CN103169727B - General-flavonoid compound in chionanthus as well as preparation method and application thereof - Google Patents
General-flavonoid compound in chionanthus as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a general-flavonoid compound in a chionanthus as well as a preparation method and an application thereof and belongs to a technology for extracting and applying effective components of Chinese medicinal herbs. The preparation method of the general-flavonoid compound in the chionanthus comprises the following steps of: extracting by using an ethanol solution, separating by adopting the macroporous-resin column chromatography, separating by adopting the silica-gel column chromatography, separating by adopting the gel column chromatography, purifying a sample by adopting the semi-preparative high-performance liquid chromatography, detecting the sample by adopting the high-performance liquid chromatography, and finally detecting the purified sample by adopting the high-performance liquid chromatography. The specific composition and content of the general-flavonoid compound in the chionanthus are detected accurately, and the antioxidant effect of the general-flavonoid compound in the chionanthus is studied systematically. The preparation method provided by the invention ensures high extraction purity and is suitable for industrial production, and the general-flavonoid compound prepared by extracting has excellent antioxidant activity.
Description
Technical field
The present invention relates to traditional Chinese medicine extyaction to be separated and application technology, particularly relate to Diffuse Coptosapelta flower Flavonoids and its preparation method and application.
Background technology
Diffuse Coptosapelta is Oleaceae Chionanthus plant, is Chinese Second Class Key Protected Plant, have another name called HUAMU, radish flower, oily root, Niu Jingzi, April snow.Diffuse Coptosapelta be mainly distributed in the Yellow River middle and lower reaches and areas to the south thereof [Yang Yumei, Hu Jiang. the breeding and culturing of chionanthus retusa Lindl et Paxt and Landscape Application [J]. Shandong forestry science and technology, 2005 (1): 56].Diffuse Coptosapelta is medicine food dual purpose plant, and record in " China's economic flora ", bud, the leaf of Oleaceae plants chionanthus retusa Lindl et Paxt have effect of relieving heat and thirst, and cure mainly heatstroke.Among the people have spring sampled-current Soviet Union tender leaf and flower, make the history of tea-drinking, be called " Oryza glutinosa scented tea " with the tea that Diffuse Coptosapelta flower makes, pleasant to the palate, some local flavor of other tool [Hu Shicai. excellent beverage plant-chionanthus retusa Lindl et Paxt and the brewed method of branch and leaf [J] thereof. forestry science and technology is developed, 1991,3:16; Fang Shaokun, Du Qingzhi, Zheng Dong. the breeding and applying [J] of chionanthus retusa Lindl et Paxt. territory greening, 2005,9:45-46].Diffuse Coptosapelta " Oryza glutinosa scented tea " is according to existing more than the 1000 year history of historical document, and Lu Yu " tea warp " says: " the raw rotten stone of upper person, the raw oak earth of middle person, lower person's SHENGHUANG soil.On the person of open country, garden person time "." Oryza glutinosa scented tea " spontaneous growth, include and the useful thing of health is enriched, vitamin, aminoacid, catechu polyphenol, caffeine, teatannin content are all higher, and drinking this tea removing food stagnancy food, clear endogenous fire, sterilization can control dysentery, pleasant dredging collateral, diuresis of relieving summer heat, goes greasy blood pressure lowering, allaying tiredness, fat-reducing skin protection.Tea grounds can control gastropathy and infantile diarrhea, has medical value.Diffuse Coptosapelta flower as a kind of tea raw material among the people of preciousness just because its special local flavor and function receive increasing concern.Specify that Diffuse Coptosapelta in existing research containing more flavone compound in spending, and prove that these materials have good antioxidation.But only extract and determine the content of wherein Flavonoids, the purity of extraction is not high, for how measuring wherein concrete contained material and proportion of composing indefinite, the defying age of Flavonoids and the effect of antioxidation how also indefinite; And the toxic solvent that more cost is higher is used in the preparation method of Flavonoids, there is harm not only bad for suitability for industrialized production and to the health of human body.
Summary of the invention
The technical problem to be solved in the present invention is, Diffuse Coptosapelta flower Flavonoids that definite ingredients is provided and its preparation method and application, make preparation method economical and convenient, be easy to suitability for industrialized production, extract the Diffuse Coptosapelta flower Flavonoids product purity obtained high, concrete material proportion of composing is clear and definite, the antioxidation definite effect of Flavonoids.
