CN103169727A - 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 Effective Component of Chinese Medicine and extract separation and application technology, relate in particular 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, has another name called HUAMU, Radix Raphani silk flower, oily root, Niu Jingzi, April snow.Diffuse Coptosapelta mainly be 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, record in " China's economic flora ", and bud, the leaf of Oleaceae plants chionanthus retusa Lindl et Paxt have the effect of relieving heat and thirst, and cure mainly heatstroke.Tender leaf and the flower had in sampled-current Soviet Union in spring among the people, make the history that tea-drinking is used, and the tea of making of the Diffuse Coptosapelta flower is called " Oryza glutinosa scented tea ", pleasant to the palate, some local flavor of other tool [Hu Shicai. the brewed method of good beverage plant-chionanthus retusa Lindl et Paxt and branch and leaf thereof [J]. forestry science and technology exploitation, 1991,3:16; Fang Shaokun, Du Qingzhi, Zheng's winter. the breeding and apply [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: " upper person gives birth to rotten stone, the living oak earth of middle person, lower person's SHENGHUANG soil.Wild Zhe Shang， garden person time "." Oryza glutinosa scented tea " spontaneous growth, include the useful thing of health abundant, vitamin, aminoacid, catechu polyphenol, caffeine, teatannin content are all higher, but drink this tea removing food stagnancy food, clear endogenous fire, sterilization, control dysentery, pleasant dredging collateral, the diuresis of relieving summer heat, go greasy blood pressure lowering, allaying tiredness, fat-reducing skin protection.Tea grounds can be controlled gastropathy and infantile diarrhea, has medical value.The Diffuse Coptosapelta flower just receives increasing concern because of its special local flavor and function as a kind of tea raw material among the people of preciousness.In existing research, clear and definite Diffuse Coptosapelta contains more flavone compound in spending, and proves that these materials have antioxidation preferably.But only extracted and measured the wherein content of Flavonoids, the purity of extraction is not high, for how measuring wherein concrete contained material and proportion of composing indefinite, how also indefinite the defying age of Flavonoids and the effect of antioxidation be; And use the more higher toxic solvent of cost in the preparation method of Flavonoids, not only bad for suitability for industrialized production and to the health of human body, harm is arranged.
Summary of the invention
The technical problem to be solved in the present invention is, Diffuse Coptosapelta flower Flavonoids of definite ingredients and its preparation method and application is provided, make the preparation method economical and convenient, be easy to suitability for industrialized production, the Diffuse Coptosapelta flower Flavonoids product purity that extraction obtains is high, concrete material proportion of composing is clear and definite, and the antioxidation effect of Flavonoids is clear and definite.
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:
Diffuse Coptosapelta flower pesticide material is dry, pulverizing, after adding mass concentration to be the 60-80% soak with ethanol, heating and refluxing extraction 3-5 hour, filtration obtains filtrate and residue, again residue be take to mass concentration as 60 ~ 80% alcohol heating reflux extraction 2 times, each 3-5 hour, merging filtrate, concentrate to obtain extractum by filtrate evaporated under reduced pressure;
(2) macroporous adsorbent resin column chromatography separates:
By the extractum mass concentration obtained, it is the 10-30% dissolve with ethanol, filter, filtrate is fully adsorbed with macroporous adsorbent resin, the dress post, the ethanol-water system eluent that is 10%, 20%, 30%, 40%, 50%, 60%, 80% and 95% by volumetric concentration successively carries out gradient elution, and collecting the ethanol volumetric concentration is 50-60% alcohol-water eluting fraction, is evaporated to and soaks paste, extractum, 50-60 ℃ of lower vacuum decompression drying, is obtained to Diffuse Coptosapelta flower Flavonoids crude product;
(3) silica gel column chromatography separates:
Adopt the silica gel column chromatography loading to separate Diffuse Coptosapelta flower Flavonoids crude product, successively with CHCl
3: CH
3the OH volume ratio is 1:9,1:7, and 1:5,1:3 is the eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects and contains the fraction section that Diffuse Coptosapelta flower Flavonoids is greater than 60%;
(4) gel column chromatography separates:
Sample after silica gel column chromatography is separated adopts gel column chromatography to separate, and adopts CHCl
3: CH
3the OH volume ratio is the 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, drying;
(5) half preparative high-performance liquid chromatographic purification:
Sample after gel column chromatography is separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta is spent Flavonoids, chromatographic condition is permaphase ODS-A, 20 * 250mm, 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: the Agilent1100 high performance liquid chromatograph,
Chromatographic column: Agilent Zorbax SB C
18chromatographic column, 4.6 * 250 mm, 5 μ m,
Mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60,
Flow velocity: 1.0 mL/min,
Column temperature: 25 ℃,
Detect 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 10-12 that the addition of ethanol is Diffuse Coptosapelta flower quality of medicinal material doubly, when the 2nd time and the 3rd reflux, the addition of ethanol be Diffuse Coptosapelta flower quality of medicinal material 8-10 doubly.
