CN102924418B - 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 ', the separation purification method of 4 '-trimethoxy flavone, β-sitosterol, β-daucosterol.
Background technology
L. similis Hemsl Lonicera similes Hemsl is the main flow kind of river pan honeysuckle flower, distribute wide, 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 in Sichuan Province, shop of Chinese medicines and medical institutions are used 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 dry honeysuckle flowers per year, realize 200,000,000 yuan of the gross output values, producing region increases income 560 yuan per capita.Japanese Honeysuckle industry has become the Main Economic mainstay industry of Nanjiang County.
In recent years, many research shows that L. similis Hemsl chlorogenic acid content is high, and 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, 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, 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, can be used for II type hyperlipidaemia and prevention of arterial atherosis; Meanwhile, 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 oxidation resistant pharmacologically active simultaneously.
At present, yet there are no separation and purification 5-hydroxyl-7 from L. similis Hemsl, 3 ', the relevant report of the 4 '-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 ', the separation purification method of 4 '-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 ', the separation purification method of 4 '-trimethoxy flavone, it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add 70-90%V/V extraction using alcohol, united extraction liquid, reclaims solvent, obtains 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, collect 90-95%v/v ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, uses hexanaphthene-ethyl acetate=20: 1～0: 1 successively, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collects elutriant, thin layer monitoring, gets 35～38 parts, reclaims after solvent, with methyl alcohol, redissolve, upper gel column, by methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallization, obtain 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone.
Wherein, in step (1), the concrete operations of described extraction using alcohol are: by concentration, be the extraction using alcohol of 90%V/V, 80%V/V, 70%V/V successively; 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 is 1: 6-10.
Wherein, in step (2), water, 80%/V ethanol, 95%V/V ethanol elution, collect 95%v/v ethanol position successively; Described macroporous adsorbent resin is D101 type macroporous adsorbent resin.
Wherein, in step (2), upper column liquid concentration is counted 0.02-0.2g/ml with crude drug, than upper column quantity, is less than 2.8g medicinal material/g dried resin.
Further, in 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 step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volume; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes.
Wherein, in 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 70-90%V/V extraction using alcohol, united extraction liquid, reclaims solvent, obtains 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, collect 90-95% ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, uses hexanaphthene-ethyl acetate=20: 1～0: 1, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collects elutriant, thin layer monitoring, get 17～22 parts, reclaim after solvent, recrystallization, obtains β-sitosterol;
In step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume.
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 70-90%V/V extraction using alcohol, united extraction liquid, reclaims solvent, obtains 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, collect 90-95% ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, uses hexanaphthene-ethyl acetate=20: 1～0: 1, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collects elutriant, thin layer monitoring, get 120～124 parts, reclaim after solvent, recrystallization, obtains β-daucosterol;
In step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volume; 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 provides while separated 5-hydroxyl-7 from L. similis Hemsl, 3 ', the method for 4 '-trimethoxy flavone, β-sitosterol and β-daucosterol, it comprises the steps:
(1) get the dry flower of L. similis Hemsl Lonicera similes Hemsl, add 70-90%V/V extraction using alcohol, united extraction liquid, reclaims solvent, obtains 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, collect 90-95%v/v ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, with hexanaphthene-ethyl acetate=20: 1～0: 1, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collect elutriant, thin layer monitoring, condition of gradient elution is as follows: hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 11.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volume; 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 streams part, reclaim after solvent, recrystallization, obtains β-daucosterol;
Get 35～38 parts, reclaim after solvent, with methyl alcohol, redissolve, upper gel column, by methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallization, obtains 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone;
Get 120～124 parts, reclaim after solvent, recrystallization, obtains 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 ', the separation purification method of 4 '-trimethoxy flavone, β-daucosterol, β-sitosterol, each compound purity obtaining is higher, for L. similis Hemsl medicinal material has searched out new use value; Wherein, 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone, β-daucosterol be first from L. similis Hemsl separation obtain.
(2) provided by the invention from L. similis Hemsl separated 5-hydroxyl-7 simultaneously, 3 ', the method of 4 '-trimethoxy flavone, β-daucosterol, β-sitosterol, can be under same courses of action, separation obtains above-mentioned three kinds of compounds successively, compares with the independent separation method of compound, and 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 antibacterial, the antiviral effect of L. similis Hemsl, may be the effective constituent that its function of reflection cures mainly, the effective separation of the present invention to this compound, also the effective substance for clear and definite L. similis Hemsl provides possibility.