To achieve these goals, the technical solution used in the present invention is: a kind of preparation method of Diffuse Coptosapelta flower Flavonoids, comprises the following steps:
(1) alcoholic solution extracts:
By Diffuse Coptosapelta flower medicinal material drying, pulverizing, adding mass concentration is after 60-80% soak with ethanol, heating and refluxing extraction 3-5 hour, filtration obtains filtrate and residue, be that 60 ~ 80% alcohol heating reflux extract 2 times by residue with mass concentration again, each 3-5 hour, merging filtrate, concentrates to obtain extractum by filtrate evaporated under reduced pressure;
(2) macroporous adsorbent resin column chromatography is separated:
Be 10-30% dissolve with ethanol by the extractum mass concentration obtained, filter, filtrate is fully adsorbed with macroporous adsorbent resin, dress post, carry out gradient elution with the ethanol-water system eluent that volumetric concentration is 10%, 20%, 30%, 40%, 50%, 60%, 80% and 95% successively, collecting ethanol contend concentration is 50-60% alcohol-water eluting fraction, is evaporated to leaching paste, by dry for extractum vacuum decompression at 50-60 DEG C, obtain Diffuse Coptosapelta flower Flavonoids crude product;
(3) silica gel column chromatography is separated:
Silica gel column chromatography loading is adopted to be separated, successively with CHCl Diffuse Coptosapelta flower Flavonoids crude product
3: CH
3oH volume ratio is 1:9,1:7,1:5,1:3 is eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects the fraction section being greater than 60% containing Diffuse Coptosapelta flower Flavonoids;
(4) gel column chromatography is separated:
Sample after being separated by silica gel column chromatography adopts gel column chromatography to be separated, and adopts CHCl
3: CH
3oH volume ratio is 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, dry;
(5) half preparative high-performance liquid chromatographic purification:
Sample after gel column chromatography being separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta spends Flavonoids, chromatographic condition is permaphase ODS-A, 20 × 250mm, and mobile phase is CH
3oH:H
2o, volume ratio is 7:3;
(6) high performance liquid chromatography detects:
Finally adopt high performance liquid chromatography to detect in the sample after purification, chromatographic condition is:
Chromatograph: Agilent1100 high performance liquid chromatograph,
Chromatographic column: Agilent Zorbax SB C
18chromatographic column, 4.6 × 250 mm, 5 μm,
Mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60,
Flow velocity: 1.0 mL/min,
Column temperature: 25 DEG C,
Determined wavelength: 350 nm.
In described step (1), the time of soak with ethanol is 8-12 hour.
In described step (1) during the 1st reflux, the addition of ethanol be the 10-12 of Diffuse Coptosapelta flower quality of medicinal material doubly, the 2nd time and the 3rd reflux time, the addition of ethanol be the 8-10 of Diffuse Coptosapelta flower quality of medicinal material doubly.
Macroporous adsorbent resin in described step (2) is D101 or AB-8 type macroporous adsorbent resin.
Diffuse Coptosapelta flower Flavonoids, be made up of Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides 5 kinds chromocor compound, mass percentage is respectively 5-8%, 8-12%, 3-6%, 30-50% and 20-30%.
The purposes of Diffuse Coptosapelta flower Flavonoids, described Diffuse Coptosapelta flower Flavonoids has antioxidant activity, has the purposes removing ultra-oxygen anion free radical, DPPH free radical, hydroxyl radical free radical and anti-peroxidation.
The beneficial effect that the present invention brings is:
1, the present invention the mode of ethanol extraction, macroporous adsorbent resin column chromatography, silica gel column chromatography, gel column chromatography and half preparative high-performance liquid chromatographic coupling is come extraction separation and purification Diffuse Coptosapelta spend in Flavonoids, DNA purity is high, impurity content is few, and after separation and purification, the content of Flavonoids is greater than 90%;
2, sample after purification uses high performance liquid chromatography to detect, accurately define material composition and proportion of composing that Diffuse Coptosapelta spends middle Flavonoids concrete, the Flavonoids definite ingredients obtained, quality controllable, record effective ingredient wherein and mass percentage is (1): Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides (5-8 %), (2): Eriodictyol-7-O-β-D-Glucose glycosides (8-12%), (3): ampelopsin-3-O-β-D-Glucose glycosides (3-6 %), (4): luteolin-7-O-β-D-Glucose glycosides (30-50%) and (5): kaempferol-3-O-β-D-Glucose glycosides (20-30%),
3, the macroporous adsorbent resin used can utilize by repeated regeneration, and extracted amount is large, the simple feature of technique to adopt organic solvent ethanol extraction to have, and with water and ethanol as solvent elution, economical and convenient, is easy to industrialization, very little to harm effect;
4, extract the Diffuse Coptosapelta flower Flavonoids obtained and there is excellent antioxidant activity, have and remove ultra-oxygen anion free radical, DPPH free radical, hydroxyl radical free radical ability and anti-peroxidation ability significantly.
Accompanying drawing explanation
Fig. 1 is the chemical structural formula of Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides;
Fig. 2 is the chemical structural formula of Eriodictyol-7-O-β-D-Glucose glycosides;
Fig. 3 is the chemical structural formula of ampelopsin-3-O-β-D-Glucose glycosides;
Fig. 4 is the chemical structural formula of luteolin-7-O-β-D-Glucose glycosides;
Fig. 5 is the chemical structural formula of kaempferol-3-O-β-D-Glucose glycosides;
Fig. 6 is the HPLC spectrogram of Flavonoids reference substance;
Fig. 7 is the HPLC spectrogram of the embodiment of the present invention 1 gained Diffuse Coptosapelta flower Flavonoids;
Fig. 8 is the HPLC spectrogram of the embodiment of the present invention 2 gained Diffuse Coptosapelta flower Flavonoids;
Fig. 9 is the HPLC spectrogram of the embodiment of the present invention 3 gained Diffuse Coptosapelta flower Flavonoids.
