology Ltd.)
1.1.2 reagent and material
Column chromatography: macroporous resin D101:(granularity 0.3-1.25mm; Tianjin recovery fine chemistry industry institute)
Thin-layer chromatography: silica gel G (Haiyang Chemical Plant, Qingdao)
Polyamide layer (the biochemical plastic molding and processing plant of City of Taizhou road and bridge tetramethyl produces)
Xylo-Mucine (Solution on Chemical Reagents in Shanghai purchasing and supply station)
1.1.3 reagent
Reagent: extract and be chemical pure with reagent, column chromatography is analytical pure with reagent
Developer: 10% ethanol solution of sulfuric acid, 0.2% vanillin food grade,1000.000000ine mesh-concentrated sulfuric acid solution, AlCl3 solution
1.1.4 crude drug source and evaluation
Test used L. similis Hemsl medicinal material and purchase in Nanjiang County, Sichuan Province, identify through professor Ma Yuying of Chengdu University of Traditional Chinese Medicine, be caprifoliaceae plant L. similis Hemsl Lonicera similis Hemsl.
1.2 method and result
1.2.1 the extraction of L. similis Hemsl and the assay of total flavones
1.2.1.1 the preparation of L. similis Hemsl extracting solution
Take by weighing L. similis Hemsl dry flower 475g, adopt literature method [71-72] to extract, namely use 90%, 80%, 70% alcohol reflux, material ratio is 1: 8, filters decompression recycling ethanol, get medicinal extract 95g, (being that 1g medicinal extract is equivalent to 5g crude drug amount), for subsequent use.
1.2.1.2 the preparation of reference substance solution
Get the about 25mg of control substance of Rutin, put in the 50ml volumetric flask, add 75% appropriate amount of ethanol, supersound process makes dissolving, lets cool, and adds 75% ethanol to scale, shakes up.Precision is measured 20ml, puts in the 50ml volumetric flask, adds 75% ethanol to scale, shakes up, and namely gets (concentration is 0.2mg/ml).
1.2.1.3 the preparation of need testing solution
Get the medicinal extract 0.5g (being equivalent to crude drug amount 2.5g) among the 2.1.1, the accurate absorption put in the 50ml volumetric flask, adds 75% appropriate amount of ethanol, and supersound process makes dissolving, lets cool, and adds 75% ethanol to scale, shakes up.Precision is measured 1ml, puts in the 50ml volumetric flask, adds 75% ethanol to scale, shakes up, and gets namely that (concentration is 0.001g crude drug/ml).
1.2.1.4 wavelength is selected
Accurate draw control substance of Rutin solution and need testing solution an amount of, with Sodium Nitrite-aluminum nitrate colour developing, blank reagent is contrast, in the interscan of 200～800nm wavelength region, result such as Fig. 1.
Above result shows that reference substance solution has maximum absorption at the 510.5nm place, and need testing solution has maximum absorption at the 509.3nm place, because the flavones kind is many in the trial-product, cause maximum absorption and reference substance that small difference is arranged, consider in reference substance and come content, so select 510nm for measuring wavelength.
1.2.1.5 the drafting of typical curve
Precision is measured reference substance solution 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml and 6.0ml, put respectively in the 25ml volumetric flask, respectively add water to 6.0ml, add 5% sodium nitrite solution 1ml, mixing, placed 6 minutes, and added 10% aluminum nitrate solution 1ml, shake up, placed 6 minutes, hydro-oxidation sodium solution 10ml adds water again and is settled to scale, shakes up, placed 15 minutes, take corresponding reagent as blank, the photograph ultraviolet visible spectrophotometry (" appendix VA of Chinese pharmacopoeia version in 2010), measure absorbancy in 510nm wavelength place.Take absorbancy (A) as ordinate zou, concentration C (μ g/ml) is X-coordinate, the drawing standard curve.Obtain regression equation: A=0.0137C+0.0008, r=0.9999, n=6.Measurement result sees Table 1, Fig. 2.