Accompanying drawing explanation
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 composition separation process figure
Fig. 8 β-sitosterol (β-sitosterol) hydrogen spectrogram
Fig. 9 β-sitosterol (β-sitosterol) carbon spectrogram
Figure 105-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone) hydrogen spectrogram
Figure 115-hydroxyl-7,3 ', 4 '-trimethoxy flavone (5-hydroxyl-7,3', 4'-trimethoxyflavone) carbon spectrogram
Figure 12 β-daucosterol (β-Daucosterin) hydrogen spectrogram
Figure 13 β-daucosterol (β-Daucosterin) carbon spectrogram
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 (be above analytical pure, Chengdu Ke Long chemical reagent factory 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
Testing L. similis Hemsl medicinal material used and purchase in Nanjiang County, Sichuan Province, through professor Ma Yuying of Chengdu University of Traditional Chinese Medicine, identify, is caprifoliaceae plant L. similis Hemsl Lonicera similis Hemsl.
Embodiment 15-hydroxyl-7,3 ', the separation purification method of 4 '-trimethoxy flavone
(1) take the dry flower 1.9Kg of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70%, material ratio is 1: 8, filters, decompression recycling ethanol, obtains medicinal extract.After measured, in this extract, contain total flavones 3.39%.
(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, reclaim after solvent, upper silica gel column chromatography (200-300 order, 350g, column volume 925ml), with hexanaphthene-ethyl acetate=20: 1～0: 1, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, every stream part 150ml, collect elutriant, thin layer monitoring, merge same stream part, get 35～38 parts, reclaim after solvent, with methyl alcohol, redissolve, go up again SephadexLH-20 gel column, by methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallization, obtain 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:
Gained compound is yellow powder, mp167～169 ℃.Under TLC ultraviolet lamp, develop the color, spray AlCl
3have yellow fluorescence, the reaction of hydrochloric acid-magnesium powder 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 can be substantially separated with other component chromatographic peaks in sample, 5-hydroxyl-7,3 ', the resolution that 4 '-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 determine number of theoretical plate with 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone meter should be not less than 4000.
Through HPLC, detect, separated 5-hydroxyl-7 that obtain in embodiment 1,3 ', 4 '-trimethoxy flavone, purity is 99.5%; In L. similis Hemsl medicinal material, 5-hydroxyl-7,3 ', the content of 4 '-trimethoxy flavone is 1.08%.
The separation purification method of embodiment 2 β-sitosterols
(1) take the dry flower 475g of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70%, material ratio is 1: 8, filters, decompression recycling ethanol, obtains medicinal extract 95g, (being that 1g medicinal extract is equivalent to 5g crude drug amount).After measured, in this extract, contain total flavones 3.39%.
(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, reclaim after solvent upper silica gel column chromatography (200-300 order, 350g), use hexanaphthene-ethyl acetate=20: 1～0: 1, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, every stream part 150ml, collect elutriant, thin layer monitoring, merges same stream part, get 17～22 parts, reclaim after solvent, recrystallization, obtains β-sitosterol (158mg).
Compound identification:
Gained compound is white plates crystallization, mp137.6～138.0 ℃.Liebermann-Burchard reaction is positive.
1in H-NMR spectrum, δ 5.25 is rare hydrogen proton signal, and δ 3.26 is for connecting proton signal on oxygen carbon.δ: 0.62,0.93,0.86,0.88,0.77,0.75 is methyl proton signal, pointing out this compound is steroid compound.
13in C-NMR spectrum, have 29 carbon signals, 140.83,120.97 two typical rare carbon signals wherein, 71.01 for connecting carbon signal, therefore determines that what containing oxygen carbon, connect is a hydroxyl, and remaining carbon signal is all 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）。By it
13c-NMR data contrast with the data of β-sitosterol in 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 decline, therefore compound 1 is accredited as β-sitosterol.