In Fig. 6-9 1,2,3,4 and 5 represents Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides respectively.
Detailed description of the invention
diffuse Coptosapelta spends the determination of middle flavone compound composition and structure
(1) Diffuse Coptosapelta spend in the extraction of each flavone compound and separation and purification
The Diffuse Coptosapelta of getting natural drying spends 10 kilograms, pulverizes, and after crossing 40 mesh sieves, extracts 3 times, each 6 days by 95 % soak with ethanol, collects the extracting solution at every turn obtained.By extracting solution concentrating under reduced pressure at lower than the temperature of 60 DEG C, obtain total extractum 3200 grams.Getting gained extractum 3000 grams is suspended in 20 L distilled water, and repeatedly extract with petroleum ether, ethyl acetate, n-butyl alcohol successively, often kind of extract consumption is 50 L, and extract is carried out concentrating under reduced pressure respectively.Obtain petroleum ether extract 2.3 grams, acetic acid ethyl ester extract 107 grams, n-butyl alcohol extract 1500 grams.
The n-butyl alcohol extract obtained is separated through silica gel column chromatography (1200 mm × 70 mm) and obtains 52 fractions.13rd section of fraction and fraction 13 carry out eluting with chloroform-methanol (volume ratio is for 2: 1) again, to get back 15 fractions, fraction 8 wherein obtains compound 3(100.3 mg through recrystallization), after recrystallization, remaining mother solution obtains compound 2(84.5 mg through PTLC); Fraction 19-22 uses chromatographic column (1200 mm × 40 mm) to be separated, with chloroform-methanol (9: 1,7: 1 after merging again, 5: 1,3: 1,2: 1,1: 1, volume ratio) carry out gradient elution, to get back 21 fractions, through semi-preparative liquid chromatography (20 mm × 250 mm, 5 μm after fraction 8-11 is wherein merged, methanol: water=40: 60, volume ratio) be separated obtain compound 1(12.1 mg).
Silicagel column (1200 mm × 70 mm) on the acetic acid ethyl ester extract obtained, with the petroleum ether-ethyl acetate (4: 1-0: 1 of variable concentrations, volume ratio) carry out gradient elution, every 500 mL of eluent receive 1 part, obtain 43 fractions with TLC detection, merging.The 36th section of fraction wherein and fraction 36 chromatographic column (1200 mm × 35 mm) are separated, with chloroform-methanol (9: 1-5: 1, volume ratio) carry out gradient elution, finally obtain compound 4 (85.3 mg) and compound 5(451 mg in conjunction with Sephadex LH-20 chromatograph (1200 mm × 40 mm) purification).
(2) Structural Identification of the flavone compound obtained is extracted
Compound 1 is Yellow amorphous powder, mp 181 ~ 183 DEG C, and the reaction of hydrochloric acid magnesium powder is positive, and molish reaction is positive, and after hydrochloric acid hydrolysis, paper chromatography detects glucose sugar and arabinose.ESI-MS
m/z: 593 [M-H]
-(molecular formula is C
27h
30o
15).
1H NMR (400MHz，DMSO-d
6) δ：12.72 (5-OH)， 7.61 (1H，d，
J = 2.0 Hz，2'-H)，7.49 (1H，dd，
J= 8.4 Hz，2.0 Hz，6'-H)，6.95 (1H， d，
J=8.4 Hz，5'-H)，6.76 (1H，d，
J= 2.0 Hz，8-H)，6.46 (1H，d，
J= 2.0 Hz， 6-H)，5.11 (1H，d，
J= 7.6 Hz，1"-H)，4.74 (1H，d，
J= 2.0 Hz，1"'-H)，3.81 (3H， s，3-OCH
3)。
13C NMR (100 MHz，DMSO-d
6) δ：156.2(C-2)，138.1(C-3)，178.3 (C-4)，161.2 (C-5)，99.4 (C-6)，163.1(C-7)，94.6(C-8)，156.2(C-9)，106.1(C-10)，121.0(C-1')，115.9(C-2')，145.4(C-3')，1491(C-4')，116.1(C-5')，120.9(C-6')， 100.0(C-1'')，73.3(C-2")，76.4(C-3")，70.0 (C-4")，75.7(C-5")，67.1(C-6")， 108.7(C-1''')，82.23(C-2"')，77.4(C-3"')，84.2 (C-4"')，61.5(C-5"')。Above data and document [Choi Y H, Lim Y H, Yeo H, et al. A flavonoid diglycoside from
lepisorus ussuriensis. Phytochemistry, 1996, that 43 (5): 1111-1114] reports is basically identical, and therefore authenticating compound 1 is Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, and structural formula as shown in Figure 1.
Compound 2 is yellow crystals (in methanol, crystallization obtains).Mp 156 ~ 158 DEG C, the reaction of hydrochloric acid magnesium powder is positive, and molish reaction is positive, and after hydrochloric acid hydrolysis, paper chromatography detects glucose sugar.ESI-MS
m/z: 449 [M-H]
-(molecular formula is C
21h
22o
11).