Macroporous adsorbent resin in described step (2) is D101 or AB-8 type macroporous adsorbent resin.
Diffuse Coptosapelta flower Flavonoids, Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--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-5 kinds of D-Glucose glycosides chromocor compound, consist of, the quality percentage composition 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 of 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 carrys out the Flavonoids of extraction separation and purification Diffuse Coptosapelta in spending by 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, 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 is used high performance liquid chromatography to detect, accurately determined that Diffuse Coptosapelta spends the concrete material composition of middle Flavonoids and proportion of composing, resulting Flavonoids definite ingredients, quality controllable, the effective ingredient and the quality percentage composition that record wherein are (1): Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--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 the repeated regeneration utilization, adopts the organic solvent ethanol extraction to have that extracted amount is large, the simple characteristics of technique, and water and ethanol is as solvent elution, economical and convenient, is easy to industrialization, very little to the harm effect;
4, extract the Diffuse Coptosapelta flower Flavonoids obtained and there is excellent antioxidant activity, there is significant removing ultra-oxygen anion free radical, DPPH free radical, hydroxyl radical free radical ability and anti-peroxidation ability.
The accompanying drawing explanation
The chemical structural formula that Fig. 1 is Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--D-Glucose glycosides;
The chemical structural formula that Fig. 2 is Eriodictyol-7-O-β-D-Glucose glycosides;
The chemical structural formula that Fig. 3 is ampelopsin-3-O-β-D-Glucose glycosides;
The chemical structural formula that Fig. 4 is luteolin-7-O-β-D-Glucose glycosides;
The chemical structural formula that Fig. 5 is kaempferol-3-O-β-D-Glucose glycosides;
The HPLC spectrogram that Fig. 6 is the Flavonoids reference substance;
The HPLC spectrogram that Fig. 7 is the embodiment of the present invention 1 gained Diffuse Coptosapelta flower Flavonoids;
The HPLC spectrogram that Fig. 8 is the embodiment of the present invention 2 gained Diffuse Coptosapelta flower Flavonoids;
The HPLC spectrogram that Fig. 9 is the embodiment of the present invention 3 gained Diffuse Coptosapelta flower Flavonoids.
In Fig. 6-9 1,2,3,4 and 5 represent respectively Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--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.
The specific embodiment
diffuse Coptosapelta is spent determining of middle flavone compound composition and structure
(1) Diffuse Coptosapelta spend in extraction and the separation and purification of each flavone compound
Take from the Diffuse Coptosapelta of right drying and spend 10 kilograms, pulverize, after crossing 40 mesh sieves, extract 3 times by 95 % soak with ethanol, each 6 days, collect the extracting solution at every turn obtained.By extracting solution concentrating under reduced pressure at the temperature lower than 60 ℃, obtain total extractum 3200 grams.Get gained extractum 3000 grams and be suspended in 20 L distilled water, repeatedly extract with petroleum ether, ethyl acetate, n-butyl alcohol successively, every kind of extract consumption is 50 L, and extract is carried out respectively to concentrating under reduced pressure.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 of obtaining separates and obtains 52 fractions through silica gel column chromatography (1200 mm * 70 mm).The 13rd section fraction is that fraction 13 be take chloroform-methanol (volume ratio is 2: 1) again and carried out eluting, 15 fractions of getting back, fraction 8 wherein obtains compound 3(100.3 mg through recrystallization), after recrystallization, remaining mother solution obtains compound 2(84.5 mg through PTLC); After fraction 19-22 merges, use again chromatographic column (1200 mm * 40 mm) to separate, with chloroform-methanol (9: 1,7: 1,5: 1,3: 1,2: 1,1: 1, volume ratio) carry out gradient elution, 21 fractions of getting back, merge fraction 8-11 wherein by half preparative liquid chromatography (20 mm * 250 mm, 5 μ m, methanol: water=40: 60, volume ratio) separate and obtain compound 1(12.1 mg).