Compound VI β-sitosterol (β-sitosterol)
The separation purification method of embodiment 3 β-daucosterol
(1) take the dry flower 475g of L. similis Hemsl Lonicera similes Hemsl, the alcohol reflux with 90%, 80%, 70%, material ratio is 1: 8, filters, decompression recycling ethanol, obtains medicinal extract 95g, (being that 1g medicinal extract is equivalent to 5g crude drug amount).After measured, in this extract, contain total flavones 3.39%.
(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, reclaim after solvent upper silica gel column chromatography (200-300 order, 350g), use hexanaphthene-ethyl acetate=20: 1～0: 1, ethyl acetate-methyl alcohol=5: 1～0: 1 system is carried out gradient elution, every stream part 150ml, collect elutriant, thin layer monitoring, merges same stream part, get 120～124 parts, reclaim after solvent, recrystallization, obtains β-daucosterol (74.5mg).
Compound identification:
Gained compound is white powder, and mp289～290 ℃, are insoluble to sherwood oil, are slightly soluble in chloroform, are dissolved in methyl alcohol, and Liebermann-Burchard reaction is positive, and supposition may be triterpene or steroid compound.
1h-NMR (400MHz, C
5d
6n): show H signal δ: 4.42 (1H, t, J=3.0Hz, H-3) on H signal δ on a double key carbon: 5.32 (1H, s, H-6) and company's oxygen carbon.At 4.57 places, occur that simple substance is bimodal, J=7.6Hz, is sugared terminal hydrogen signal simultaneously.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, the existence of 121.99 fignal center explanation ethylene linkages, δ 62.84～102.61 has 6 carbon, show to exist a hexose fragment, wherein sugared end group carbon δ 102.61 with
1δ 4.57(1H in H-NMR, d, J=7.6Hz) corresponding, show that hexose is beta configuration.Above spectral data and bibliographical information are basically identical, simultaneously consistent with β-daucosterol reference substance thin-layer chromatography Rf value and colour developing behavior, and mixed melting point does not decline.Therefore compound IX is accredited as β-daucosterol.
Compound IX β-daucosterol (β-Daucosterin)
According to the literature, the main effective constituent of Japanese Honeysuckle class medicinal material is organic acid, flavonoid and saponins, and wherein organic acid be take chlorogenic acid as main, its extract separated, content assaying method is relatively ripe, but does not have a specificity for evaluating the quality of Japanese Honeysuckle.And by L. similis Hemsl flavonoid, saponin component being carried out to qualitative identification, the reaction of saponin component qualitative identification is not obvious, illustrates that saponin component content contained in L. similis Hemsl is very micro-or do not contain specific examples of such components.The extraction that the present invention carries out flavones ingredient by macroporous resin chromatogram, polymeric amide chromatogram, column chromatography chromatogram and gel column chromatography isochrome spectrometry to L. similis Hemsl is separated, prepares reference substance, and thin layer is differentiated and the research of content assaying method.
1.1 instruments, 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: TD6001(Tianjin Tian Yu instrument plant)
Dark box type ultraviolet transilluminator: ZF-90(Shanghai Gu Cun electric light instrument plant)
Ultraviolet-visible spectrophotometer: UV1100(Shanghai Tian Mei 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 (road and bridge tetramethyl biochemical plastic molding and processing plant in City of Taizhou 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
Testing L. similis Hemsl medicinal material used and purchase in Nanjiang County, Sichuan Province, through professor Ma Yuying of Chengdu University of Traditional Chinese Medicine, identify, is caprifoliaceae plant L. similis Hemsl Lonicera similis Hemsl.
1.2 methods 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 L. similis Hemsl dry flower 475g, adopt literature method [71-72] to extract, use 90%, 80%, 70% alcohol reflux, material ratio is 1: 8, filters decompression recycling ethanol, obtain medicinal extract 95g, (being that 1g medicinal extract is equivalent to 5g crude drug amount), standby.
1.2.1.2 the preparation of reference substance solution
Get the about 25mg of control substance of Rutin, put in 50ml volumetric flask, add 75% appropriate amount of ethanol, supersound process makes to dissolve, and lets cool, and adds 75% ethanol to scale, shakes up.Precision measures 20ml, puts in 50ml volumetric flask, adds 75% ethanol to scale, shakes up, and obtains (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) in 2.1.1, accurate absorption, puts in 50ml volumetric flask, adds 75% appropriate amount of ethanol, and supersound process makes to dissolve, and lets cool, and adds 75% ethanol to scale, shakes up.Precision measures 1ml, puts in 50ml volumetric flask, adds 75% ethanol to scale, shakes up, and obtains (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 appropriate, with the colour developing of Sodium Nitrite-aluminum nitrate, blank reagent is contrast, in the interscan of 200～800nm wavelength region, result is as Fig. 1.