1H-NMR(DMSO-d
6，400 MHz) δ：6.91(1H，s， H-5')，6.72(2H，s，H-2'，6')，6.19(1H，s，H-6)，6.1(1H，s，H-8)，5.33(1H，dd，H-2)，2.76(1H，dd，
J=17.1 Hz，H-3)，4.98(1H，d，glc-1')。
13C-NMR(100 MHz，DMSO-d
6) δ：80.7(C-2)，44.1(C-3)，198.5(C-4)，164.9(C-5)，97.9(C-6)， 167.0(C-7)，96.9(C-8)，164.5(C-9)，103.75(C-10)，131.5(C-1')，116.2(C-2')， 146.9(C-3')，114.7(C-4')，119.3(C-5')，104.9(C-6')，101.2(C-1")，74.6(C-2")， 77.8(C-3")，71.1(C-4")，78.2(C-5")，62.3(C-6")。Above data and document [Zhao Lei, Wu Dinghui, Yu Xiaohui, Deng. the flavanone constituents [J] in tasteful and refined Cuculus polioephalus. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2010,35 (6): 722-724] relatively more consistent, therefore authenticating compound 2 is Eriodictyol-7-O-β-D-Glucose glycosides, and structural formula as shown in Figure 2.
Compound 3 is yellow powder (in methanol, crystallization obtains), mp 193 ~ 195 DEG C.The reaction of hydrochloric acid magnesium powder is positive, and molish reaction is positive, and after hydrochloric acid hydrolysis, paper chromatography detects glucose.ESI-MS
m/z: 479 [M-H]
-(molecular formula is C
21h
20o
13).
1H-NMR(DMSO-d
6，400 MHz) δ：12.63(1H，s， 5-OH)，8.68(1H，s，3'-OH)，7.81(1H，s，4'-OH)，7.23 (1H，s，H-2'，6')，6.37 (1H，d，
J=1.9 Hz，H-8)，6.21 (1H，d，
J=1.9 Hz，H-6)，5.47 (1H，d，
J=7.6 Hz，H-1'')。
13C-NMR(DMSO-d
6，100 MHz) δ：156.4 (C-2)，133.4 (C-3)，177.4 (C-4)，161.3 (C-5)，98.9 (C-6)，164.6 (C-7)，93.5(C-8)，156.2(C-9)，103.7(C-10)，120.1 (C-1')，108.6 (C-2')，145.5(C-3')，136.7 (C-4')，145.5(C-5')，108.6 (C-6')，100.9 (C-1'')，73.9 (C-2")，76.6 (C-3")，69.8 (C-4")，77.6 (C-5")，61.1 (C-6")。Above data and bibliographical information basically identical [Zhao Liqin, Zhang Xiaoping, Zhang Chaofeng. the isolation identification [J] of Radix Polygoni aubertii chemical composition, Food Science, 2012,33 (7): 1-5; Li Chunmei, Wang Tao; a Yi, Gao Xiumei, Deng. Isolation and ldentification (I) [J] of Chinese medicine Flos abelmoschi manihot chemical composition. Shenyang Pharmaceutical University's journal, 2010,27 (9): 711-714], authenticating compound 3 is ampelopsin-3-O-β-D-Glucose glycosides, and structural formula as shown in Figure 3.
Compound 4 is yellow powder (in methanol, crystallization obtains), mp 259 ~ 261 DEG C.The reaction of hydrochloric acid magnesium powder is positive, and molish reaction is positive, and after hydrochloric acid hydrolysis, paper chromatography detects glucose sugar.ESI-MS
m/z: 447 [M-H]
-(molecular formula is C
21h
20o
11).
1H-NMR(Pyridine-d
5，400 MHz) δ：12.11(1H，s， 5-OH)，8.71(1H，s，3'-OH)，7.89(1H，s，4'-OH)，7.49(1H，dd，
J=8.7，1.7 Hz， H-6')，7.27(1H，d，
J=1.7 Hz，H-2')，6.98(1H，d，
J=8.7 Hz，H-5')，6.92(1H， s，H-3)，6.83(1H，d，
J=1.9 Hz，H-8)，5.82(1H，d，
J=1.9 Hz，H-6)。
13C-NMR(Pyridine-d
5，100 MHz) δ：164.0 (C-2)，101.8(C-3)，182.8 (C-4)，157.9 (C-5)，100.6(C-6)，165.3(C-7)，95.3(C-8)，162.6 (C-9)，104.1(C-10)，122.7(C-1')， 113.30(C-2')，147.8(C-3')，150.1(C-4')，116.9 (C-5')，119.7 (C-6')，95.3(C-1")， 79.2(C-2")，78.5(C-3")，74.8(C-4")，71.1(C-5")，62.3(C-6")。Above data and bibliographical information basically identical [dragon flies, Deng Liang, Chen Yang. the chemical constitution study [J] of Ligustrum japonicum Thunb.flower. West China pharmaceutical journal, 2011,26 (2): 97-100], authenticating compound
4for luteolin-7-O-β-D-Glucose glycosides, structural formula as shown in Figure 4.
Compound 5 is yellow powder (in methanol, crystallization obtains).mp 229~231℃。The reaction of hydrochloric acid magnesium powder is positive, and molish reaction is positive, and after hydrochloric acid hydrolysis, paper chromatography detects glucose sugar.ESI-MS
m/z: 447 [M-H]
-(molecular formula is C
21h
20o
11).