Silicagel column on the acetic acid ethyl ester extract of obtaining (1200 mm * 70 mm), petroleum ether-ethyl acetate (4: 1-0: 1 with variable concentrations, volume ratio) carry out gradient elution, every 500 mL of eluent receive 1 part, with TLC detection, merging, obtain 43 fractions.The 36th section fraction wherein is that fraction 36 use chromatographic columns (1200 mm * 35 mm) separate, with chloroform-methanol (9: 1-5: 1, volume ratio) carry out gradient elution, finally in conjunction with Sephadex LH-20 chromatograph (1200 mm * 40 mm) purification, obtain compound 4 (85.3 mg) and compound 5(451 mg).
(2) extract the Structural Identification of the flavone compound obtained
Compound 1 is yellow amorphous powder, 181 ~ 183 ℃ of mp, and the reaction of hydrochloric acid magnesium powder is positive, and the 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, 1996,43 (5): 1111-1114] report basically identical, so authenticating compound 1 be (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L-Arab-D-Glucose glycosides, structural formula is as shown in Figure 1.
Compound 2 is yellow crystals (in methanol, crystallization obtains).156 ~ 158 ℃ of mp, the reaction of hydrochloric acid magnesium powder is positive, and the 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] 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), 193 ~ 195 ℃ of mp.The reaction of hydrochloric acid magnesium powder is positive, and the 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 of Radix Polygoni aubertii chemical composition [J], Food Science, 2012,33 (7): 1-5; Li Chunmei, Wang Tao, a Yi, Gao Xiumei, etc. the separation of Chinese medicine Flos abelmoschi manihot chemical composition and evaluation (I) [J]. 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), 259 ~ 261 ℃ of mp.The reaction of hydrochloric acid magnesium powder is positive, and the 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 of Ligustrum japonicum Thunb.flower [J]. 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 the 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 of Ligustrum japonicum Thunb.flower [J]. the 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
The western Funiu Shan Mountain Diffuse Coptosapelta in Henan of getting after drying and crushing is spent 1 kilogram, add 12 kilograms of mass concentrations, 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, and each 4 hours, merging filtrate, reduction vaporization is concentrated, reclaims ethanol and obtains extractum 126 grams.By ethanol extract 10% dissolve with ethanol, filter, filtrate is fully adsorbed with 1000 gram D101 type macroporous adsorbent resins, the 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 and soak paste, extractum, 50 ℃ of lower vacuum decompression dryings, is obtained to dry total flavones powder 10 grams.Sample after macroporous adsorbent resin column chromatography is separated adopts the silica gel column chromatography loading to separate, with CHCl
3: CH
3the OH volume ratio is 1:9,1:7, and 1:5,1:3 is the eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects and contains the fraction section that Diffuse Coptosapelta flower Flavonoids is greater than 60%, sample after silica gel column chromatography is separated adopts gel column chromatography to separate, and adopts CHCl
3: CH
3the OH volume ratio is the 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, drying, sample after gel column chromatography is separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta is spent Flavonoids, chromatographic condition is permaphase ODS-A, 20 * 250 mm, 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 μ m), mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60, flow velocity: 1.0 mL/min, column temperature: 25 ℃, detect wavelength: 350 nm, high performance liquid chromatography records wherein Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, the quality percentage composition 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 are respectively the chemical structural formulas of these 5 kinds of chromocor compounds), general flavone content is greater than 93.2%, as shown in Figure 7, Fig. 6 is by Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, these 5 kinds of compounds of luteolin-7-O-β-D-Glucose glycosides and kaempferol-3-O-β-D-Glucose glycosides are mixed with mixture product in contrast according to finite concentration, the high-efficient liquid phase chromatogram of the reference substance that test obtains.