Above result shows, reference substance solution has maximum absorption at 510.5nm place, and need testing solution has maximum absorption at 509.3nm place, because flavones kind in trial-product is many, cause maximum absorption and reference substance to have small difference, consider in reference substance and carry out content, therefore select 510nm for measuring wavelength.
1.2.1.5 the drafting of typical curve
Precision measures reference substance solution 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml and 6.0ml, put respectively in 25ml volumetric flask, respectively add water to 6.0ml, add 5% sodium nitrite solution 1ml, mix, place 6 minutes, add 10% aluminum nitrate solution 1ml, shake up, place 6 minutes, hydro-oxidation sodium solution 10ml, add again water and be settled to scale, shake up, place 15 minutes, take corresponding reagent as blank, according to ultraviolet visible spectrophotometry (appendix V A of < < Chinese Pharmacopoeia > > version in 2010), in 510nm wavelength place, measure absorbancy.The absorbancy (A) of take is ordinate zou, and concentration C (μ g/ml) is X-coordinate, drawing standard curve.Obtain regression equation: A=0.0137C+0.0008, r=0.9999, n=6.Measurement result in Table 1, Fig. 2.
Table 1 control substance of Rutin Specification Curve of Increasing result
Measurement result shows, control substance of Rutin solution is in 8.0179～48.1074 μ g/ml concentration ranges, and absorbancy and concentration are good linear relation.
1.2.1.6 the mensuration of total flavones in L. similis Hemsl extract
Get need testing solution appropriate, with reference to method in 1.2.1.5, measure, absorbancy is 0.465, with absorbancy, brings typical curve into, and general flavone content calculates with rutin Equivalent, and the content of total flavones is 3.39%.
1.2.2 macroporous resin purification L. similis Hemsl extract technical study
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, 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 in 1.2.1.1 is appropriate, 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, standby.
1.2.2.2 the screening of resinous type
The macroporous resin kind of using both at home and abroad is at present numerous, and macroporous resin can be divided into the nonpolar and large class of polarity two, according to the size of polarity, can also be divided into low-pole, middle polarity and strong polarity etc.All have commodity production both at home and abroad, 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.It is comprehensive evaluation index that adsorptive capacity and the resolution factor of L. similis Hemsl total flavones take in this experiment, to determine the macroporous resin type of purifying L. similis Hemsl general extractive.All kinds of macroporous resin physicalies are in Table 2.
All kinds of macroporous resin physical-property parameters of table 2
(1) mensuration of macroporous adsorbent resin pre-treatment settling ratio
Get various resin appropriate, dry the w that weighs, add 95% alcohol immersion resin 24h, abundant swelling, and constantly stir, get rid of bubble, wet method dress post, with 95% ethanol elution, to effluent liquid, mix (1:3) muddiness that is not white in color with water, then be washed till without alcohol taste with distilled water, until resin, in water, after sedimentation completely, measure its volume V, 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 performance
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), standing 24 hours, jolting constantly, after it is fully adsorbed, filter, according to the method in 1.6, measure the content that remains total flavones in filtrate, calculate adsorptive capacity; Macroporous resin after adsorption equilibrium is transferred in young shape bottle, with 95% ethanol desorb, measures the content of total flavones in desorption efficiency, calculate adsorption rate.The results are shown in Table 4, Fig. 3.
The various resin of table 4 is to the adsorption rate of L. similis Hemsl total flavones and desorption efficiency
Note: 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 stripping liquid (mg)/adsorptive capacity (mg)] * 100%
From table 4 and Fig. 3 result, by five kinds of different macroporous resins are investigated, find that LD605 adsorptive power is the strongest, but be difficult to be desorbed, under given experiment condition, to total flavones, absorption has larger adsorptive capacity to D101 macroporous resin, and most desorbs can be got off.Therefore select D101 macroporous resin purification L. similis Hemsl extract.