1H-NMR(DMSO-d
6，400 MHz) δ：6.2(1H， d，
J=2.0 Hz)，6.43(1H，d，
J=2.0 Hz)，8.07(1H，dd，
J=2.0 Hz，7.2 Hz)，6.86(1H，dd，
J=2.0 Hz，7.0 Hz)， 6.86(1H，dd，
J=2.0 Hz，7.0 Hz)，8.07(1H，dd，
J=2.0 Hz， 7.2 Hz)，5.35(1H，d，
J=7.6 Hz)，12.61(1H，s)，10.87(1H，s)。
13C-NMR(DMSO-d
6， 100 MHz) δ：156.3(C-2)，133.1(C-3)，177.4(C-4)，161.1(C-5)，98.71(C-6)， 164.3(C-7)，93.65 (C-8)，156.1(C-9)，103.8(C-10)，120.8(C-1')，130.8(C-2')， 115.0(C-3')，159.9 (C-4')，115.0(C-5')，130.9(C-6')，100.8(C-1'')，74.17(C-2")， 76.36(C-3")，69.83(C-4")，77.46 (C-5")，60.77(C-6")。Above data and bibliographical information basically identical [dragon flies, Deng Liang, Chen Yang. the chemical constitution study [J] of Ligustrum japonicum Thunb.flower. West China pharmaceutical journal, 2011,26 (2): 97-100], authenticating compound 5 is kaempferol-3-O-β-D-Glucose glycosides, and structural formula as shown in Figure 5.
embodiment 1
Get the western Funiu Shan Mountain Diffuse Coptosapelta in the Henan after drying and crushing and spend 1 kilogram, add 12 kilograms of mass concentration 60% soak with ethanol after 8 hours, heating and refluxing extraction 4 hours, filtration obtains filtrate and residue, residue adds 10 kilogram of 80% alcohol reflux 2 times again, each 4 hours, merging filtrate, reduction vaporization concentrates, and reclaims ethanol and obtains extractum 126 grams.Ethanol extract is used 10% dissolve with ethanol, filter, filtrate is fully adsorbed with 1000 grams of D101 type macroporous adsorbent resins, dress post, with alcohol-water (10%, 20%, 30%, 40%, 50%, 60%, 80% and 95%) gradient elution, collect 50-60% ethanol elution fraction, be evaporated to leaching paste, by dry for extractum vacuum decompression at 50 DEG C, obtain 10 grams, dry total flavones powder.Sample after being separated by macroporous adsorbent resin column chromatography adopts silica gel column chromatography loading to be separated, with CHCl
3: CH
3oH volume ratio is 1:9,1:7,1:5,1:3 is eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects the fraction section being greater than 60% containing Diffuse Coptosapelta flower Flavonoids, sample after being separated by silica gel column chromatography adopts gel column chromatography to be separated, and adopts CHCl
3: CH
3oH volume ratio is 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, dry, sample after gel column chromatography being separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta spends Flavonoids, chromatographic condition is permaphase ODS-A, 20 × 250 mm, and mobile phase is CH
3oH:H
2o, volume ratio is 7:3, finally adopt high performance liquid chromatography to detect in the sample after purification, chromatographic condition is: chromatograph: Agilent1100 high performance liquid chromatograph, chromatographic column: Agilent Zorbax SB C
18chromatographic column (4.6 × 250 mm, 5 μm), mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60, flow velocity: 1.0 mL/min, column temperature: 25 DEG C, determined wavelength: 350 nm, high performance liquid chromatography records wherein Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, the mass percentage of luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides flavone compound is respectively 6.2%, 10.1%, 5.2%, 46.5%, 25.2%(Fig. 1, Fig. 2, Fig. 3, Fig. 4 and Fig. 5 is the chemical structural formula of these 5 kinds of chromocor compounds respectively), general flavone content is greater than 93.2%, as shown in Figure 7, Fig. 6 is by Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, luteolin-7-O-β-D-Glucose glycosides and these 5 kinds of compounds of kaempferol-3-O-β-D-Glucose glycosides are mixed with mixture product in contrast according to finite concentration, test the high-efficient liquid phase chromatogram of the reference substance obtained.