Table 1 control substance of Rutin Specification Curve of Increasing result
Measurement result shows that control substance of Rutin solution is in 8.0179 ~ 48.1074 μ g/ml concentration ranges, and absorbancy and concentration are the good linear relation.
1.2.1.6 the mensuration of total flavones in the L. similis Hemsl extract
It is an amount of to get need testing solution, measures with reference to method among the 1.2.1.5, and absorbancy is 0.465, brings typical curve into absorbancy, and general flavone content calculates with the rutin Equivalent, and the content of total flavones is 3.39%.
1.2.2 macroporous resin purification L. similis Hemsl extract technical study
The Amberlyst process technical qualification are had relatively high expectations, and its adsorption effect is subject to the impact of the factors such as macroporous adsorbent resin type and character, sample solution concentration, flow velocity, eluent.The research of this experiment is screened resinous type by the investigation of Static Adsorption performance, and to upper column liquid concentration, investigate than the consumption of upper column quantity, eluting solvent and eluting solvent, final condition and the parameter of determining purification with macroreticular resin L. similis Hemsl general extractive is for the separation and purification of L. similis Hemsl provides reference frame.
1.2.2.1 the preparation of upper prop liquid
The L. similis Hemsl medicinal extract of getting among the 1.2.1.1 is an amount of, and adding certain water gaging, to be diluted to crude drug concentration be 0.1g/ml, centrifugal 20min(3000r/min), get supernatant liquor quantitatively to certain volume, for subsequent use.
1.2.2.2 the screening of resinous type
The macroporous resin kind of using at present both at home and abroad is numerous, and macroporous resin can be divided into nonpolar and polarity two large classes, can also be divided into low-pole, middle polarity and polarity etc. by force according to the size of polarity.Commodity production is all arranged both at home and abroad, and domestic macroporous adsorbent resin is mainly used in the separation and purification of food and Chinese medicine.The resin of domestic product mainly contains the D series of Tianjin agricultural chemicals limited-liability company, Shanghai chemical reagent work 101,102,402 etc., and the product D of Chemical Plant of Nankai Univ. series, H are serial, AB-8 and the SIP of Shanghai Institute of Pharmaceutical Industry series etc.This experiment is take the adsorptive capacity of L. similis Hemsl total flavones and resolution factor as comprehensive evaluation index, to determine the macroporous resin type of purifying L. similis Hemsl general extractive.All kinds of macroporous resin physicalies see Table 2.
All kinds of macroporous resin physical-property parameters of table 2
(1) mensuration of macroporous adsorbent resin pre-treatment settling ratio
It is an amount of to get various resin, dries the w that weighs, and adds 95% alcohol immersion resin 24h, abundant swelling, and constantly stir, get rid of bubble, wet method dress post, with 95% ethanol elution, mix (1:3) muddiness that is not white in color with water to effluent liquid, be washed till without the alcohol flavor with distilled water again, in water, measure its volume V after the sedimentation fully until resin, ρ=W/V the results are shown in Table 3.
The settled density table of the various resin of table 3
(2) investigation of dissimilar macroporous resin Static Adsorption performances
Measuring various types of macroporous resins (dry weight is about 1g) puts in the tool plug Erlenmeyer flask of 150ml, precision adds L. similis Hemsl need testing solution 90ml(crude drug concentration 0.1g/ml respectively), left standstill 24 hours, constantly jolting, after it is fully adsorbed, filter, measure the content that remains total flavones in the filtrate according to the method in 1.6, calculate adsorptive capacity; Macroporous resin after the adsorption equilibrium is transferred in the young shape bottle, with 95% ethanol desorb, measures the content of total flavones in the desorption efficiency, calculate adsorption rate.The results are shown in Table 4, Fig. 3.
The various resin of table 4 is to adsorption rate and the desorption efficiency of L. similis Hemsl total flavones
Annotate: general flavone content (mg) (as follows) in general flavone content (mg)-leakage fluid in (1) adsorptive capacity=upper prop liquid
(2) adsorption rate=[general flavone content (mg) in adsorptive capacity (mg)/upper prop liquid] * 100%
(3) desorption efficiency (%)=[general flavone content in the stripping liquid (mg)/adsorptive capacity (mg)] * 100%
From table 4 and Fig. 3 result as can be known, by five kinds of different macroporous resins are investigated, find that the LD605 adsorptive power is the strongest, but be difficult to be desorbed, absorption has larger adsorptive capacity to the D101 macroporous resin to total flavones under the experiment condition of giving, and can get off most desorbs.So select D101 macroporous resin purification L. similis Hemsl extract.