1.2.2.3 go up the investigation of column liquid concentration
Take a certain amount of L. similis Hemsl medicinal extract, be diluted to respectively different concns, by being equipped with in the absorption column of D101 macroporous resin (the about 4g of dry weight), with 2～3mL/min flow velocity, adsorb, collect effluent liquid, measure the content of total flavones, calculate the ratio adsorptive capacity of macroporous resin.As table 5, shown in Fig. 4.
Table 5 sample solution concentration is investigated
Note: than adsorptive capacity=adsorptive capacity (mg)/resin dry weight (g) (as follows)
Result shows, when upper prop liquid crude drug concentration is 0.10g/ml, D101 macroporous resin is 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 than the investigation of upper column quantity
The L. similis Hemsl extracting solution of 180ml (crude drug concentration is 0.1g/ml) is passed through to 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 effluent liquid, calculate it and leak percentage, result is as shown in table 6, and draws dynamic leakage curve, sees Fig. 5.
Table 6 is than the investigation of upper column quantity
Note: "-" represents not detect, than applied sample amount=crude drug amount (g)/resin dry weight (g)
Interpretation of result: during by 14 parts of known upper loadings to the of measurement result, cumulative percentage rate surpasses 5%, can find out that the L. similis Hemsl total flavones of the 14th part of leakage starts to enlarge markedly from leakage plot, illustrates that resin column starts to adsorb the total flavones in liquid completely.For L. similis Hemsl total flavones retains completely, using 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, the total flavones of take is 3.39% at medicinal material content, and 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, in 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, than upper column quantity 2.74g/g (medicinal material/dried resin) upper prop, adsorbs.
In sum, the invention provides separation and purification 5-hydroxyl-7 from L. similis Hemsl, 3 ', the separation purification method of 4 '-trimethoxy flavone, β-daucosterol, β-sitosterol, each compound purity obtaining is higher, for L. similis Hemsl has searched out new use value, the while is also for the effective substance of clear and definite L. similis Hemsl provides possibility.
Claims (9)
1.5-hydroxyl-7,3 ', the separation purification method of 4 '-trimethoxy flavone, is characterized in that: it comprises the steps:
(1) get L. similis Hemsl
lonicera similesthe dry flower of Hemsl, adds 70-90%V/V extraction using alcohol, and united extraction liquid reclaims solvent, obtains L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper D101 type macroporous adsorptive resins, water, 80%/V ethanol, 95%V/V ethanol elution, collect 95%v/v ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, use successively hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ 0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collect elutriant, thin layer monitoring, gets 35 ~ 38 parts, reclaim after solvent, with methyl alcohol, redissolve, upper SephadexLH-20 type gel column, by methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallizing methanol, obtains 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone.
2. separation purification method according to claim 1, is characterized in that: in step (1), the concrete operations of described extraction using alcohol are: by concentration, be the extraction using alcohol of 90%V/V, 80%V/V, 70%V/V successively.
3. separation purification method according to claim 2, it is characterized in that: in step (1), the concrete grammar of extraction using alcohol is: use respectively the alcohol reflux of 90%V/V, 80% V/V, 70% V/V, the extraction using alcohol of each concentration 1-2 time, each 1-3 hour, material ratio is 1: 6-10.
4. separation purification method according to claim 1, is characterized in that: in step (2),
Upper column liquid concentration is counted 0.02-0.2g/ml with crude drug, than upper column quantity, is less than 2.8g medicinal material/g dried resin.
5. preparation method according to claim 4, is characterized in that: in 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, is characterized in that: in step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 1,1.07 times of column volume, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volume; Hexanaphthene-ethyl acetate=4:1,2.0 times of column volumes.
7. the separation purification method of β-sitosterol, is characterized in that: it comprises the steps:
(1) get L. similis Hemsl
lonicera similesthe dry flower of Hemsl, adds 70-90%V/V extraction using alcohol, and united extraction liquid reclaims solvent, obtains L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper D101 type macroporous adsorptive resins, water, 80%/V ethanol, 95%V/V ethanol elution, collect 95% ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, uses hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ 0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collects elutriant, thin layer monitoring, get 17 ~ 22 parts, reclaim after solvent, recrystallization, obtains β-sitosterol;
In step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 1 1.07 times of column volumes, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume.