embodiment 2
Get the western Funiu Shan Mountain Diffuse Coptosapelta in the Henan after drying and crushing and spend 2 kilograms, add 22 kilograms of mass concentration 70% soak with ethanol after 11 hours, heating and refluxing extraction 5 hours, filtration obtains filtrate and residue, residue adds 18 kilogram of 70% alcohol reflux 2 times again, each 5 hours, merging filtrate, reduction vaporization concentrates, and reclaims ethanol and obtains extractum 284 grams.Ethanol extract is used 20% dissolve with ethanol, filter, filtrate is fully adsorbed with 2000 grams of AB-8 type macroporous adsorbent resins, dress post, with alcohol-water (10%, 20%, 30%, 40%, 50%, 60%, 80% and 95%) gradient elution, collect 50-60% ethanol elution fraction, be evaporated to leaching paste, by dry for extractum vacuum decompression at 60 DEG C, obtain 18.6 grams, dry total flavones powder.Sample after being separated by macroporous adsorbent resin column chromatography adopts silica gel column chromatography loading to be separated, with CHCl
3: CH
3oH volume ratio is 1:9,1:7,1:5,1:3 is eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects the fraction section being greater than 60% containing Diffuse Coptosapelta flower Flavonoids, sample after being separated by silica gel column chromatography adopts gel column chromatography to be separated, and adopts CHCl
3: CH
3oH volume ratio is 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, dry, sample after gel column chromatography being separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta spends Flavonoids, chromatographic condition is permaphase ODS-A, 20 × 250 mm, and mobile phase is CH
3oH:H
2o, volume ratio is 7:3, finally adopt high performance liquid chromatography to detect in the sample after purification, chromatographic condition is: chromatograph: Agilent1100 high performance liquid chromatograph, chromatographic column: Agilent Zorbax SB C
18chromatographic column (4.6 × 250 mm, 5 μm), mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60, flow velocity: 1.0 mL/min, column temperature: 25 DEG C, determined wavelength: 350 nm, high performance liquid chromatography records wherein Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, the mass percentage of luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides flavone compound is respectively 8.0%, 9.1%, 5.0%, 44.1%, 27.2%, general flavone content is greater than 93.4%, as shown in Figure 8.
embodiment 3
Get the western Funiu Shan Mountain Diffuse Coptosapelta in the Henan after drying and crushing and spend 4 kilograms, add 40 kilograms of mass concentration 80% soak with ethanol after 12 hours, heating and refluxing extraction 3 hours, filtration obtains filtrate and residue, residue adds 32 kilogram of 60% alcohol reflux 2 times again, each 3 hours, merging filtrate, reduction vaporization concentrates, and reclaims ethanol and obtains extractum 605 grams.Ethanol extract is used 30% dissolve with ethanol, filter, filtrate is fully adsorbed with 5000 grams of D101 type macroporous adsorbent resins, dress post, with alcohol-water (10%, 20%, 30%, 40%, 50%, 60%, 80% and 95%) gradient elution, collect 50-60% ethanol elution fraction, be evaporated to leaching paste, by dry for extractum vacuum decompression at 55 DEG C, obtain 37.6 grams, dry total flavones powder.Sample after being separated by macroporous adsorbent resin column chromatography adopts silica gel column chromatography loading to be separated, with CHCl
3: CH
3oH volume ratio is 1:9,1:7,1:5,1:3 is eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects the fraction section being greater than 60% containing Diffuse Coptosapelta flower Flavonoids, sample after being separated by silica gel column chromatography adopts gel column chromatography to be separated, and adopts CHCl
3: CH
3oH volume ratio is 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, dry, sample after gel column chromatography being separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta spends Flavonoids, chromatographic condition is permaphase ODS-A, 20 × 250mm, and mobile phase is CH
3oH:H
2o, volume ratio is 7:3, finally adopt high performance liquid chromatography to detect in the sample after purification, chromatographic condition is: chromatograph: Agilent1100 high performance liquid chromatograph, chromatographic column: Agilent Zorbax SB C
18chromatographic column (4.6 × 250 mm, 5 μm), mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60, flow velocity: 1.0 mL/min, column temperature: 25 DEG C, determined wavelength: 350 nm, high performance liquid chromatography records wherein Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, the mass percentage of luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides flavone compound is respectively 6.9%, 11.3%, 4.9 %, 45.1%, 22.2%, general flavone content is greater than 90.4%, as shown in Figure 9.
pharmacological research
Test material:
(mass percentage of Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides is respectively 8.0% to Diffuse Coptosapelta flower total flavones product, 9.1%, 5.0%, 44.1%, 27.2%, general flavone content is greater than 93.4%.Preparation method is shown in embodiment 2).
Sodium nitrite, aluminum nitrate, sodium hydroxide, 1,1-diphenyl-2-picryl phenylhydrazine (DPPH), nicotinamide adenine dinucleotide reduced (NADH), azophenlyene methosulfate (PMS), chlorination nitro tetrazole (NBT), dehydrated alcohol, hydrogen peroxide, ascorbic acid (VC), salicylic acid, Tris (Tris), trichloroacetic acid (TCA), thiobarbituricacidα-(TBA), hydrochloric acid (HCl) are analytical pure, soybean lecithin (>90%).
Assay method:
(1) ultra-oxygen anion free radical (O is removed
- 2) assay method
With pH8.0, Diffuse Coptosapelta flower total flavones product is diluted to different Concentraton gradient by the Tris-HCl buffer of concentration 50 mol/L, respectively get the different gradient flavonoids solution of 1.5 mL, then 0.5 mL NBT(300 μm ol/L is added successively, Tris-HCl buffer with pH8.0), 0.5 mL NADH(468 μm ol/L, Tris-HCl buffer with pH8.0), 0.5 mL PMS(60 μm ol/L, Tris-HCl buffer with pH8.0), water-bath 5 minutes in 25 DEG C after mixing, take out and measure absorbance at wavelength 560 nm place, blank group replaces flavonoids solution with buffer.The formula calculating ultra-oxygen anion free radical clearance rate is as follows:
E(O
- 2·)(%)=(1－A
1/A
0)×100
In formula: E (O
- 2) for flavone is to O
- 2clearance rate (%); A
1for flavone absorbance; A
0for blank absorbency.