1.2.2.3 the investigation of upper column liquid concentration
Take by weighing a certain amount of L. similis Hemsl medicinal extract, be diluted to respectively different concns, in the absorption column that D101 macroporous resin (the about 4g of dry weight) is housed, adsorb with 2 ~ 3mL/min flow velocity, collect effluent liquid, measure the content of total flavones, calculate the ratio adsorptive capacity of macroporous resin.Such as table 5, shown in Figure 4.
Table 5 sample solution concentration is investigated
Annotate: than adsorptive capacity=adsorptive capacity (mg)/resin dry weight (g) (as follows)
The result shows that when upper prop liquid crude drug concentration was 0.10g/ml, the D101 macroporous resin was maximum to the ratio adsorptive capacity of L. similis Hemsl total flavones, is 97.55mg/g, and therefore selecting upper prop liquid crude drug concentration is 0.10g/ml.
1.2.2.4 the investigation than upper column quantity
The L. similis Hemsl extracting solution (crude drug concentration is 0.1g/ml) of 180ml is passed through D101 macroporous resin (the about 4g of dry weight), upper column separating purification, Fractional Collections effluent liquid, every part of 10ml.Measure the content of total flavones in the effluent liquid, calculate it and leak percentage, the result is as shown in table 6, and draws the dynamic leakage curve, sees Fig. 5.
Table 6 is than the investigation of upper column quantity
Annotate: "-" expression does not detect, than applied sample amount=crude drug amount (g)/resin dry weight (g)
Interpretation of result: the cumulative percentage rate surpasses 5% when going up as can be known 14 parts of loadings to the by measurement result, can find out that from leakage plot the L. similis Hemsl total flavones of the 14th part of leakage begins to enlarge markedly, and illustrates that resin column begins to adsorb fully the total flavones in the liquid.For the L. similis Hemsl total flavones keeps fully, with the amount of maximum applied sample amount 80% as applied sample amount, i.e. 110mL.In the ratio adsorptive capacity of total flavones resin for 92.92mg/g, take total flavones at medicinal material content as 3.39%, the ratio upper column quantity that calculates resin is 2.74g/g (medicinal material/dried resin).
1.2.2.5 brief summary
With Static Adsorption capacity and resolution factor screening resinous type, on the dynamic adsorption condition, the processing parameter that research affects purifying as above sample liquid concentration, than upper column quantity etc., the technique of finally having determined macroporous resin purification L. similis Hemsl extract is: D101 macroporous resin purification L. similis Hemsl extract, upper prop liquid crude drug concentration is 0.10g/ml, adsorbs than upper column quantity 2.74g/g (medicinal material/dried resin) upper prop.
In sum, the invention provides separation and purification 5-hydroxyl-7 from L. similis Hemsl, 3 ', 4 '-separation purification method of trimethoxy flavone, β-daucosterol, β-sitosterol, each compound purity that obtains is higher, for L. similis Hemsl has searched out new use value, the while also provides possibility for the effective substance of clear and definite L. similis Hemsl.
Claims (10)
1.5-hydroxyl-7,3 ', 4 '-separation purification method of trimethoxy flavone, it is characterized in that: it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95%v/v ethanol position successively;
(3) get gained ethanol position, reclaim solvent after, upper silica gel column chromatography is used hexanaphthene-ethyl acetate=20: 1 ~ 0: 1 successively, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, and the elutriant of per 0.16 column volume is 1 stream part, collects elutriant, the thin layer monitoring is got 39 ~ 59 parts, behind the recovery solvent, redissolve with methyl alcohol, go up again gel column, use methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallization, namely get 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone.