8. the separation purification method of β-daucosterol, is characterized in that: it comprises the steps:
(1) get L. similis Hemsl
lonicera similesthe dry flower of Hemsl, adds 70-90%V/V extraction using alcohol, and united extraction liquid reclaims solvent, obtains L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper D101 type macroporous adsorptive resins, water, 80%/V ethanol, 95%V/V ethanol elution, collect 95% ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, uses hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ 0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collects elutriant, thin layer monitoring, get 120 ~ 124 parts, reclaim after solvent, recrystallization, obtains β-daucosterol;
In step (3), condition of gradient elution is as follows:
Hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 1,1.07 times of column volume, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volume; 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.
9. separated 5-hydroxyl-7 simultaneously from L. similis Hemsl, 3 ', the method for 4 '-trimethoxy flavone, β-sitosterol and β-daucosterol, is characterized in that: it comprises the steps:
(1) get L. similis Hemsl
lonicera similesthe dry flower of Hemsl, adds 70-90%V/V extraction using alcohol, and united extraction liquid reclaims solvent, obtains L. similis Hemsl general extractive medicinal extract;
(2) get L. similis Hemsl general extractive medicinal extract, add water preparation upper prop liquid, upper D101 type macroporous adsorptive resins, water, 80%/V ethanol, 95%V/V ethanol elution, collect 95%v/v ethanol position successively;
(3) get gained ethanol position, reclaim after solvent, upper silica gel column chromatography, with hexanaphthene-ethyl acetate=20: 1 ~ 0: 1, ethyl acetate-methyl alcohol=5: 1 ~ 0: 1 system is carried out gradient elution, the elutriant of every 0.16 column volume is 1 stream part, collect elutriant, thin layer monitoring, condition of gradient elution is as follows: hexanaphthene-ethyl acetate=20: 1,1.07 times of column volume; Hexanaphthene-ethyl acetate=10: 1,1.07 times of column volume, hexanaphthene-ethyl acetate=8: 1,3.3 times of column volume; Hexanaphthene-ethyl acetate=6: 1; 1.05 column volume doubly; Hexanaphthene-ethyl acetate=5: 1,2.05 times of column volume; 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 streams part, reclaim after solvent, recrystallization, obtains β-sitosterol;
Get 35 ~ 38 parts, reclaim after solvent, with methyl alcohol, redissolve, upper SephadexLH-20 type gel column, by methanol-eluted fractions, collect elutriant, reclaim solvent, recrystallizing methanol, obtains 5-hydroxyl-7,3 ', 4 '-trimethoxy flavone;
Get 120 ~ 124 parts, reclaim after solvent, recrystallization, obtains β-daucosterol.
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Non-Patent Citations (11)
| Title |
|---|
| DNA topoisomerase I inhibitors: Cytotoxic flavones from Lethedon tannaensis;Zahir A et al;《Journal of natural products》;19961231;第59卷(第7期);全文 * |
| Hideaki K et al.Studies on the Saponins of Lonicera japonica THUNB.《Chemical & pharmaceutical bulletin》.1988,第36卷(第12期),全文. |
| Hideaki K et al.Studies on the Saponins of Lonicera japonica THUNB.《Chemical & * |
| pharmaceutical bulletin》.1988,第36卷(第12期),全文. * |
| Zahir A et al.DNA topoisomerase I inhibitors: Cytotoxic flavones from Lethedon tannaensis.《Journal of natural products》.1996,第59卷(第7期),全文. |
| 刑俊波 et al.忍冬花蕾化学成分研究.《中国新药杂志》.2002,第11卷(第11期),全文. |
| 忍冬花蕾化学成分研究;刑俊波 et al;《中国新药杂志》;20021231;第11卷(第11期);全文 * |
| 李永梅 et al.细毡毛忍冬花蕾化学成分研究.《中 国 中 药 杂 志》.2001,第26卷(第1期),全文. |
| 王天志 et al.细毡毛忍冬花蕾挥发油成分研究.《中药材》.1999,第22卷(第11期),全文. |
| 细毡毛忍冬花蕾化学成分研究;李永梅 et al;《中 国 中 药 杂 志》;20010131;第26卷(第1期);全文 * |
| 细毡毛忍冬花蕾挥发油成分研究;王天志 et al;《中药材》;19991130;第22卷(第11期);全文 * |
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