embodiment 2
The western Funiu Shan Mountain Diffuse Coptosapelta in Henan of getting after drying and crushing is spent 2 kilograms, add 22 kilograms of mass concentrations, 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, and each 5 hours, merging filtrate, reduction vaporization is concentrated, reclaims ethanol and obtains extractum 284 grams.By ethanol extract 20% dissolve with ethanol, filter, filtrate is fully adsorbed with 2000 gram AB-8 type macroporous adsorbent resins, the 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 and soak paste, extractum, 60 ℃ of lower vacuum decompression dryings, is obtained to dry total flavones powder 18.6 grams.Sample after macroporous adsorbent resin column chromatography is separated adopts the silica gel column chromatography loading to separate, with CHCl
3: CH
3the OH volume ratio is 1:9,1:7, and 1:5,1:3 is the eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects and contains the fraction section that Diffuse Coptosapelta flower Flavonoids is greater than 60%, sample after silica gel column chromatography is separated adopts gel column chromatography to separate, and adopts CHCl
3: CH
3the OH volume ratio is the 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, drying, sample after gel column chromatography is separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta is spent Flavonoids, chromatographic condition is permaphase ODS-A, 20 * 250 mm, 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 μ m), mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60, flow velocity: 1.0 mL/min, column temperature: 25 ℃, detect wavelength: 350 nm, high performance liquid chromatography records wherein Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, the quality percentage composition 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
The western Funiu Shan Mountain Diffuse Coptosapelta in Henan of getting after drying and crushing is spent 4 kilograms, add 40 kilograms of mass concentrations, 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, and each 3 hours, merging filtrate, reduction vaporization is concentrated, reclaims ethanol and obtains extractum 605 grams.By ethanol extract 30% dissolve with ethanol, filter, filtrate is fully adsorbed with 5000 gram D101 type macroporous adsorbent resins, the 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 and soak paste, extractum, 55 ℃ of lower vacuum decompression dryings, is obtained to dry total flavones powder 37.6 grams.Sample after macroporous adsorbent resin column chromatography is separated adopts the silica gel column chromatography loading to separate, with CHCl
3: CH
3the OH volume ratio is 1:9,1:7, and 1:5,1:3 is the eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects and contains the fraction section that Diffuse Coptosapelta flower Flavonoids is greater than 60%, sample after silica gel column chromatography is separated adopts gel column chromatography to separate, and adopts CHCl
3: CH
3the OH volume ratio is the 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, drying, sample after gel column chromatography is separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta is spent Flavonoids, chromatographic condition is permaphase ODS-A, 20 * 250mm, 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 μ m), mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60, flow velocity: 1.0 mL/min, column temperature: 25 ℃, detect wavelength: 350 nm, high performance liquid chromatography records wherein Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--D-Glucose glycosides, Eriodictyol-7-O-β-D-Glucose glycosides, ampelopsin-3-O-β-D-Glucose glycosides, the quality percentage composition 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:
(the quality percentage composition of Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--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 methyl ester sulfate (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) remove ultra-oxygen anion free radical (O
- 2) assay method
Use pH8.0, the Tris-HCl buffer of concentration 50 mol/L is diluted to different Concentraton gradient by Diffuse Coptosapelta flower total flavones product, respectively get the different gradient flavonoids solutions of 1.5 mL, then add successively 0.5 mL NBT(300 μ mol/L, Tris-HCl buffer preparation with pH8.0), 0.5 mL NADH(468 μ mol/L, Tris-HCl buffer preparation with pH8.0), 0.5 mL PMS(60 μ mol/L, Tris-HCl buffer preparation with pH8.0), water-bath 5 minutes in 25 ℃ after mixing, taking-up is measured absorbance at wavelength 560 nm places, the blank group replaces flavonoids solution with buffer.The formula that calculates the ultra-oxygen anion free radical clearance rate is as follows:
E(O
- 2·)(%)=(1－A
1/A
0)×100
In formula: E (O
- 2) be that flavone is to O
- 2clearance rate (%); A
1for the flavone absorbance; A
0for blank absorbency.