(2) assay method of DPPH free radical is removed
Diffuse Coptosapelta flower total flavones product is diluted to different Concentraton gradient, respectively get the flavonoids solution of the above-mentioned concentration of 2 mL in test tube, add the DPPH solution that 2 mL concentration are 0.04 mg/mL again, mix homogeneously, react 20 minutes, 3500 r/min centrifugalize 10 minutes, getting supernatant at wavelength 517 nm place its absorbance of survey is A
i; Separately respectively get the flavonoids solution of the above-mentioned concentration of 2 mL in test tube, add dehydrated alcohol 2 mL respectively, react 20 minutes, 3500 r/min centrifugalize 10 minutes, getting supernatant at wavelength 517 nm place its absorbance of survey is A
j; React as reference using 2 mL 0.04 mg/mL DPPH and 2 mL dehydrated alcohol, its absorbance is designated as A
0.The formula calculating DPPH free radical scavenging activity is as follows:
E(DPPH)(%)=[1－(A
i-A
j)/A
0]×100
In formula: E (DPPH) is for flavone is to the clearance rate (%) of DPPH free radical; A
0it is the absorbance of 2 mL DPPH solution+2 mL dehydrated alcohol; A
iit is the absorbance of 2 mL DPPH solution+2 mL flavonoids solutions; A
jit is the absorbance of 2 mL dehydrated alcohol+2 mL flavone.
(3) assay method of hydroxyl radical free radical (OH) is removed
Diffuse Coptosapelta flower total flavones product distilled water is mixed with variable concentrations gradient, respectively gets the flavonoids solution of the above-mentioned concentration of 2mL, add the FeSO of 2 mL 6 mmol/L successively
4, 2 mL 6mmol/L H
2o
2, leave standstill 10 minutes after mixing, then add 2 mL 6 mmol/L salicylic acid, mixing, leave standstill 30 minutes, survey its absorbance at wavelength 510 nm place and be designated as A
i, when replacing absorbance during salicylic acid to be designated as A with distilled water
j.Blank group replaces flavonoids solution absorbance to be designated as A with distilled water
0.The formula calculating hydroxyl radical free radical clearance rate is as follows:
E(·OH)(%)= [1－(A
i-A
j)/A
0]×100
In formula: the clearance rate (%) that E (OH) is hydroxyl radical free radical; A
ifor the absorbance of Diffuse Coptosapelta flower total flavones product; A
0for blank absorbency; A
jthe absorbance of Diffuse Coptosapelta flower total flavones product when reacting for participating in without salicylic acid.
(4) mensuration of anti-peroxidation activity
Diffuse Coptosapelta flower total flavones product solution 1.00 mL of different quality concentration is added, lecithin soln 1.0 mL, 0.4 mmol/L FeSO in 10 mL color comparison tubes
41.0 mL, mixing, place 60 minutes in 37 DEG C of water-baths, add trichloroacetic acid (TCA)-thiobarbituricacidα-(TBA)-hydrochloric acid (HC1) again and mix 2.0 mL, 100 DEG C of water-baths cool after 15 minutes rapidly, with 3000 r/min centrifugal 10 minutes, get supernatant and measure absorbance at wavelength 532 nm place, make reference with distilled water, VC is contrast, calculates suppression ratio.
Suppression ratio/%=[1-(A
x-A
x0)/A
0] × 100
In formula: A
0for the absorbance of blank liquid; A
xfor adding the absorbance after flavonoids solution; A
x0for not adding the flavonoids solution absorbance of lecithin.
Result:
(1) ultra-oxygen anion free radical (O is removed
2 -)
| Total flavones concentration (mg/mL) | 0.01 | 0.02 | 0.04 | 0.08 | 0.16 |
| VC clearance rate | 25% | 29% | 37% | 51% | 83% |
| Total ketone clearance rate | 17% | 19% | 26% | 39% | 53% |
Within the scope of selected mass concentration, Diffuse Coptosapelta flower total flavones has the ability removing ultra-oxygen anion free radical.Along with the increase of Diffuse Coptosapelta flower total flavones mass concentration, the scavenging action of ultra-oxygen anion free radical is strengthened, presents dose-effect relationship.Although the removing O of Diffuse Coptosapelta flower total flavones
- 2effect is strong not as VC, but it has shown very strong removing O
- 2effect.
(2) DPPH free radical is removed
| Total flavones concentration (μ g/mL) | 5 | 10 | 15 | 20 | 25 |
| VC clearance rate | 46% | 73% | 87% | 90% | 91% |
| Total ketone clearance rate | 41% | 57% | 71% | 80% | 83% |
DPPH free radical is a kind of very stable free radical centered by nitrogen, shows that test medicine has the valid density reducing hydroxyl radical free radical, alkane free radical or peroxy radical and the effect interrupting lipid peroxidation chain reaction to its effect removed.Although it is strong not as VC that Diffuse Coptosapelta flower total flavones removes DPPH free radical effect, Diffuse Coptosapelta flower total flavones still shows the ability of very strong removing DPPH free radical.