2. separation purification method according to claim 1, it is characterized in that: in the step (1), the concrete operations of described extraction using alcohol are: be the extraction using alcohol of 90%V/V, 80%V/V, 70%V/V successively with concentration; Preferably, the concrete grammar of extraction using alcohol is: use respectively the alcohol reflux of 90%V/V, 80%V/V, 70%V/V, and the extraction using alcohol of each concentration 1-2 time, each 1-3 hour, material ratio was 1: 6-10.
3. separation purification method according to claim 1 is characterized in that: in the step (2), water, 80%/V ethanol, 95%V/V ethanol elution are collected 95%v/v ethanol position successively; Described macroporous adsorbent resin is D101 type macroporous adsorbent resin.
4. separation purification method according to claim 1 is characterized in that: in the step (2),
Upper column liquid concentration is counted 0.02-0.2g/ml with crude drug, than upper column quantity less than 2.8g medicinal material/g dried resin.
5. preparation method according to claim 4, it is characterized in that: in the step (2), upper column liquid concentration is counted 0.10g/ml with crude drug, than upper column quantity 2.74g medicinal material/g dried resin.
6. separation purification method according to claim 1, it is characterized in that: in the step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volumes; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes.
7. separation purification method according to claim 1, it is characterized in that: in the step (3), described gel column is SephadexLH-20 type gel column; Recrystallization solvent is methyl alcohol.
8. the separation purification method of β-sitosterol, it is characterized in that: it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95% ethanol position successively;
(3) get gained ethanol position, after reclaiming solvent, upper silica gel column chromatography, usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, the elutriant of per 0.16 column volume is 1 stream part, collects elutriant, the thin layer monitoring, get 17 ~ 22 parts, after reclaiming solvent, recrystallization namely gets β-sitosterol;
In the step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 1 1.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes.
9. the separation purification method of β-daucosterol, it is characterized in that: it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95% ethanol position successively;
(3) get gained ethanol position, after reclaiming solvent, upper silica gel column chromatography, usefulness hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, the elutriant of per 0.16 column volume is 1 stream part, collects elutriant, the thin layer monitoring, get 120 ~ 124 parts, after reclaiming solvent, recrystallization namely gets β-daucosterol;
In the step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 1 1.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volumes; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes; Hexanaphthene-ethyl acetate=3:1,4.05 times of column volumes; Hexanaphthene-ethyl acetate=2:1,1.67 times of column volumes; Hexanaphthene-ethyl acetate=1:1,2.12 times of column volumes; 1.62 times of column volumes of ethyl acetate.
10. from L. similis Hemsl, separate simultaneously 5-hydroxyl-7,3 ', 4 '-method of trimethoxy flavone, β-sitosterol and β-daucosterol, it is characterized in that: it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add the 70-90%V/V extraction using alcohol, united extraction liquid reclaims solvent, namely gets L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper macroporous adsorptive resins, water, 80%/V ethanol, 90-95%V/V ethanol elution are collected 90-95%v/v ethanol position successively;
(3) get gained ethanol position, after reclaiming solvent, upper silica gel column chromatography, with hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ system carried out gradient elution in 0: 1, and the elutriant of per 0.16 column volume is 1 stream part, collect elutriant, the thin layer monitoring, condition of gradient elution is as follows: hexanaphthene-ethyl acetate=20: 1,1.07 times of column volumes; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volumes; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volumes; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes; Hexanaphthene-ethyl acetate=3:1,4.05 times of column volumes; Hexanaphthene-ethyl acetate=2:1,1.67 times of column volumes; Hexanaphthene-ethyl acetate=1:1,2.12 times of column volumes; 1.62 times of column volumes of ethyl acetate;
(4) get 17 ~ 22 parts of stream parts, behind the recovery solvent, recrystallization namely gets β-sitosterol;
Get 39 ~ 59 parts, reclaim solvent after, redissolve with methyl alcohol, go up again gel column, use methanol-eluted fractions, the collection elutriant, the recovery solvent, recrystallization, namely get 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone;
Get 120 ~ 124 parts, behind the recovery solvent, recrystallization namely gets β-daucosterol.
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