(2) remove the assay method of DPPH free radical
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 again the DPPH solution that 2 mL concentration are 0.04 mg/mL, mix homogeneously, react 20 minutes, 3500 r/min centrifugalize 10 minutes, getting supernatant is A at wavelength 517 nm places its absorbances of survey
i; The another flavonoids solution of respectively getting the above-mentioned concentration of 2 mL, in test tube, adds respectively dehydrated alcohol 2 mL, reacts 20 minutes, and 3500 r/min centrifugalize 10 minutes, getting supernatant is A at wavelength 517 nm places its absorbances of survey
j; Using 2 mL 0.04 mg/mL DPPH and the reaction of 2 mL dehydrated alcohol as reference, and its absorbance is designated as A
0.The formula that calculates the DPPH free radical scavenging activity is as follows:
E(DPPH)(%)=[1－(A
i-A
j)/A
0]×100
In formula: E (DPPH) is the clearance rate (%) of flavone to the 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) remove the assay method of hydroxyl radical free radical (OH)
Diffuse Coptosapelta flower total flavones product is mixed with to the variable concentrations gradient with distilled water, respectively gets the flavonoids solution of the above-mentioned concentration of 2mL, add successively the FeSO of 2 mL 6 mmol/L
4, 2 mL 6mmol/L H
2o
2, mix latter standing 10 minutes, then add 2 mL 6 mmol/L salicylic acid, mix, standing 30 minutes, survey its absorbance at wavelength 510 nm places and be designated as A
i, the absorbance when with distilled water, replacing salicylic acid is designated as A
j.The blank group replaces the flavonoids solution absorbance to be designated as A with distilled water
0.The formula that calculates the 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
iabsorbance for Diffuse Coptosapelta flower total flavones product; A
0for blank absorbency; A
jthe absorbance of Diffuse Coptosapelta flower total flavones product while reacting for participating in without salicylic acid.
(4) mensuration of anti-peroxidation activity
Diffuse Coptosapelta flower total flavones product solution 1.00 mL that add different quality concentration in 10 mL color comparison tubes, lecithin soln 1.0 mL, 0.4 mmol/L FeSO
41.0 mL, mix, in 37 ℃ of water-baths, place 60 minutes, add trichloroacetic acid (TCA)-thiobarbituricacidα-(TBA)-hydrochloric acid (HC1) to mix 2.0 mL, 100 ℃ of water-baths are cooling rapidly after 15 minutes again, with 3000 r/min centrifugal 10 minutes, get supernatant and measure absorbance at wavelength 532 nm places, make reference with distilled water, VC is contrast, calculates suppression ratio.
Suppression ratio/%=[1-(A
x-A
x0)/A
0] * 100
In formula: A
0absorbance for blank liquid; A
xfor adding the absorbance after flavonoids solution; A
x0for not adding the flavonoids solution absorbance of lecithin.
?
Result:
(1) remove ultra-oxygen anion free radical (O
2 -)
| Total flavones concentration (mg/mL) | 0.01 | 0.02 | 0.04 | 0.08 | 0.16 |
| The VC clearance rate | 25% | 29% | 37% | 51% | 83% |
| Total ketone clearance rate | 17% | 19% | 26% | 39% | 53% |
In selected mass concentration scope, Diffuse Coptosapelta flower total flavones has the ability of removing ultra-oxygen anion free radical.Along with the increase of Diffuse Coptosapelta flower total flavones mass concentration, the scavenging action enhancing to ultra-oxygen anion free radical, present 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) remove the DPPH free radical
| Total flavones concentration (μ g/mL) | 5 | 10 | 15 | 20 | 25 |
| The VC clearance rate | 46% | 73% | 87% | 90% | 91% |
| Total ketone clearance rate | 41% | 57% | 71% | 80% | 83% |
The DPPH free radical is a kind of very stable free radical centered by nitrogen, and the effect of its removing is shown to tested medicine has the valid density that reduces hydroxyl radical free radical, alkane free radical or peroxy radical and the effect that interrupts the lipid peroxidation chain reaction.Although it is strong not as VC that Diffuse Coptosapelta flower total flavones is removed DPPH free radical effect, Diffuse Coptosapelta flower total flavones still shows the ability of very strong removing DPPH free radical.
(3) remove hydroxyl radical free radical (OH)
| Total flavones concentration (mg/mL) | 0.05 | 0.1 | 0.2 | 0.4 | 0.8 |
| The 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 the OH effect and is compared with VC, and Diffuse Coptosapelta flower total flavones has the ability of removing OH, but a little less than its effect.
(4) anti-peroxidation activity
| Total flavones concentration (μ g/mL) | 50 | 100 | 150 | 200 | 250 |
| The 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 enhancing of flavone mass concentration.In the quality of experiments concentration range, not measuring VC has the anti-peroxidation ability, may be because VC belongs to the water solublity polar substances, mainly concentrates on water in the emulsified fatty substance system, thereby affects its inhibitory action.