(3) hydroxyl radical free radical (OH) is removed
| Total flavones concentration (mg/mL) | 0.05 | 0.1 | 0.2 | 0.4 | 0.8 |
| VC clearance rate | 45% | 49% | 66% | 74% | 92% |
| Total ketone clearance rate | 21% | 34% | 47% | 56% | 71% |
Hydroxyl radical free radical is the strongest oxidant, almost can react with all cells composition in vivo, very large to body harm.Diffuse Coptosapelta flower total flavones is removed OH effect and is compared with VC, and Diffuse Coptosapelta flower total flavones has the ability removing OH, but its effect is more weak.
(4) anti-peroxidation is active
| Total flavones concentration (μ g/mL) | 50 | 100 | 150 | 200 | 250 |
| VC clearance rate | Nothing | Nothing | Nothing | Nothing | Nothing |
| Total ketone clearance rate | 39% | 47% | 51% | 60% | 63% |
Lipid peroxidation injury is relevant with the generation of numerous disease, as tumor, aging, cardiovascular and cerebrovascular vessel, autoimmune disease etc.Diffuse Coptosapelta flower total flavones is to Fe
2+the lecithin liposome peroxidating caused has inhibitory action, along with the increase suppression ratio of flavone mass concentration strengthens.In quality of experiments concentration range, not measuring VC has anti-peroxidation ability, may be because VC belongs to watersoluble polar material, mainly concentrate on aqueous phase, thus affect its inhibitory action in emulsified fatty substance system.
Claims (1)
1. a preparation method for Diffuse Coptosapelta flower Flavonoids, is characterized in that: comprise the following steps:
(1) alcoholic solution extracts:
By Diffuse Coptosapelta flower medicinal material drying, pulverizing, adding mass concentration is after 60-80% soak with ethanol 8-12 hour, heating and refluxing extraction 3-5 hour, filtration obtains filtrate and residue, be that 60-80% alcohol heating reflux extracts 2 times again with mass concentration by residue, each 3-5 hour, merging filtrate, filtrate evaporated under reduced pressure is concentrated to obtain extractum, during the 1st reflux, the addition of ethanol be the 10-12 of Diffuse Coptosapelta flower quality of medicinal material doubly, the 2nd time and the 3rd reflux time, the addition of ethanol be the 8-10 of Diffuse Coptosapelta flower quality of medicinal material doubly;
(2) macroporous adsorbent resin column chromatography is separated:
Be 10-30% dissolve with ethanol by the extractum mass concentration obtained, filter, filtrate D101 or AB-8 type macroporous adsorbent resin are fully adsorbed, dress post, carry out gradient elution with the ethanol-water system eluent that volumetric concentration is 10%, 20%, 30%, 40%, 50%, 60%, 80% and 95% successively, collecting ethanol contend concentration is 50-60% alcohol-water eluting fraction, is evaporated to leaching paste, by dry for extractum vacuum decompression at 50-60 DEG C, obtain Diffuse Coptosapelta flower Flavonoids crude product;
(3) silica gel column chromatography is separated:
Silica gel column chromatography loading is adopted to be separated, successively with CHCl Diffuse Coptosapelta flower Flavonoids crude product
3: CH
3oH volume ratio is 1:9,1:7,1:5,1:3 is eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects the fraction section being greater than 60% containing Diffuse Coptosapelta flower Flavonoids;
(4) gel column chromatography is separated:
Sample after being separated by silica gel column chromatography adopts gel column chromatography to be separated, and adopts CHCl
3: CH
3oH volume ratio is 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, dry;
(5) half preparative high-performance liquid chromatographic purification:
Sample after gel column chromatography being separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta spends Flavonoids, chromatographic condition is permaphase ODS-A, 20 × 250mm, and mobile phase is CH
3oH:H
2o, volume ratio is 7:3;
(6) high performance liquid chromatography detects:
Finally adopt high performance liquid chromatography to detect in the sample after purification, chromatographic condition is:
Chromatograph: Agilent1100 high performance liquid chromatograph,
Chromatographic column: Agilent Zorbax SB C
18chromatographic column, 4.6 × 250 mm, 5 μm,
Mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60,
Flow velocity: 1.0 mL/min,
Column temperature: 25 DEG C,
Determined wavelength: 350 nm;
The Diffuse Coptosapelta flower Flavonoids adopting said method to prepare, be made up of Quercetin-3-methoxyl group-7-O-α-L-Arab (1-6)-β-D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides, mass percentage is respectively 5-8%, 8-12%, 3-6%, 30-50% and 20-30%, and above-mentioned total mass percentage is 100%.
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| CN101475619A (en) * | 2009-02-09 | 2009-07-08 | 沈阳药科大学 | Preparation and use of Linaria vulgaris flavone and total flavone thereof |
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| CN101475619A (en) * | 2009-02-09 | 2009-07-08 | 沈阳药科大学 | Preparation and use of Linaria vulgaris flavone and total flavone thereof |
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