Claims (6)
1. the preparation method of Diffuse Coptosapelta flower Flavonoids is characterized in that: comprise the following steps:
(1) alcoholic solution extracts:
Diffuse Coptosapelta flower pesticide material is dry, pulverizing, after adding mass concentration to be the 60-80% soak with ethanol, heating and refluxing extraction 3-5 hour, filtration obtains filtrate and residue, again residue be take to mass concentration as 60 ~ 80% alcohol heating reflux extraction 2 times, each 3-5 hour, merging filtrate, concentrate to obtain extractum by filtrate evaporated under reduced pressure;
(2) macroporous adsorbent resin column chromatography separates:
By the extractum mass concentration obtained, it is the 10-30% dissolve with ethanol, filter, filtrate is fully adsorbed with macroporous adsorbent resin, the dress post, the ethanol-water system eluent that is 10%, 20%, 30%, 40%, 50%, 60%, 80% and 95% by volumetric concentration successively carries out gradient elution, and collecting the ethanol volumetric concentration is 50-60% alcohol-water eluting fraction, is evaporated to and soaks paste, extractum, 50-60 ℃ of lower vacuum decompression drying, is obtained to Diffuse Coptosapelta flower Flavonoids crude product;
(3) silica gel column chromatography separates:
Adopt the silica gel column chromatography loading to separate Diffuse Coptosapelta flower Flavonoids crude product, successively with CHCl
3: CH
3the OH volume ratio is 1:9,1:7, and 1:5,1:3 is the eluent gradient eluting, adopts rutin in contrast, with the content of ultraviolet spectroscopy Diffuse Coptosapelta flower Flavonoids, collects and contains the fraction section that Diffuse Coptosapelta flower Flavonoids is greater than 60%;
(4) gel column chromatography separates:
Sample after silica gel column chromatography is separated adopts gel column chromatography to separate, and adopts CHCl
3: CH
3the OH volume ratio is the 2:1 isocratic elution, collects eluent, concentrating under reduced pressure, drying;
(5) half preparative high-performance liquid chromatographic purification:
Sample after gel column chromatography is separated adopts half preparative high-performance liquid chromatographic purification, collects eluate, concentrating under reduced pressure, and dry that Diffuse Coptosapelta is spent Flavonoids, chromatographic condition is permaphase ODS-A, 20 * 250mm, 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: the Agilent1100 high performance liquid chromatograph,
Chromatographic column: Agilent Zorbax SB C
18chromatographic column, 4.6 * 250 mm, 5 μ m,
Mobile phase: methanol-acetonitrile-0.4% glacial acetic acid, volume ratio is 30:10:60,
Flow velocity: 1.0 mL/min,
Column temperature: 25 ℃,
Detect wavelength: 350 nm.
2. a kind of Diffuse Coptosapelta as claimed in claim 1 is spent the preparation method of Flavonoids, it is characterized in that: in described step (1), the time of soak with ethanol is 8-12 hour.
3. a kind of Diffuse Coptosapelta as claimed in claim 1 is spent the preparation method of Flavonoids, it is characterized in that: in described step (1) during the 1st reflux, the 10-12 that the addition of ethanol is Diffuse Coptosapelta flower quality of medicinal material doubly, when the 2nd time and the 3rd reflux, the addition of ethanol be Diffuse Coptosapelta flower quality of medicinal material 8-10 doubly.
4. a kind of Diffuse Coptosapelta as claimed in claim 1 is spent the preparation method of Flavonoids, and it is characterized in that: the macroporous adsorbent resin in described step (2) is D101 or AB-8 type macroporous adsorbent resin.
5. the Diffuse Coptosapelta flower Flavonoids prepared by the described method of claim 1, it is characterized in that: Arabic (the 1-6)-β of Quercetin-3-methoxyl group-7-O-α-L--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-5 kinds of D-Glucose glycosides chromocor compound, consist of, the quality percentage composition is respectively 5-8%, 8-12%, 3-6%, 30-50% and 20-30%.
6. the purposes of a Diffuse Coptosapelta as claimed in claim 5 flower Flavonoids, it is characterized in that: described Diffuse Coptosapelta flower Flavonoids has antioxidant activity, has the purposes of removing ultra-oxygen anion free radical, DPPH free radical, hydroxyl radical free radical and anti-peroxidation.
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| CN108623548B (en) * | 2018-05-02 | 2022-05-06 | 齐齐哈尔大学 | Method for separating chiral eriodictyol from peanut shells and application of method |
| CN115531443A (en) * | 2022-04-11 | 2022-12-30 | 连云港市林业技术指导站 | Method for increasing content of total flavonoids in Chinese fringetree leaf extract |
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