CN104557520B - A kind of method of separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis - Google Patents
A kind of method of separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis Download PDFInfo
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- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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Abstract
The present invention relates to a kind of method of separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis, is with Chinese propolis for raw material, through following step: the preparation of (1) Chinese Water extracts of propolis; (2) alcohol precipitation; (3) macroporous adsorbent resin roughing out; (4) Semipreparative chromatography separation and purification.Carry out separation and purification with Semipreparative chromatography, moving phase is methanol-water, obtains 3 kinds of highly purified compositions, is coffic acid, forulic acid and isoferulic acid respectively through qualification.The environmental protection of this technological process, to environment without serious harm, comprehensive cost is low.
Description
Technical field
The invention belongs to chemical field, specifically relate to a kind of method of separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis.
Background technology
China's propolis has pharmacologically active widely, as antitumor, antibacterial, antiviral, anti-inflammatory, anti-oxidant, reducing blood-fat, hypoglycemic etc., is therefore widely used in food and healthcare products industry.The chemical composition of China's propolis is extremely complicated, and up to the present, isolation identification goes out more than 20 classes more than 300 and plants chemical composition at least, and comprise the materials such as phenolic acid, flavones, amino acid, steroid, wherein phenolic acid and flavones ingredient are main active ingredient.The China fat-soluble position of propolis is mainly containing flavones ingredient, and water soluble part is then mainly containing organic acid substance (as coffic acid, forulic acid, isoferulic acid etc.).Coffic acid has cholagogic, hemostasis, effect that liter is white, and it has significant curative effect to various acute bacterial infection disease, leukopenia caused by radiation and chemotherapy; Forulic acid has anti-platelet aggregation, reducing blood-fat, prevents and treats coronary heart disease, scavenging free radicals, antisepsis and anti-inflammation, antitumor, the effect such as anti-mutation, increase immunologic function; Isoferulic acid has very strong anti-inflammatory activity.
Because propolis is soluble in ethanol, the report therefore about the chemical constitution study at the fat-soluble position of Chinese propolis is very many, and the research of the chemical composition of water soluble part report is relatively less.In order to realize the comprehensive utilization to Chinese propolis, the invention provides a kind of method of simple and quick separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis.
Summary of the invention
The object of the present invention is to provide a kind of method of simple and quick separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis.
The solution of the present invention is as follows:
From Chinese Water extracts of propolis, the method for separation and purification coffic acid, forulic acid and isoferulic acid, the steps include:
(1) preparation of Chinese Water extracts of propolis: by freezing for Chinese propolis, pulverizes, and extract with distilled water heating, filter, Aqueous extracts naturally cools to room temperature, and the beeswax removing of being solidified on upper strata, lower aqueous solution is about 1.2 through being evaporated to relative density.
(2) alcohol precipitation: adding 95% ethanol to ethanol content in above-mentioned concentrated solution is about 70%, and hold over night, gets supernatant concentration and obtain medicinal extract.
(3) macroporous adsorbent resin roughing out: upper macroporous adsorptive resins after above-mentioned medicinal extract is dissolved with suitable quantity of water, first wash with water, then be eluent gradient wash-out with 20%, 40%, 60%, 80%, 95% ethanol-water solution successively, collect 20% ethanol elution part, concentrated, obtain sample 1.
(4) Semipreparative chromatography separation and purification: by sample 1 Semipreparative chromatography separation and purification, chromatographic column is C
18sMB100 post (400mm × 25.4mmI.D., 10 μm), moving phase is methanol-water, and determined wavelength is 280nm, collects target components cut, by the cut concentrating under reduced pressure obtained, namely obtains the monomeric compound that will be separated.
Foregoing method, preferred scheme is, when step (1) heating is extracted, extraction time is 0.5-2.5 hour, and extraction time is 2-6 time.More preferably, extraction time is 2 hours, and extraction time is 4 times.
Foregoing method, preferred scheme is, during step (1) heating and refluxing extraction, the consumption of distilled water is 3 times amount-10 times amount (preferably 8 times amount).
Foregoing method, preferred scheme is, the temperature 60 C-90 DEG C (preferably 80 DEG C) when step (1) heating is extracted.
Foregoing method, preferred scheme is, the model of the macroporous adsorbent resin of the HPD series that step (3) roughing out to Chinese Water extracts of propolis is used have 100,400,500,600,722 and 826(preferably 826).
Foregoing method, preferred scheme is, the concentration of step (4) Semipreparative chromatography separation and purification methyl alcohol used is 10%-20%(preferably 15%).
Foregoing method, preferred scheme is, step (4) separation and purification methanol-water solution carries out wash-out, and the flow velocity of elutriant is the preferred 25mL/min of 20-30mL/min().
The present invention relates to a kind of method of separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis, step is: the preparation of (1) Chinese Water extracts of propolis: get the Chinese propolis crushed, extract with distilled water heating, filter, Aqueous extracts naturally cooling, the beeswax that removing upper strata is solidified, extracting solution is about 1.2 through being evaporated to relative density.(2) alcohol precipitation: adding 95% ethanol to ethanol content in above-mentioned concentrated solution is about 70%, and hold over night, gets supernatant concentration and obtain medicinal extract.(3) macroporous adsorbent resin roughing out: upper macroporous adsorptive resins after above-mentioned medicinal extract is dissolved with suitable quantity of water, first wash with water, be eluent gradient wash-out with 20%, 40%, 60%, 80%, 95% ethanol-water solution successively again, collect 20% ethanol elution part, concentratedly to obtain sample 1.(4) Semipreparative chromatography separation and purification: by sample 1 Semipreparative chromatography separation and purification, chromatographic column is C
18sMB100 post (400mm × 25.4mmI.D., 10 μm), moving phase is methanol-water, and determined wavelength is 280nm, collects target components cut, by the cut concentrating under reduced pressure obtained, namely obtains the monomeric compound that will be separated.This technological process environmental protection, to environment without serious harm, comprehensive cost is low.
The method of the present invention separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis, first by the method for water extract-alcohol precipitation, water-soluble chemical component is extracted, fat-soluble component can be made not to be dissolved as far as possible, and polyose components settle is separated removing; Carry out roughing out with macroporous adsorbent resin to Chinese propolis water soluble part again, can realize grouping to compound through roughing out, this point can be found out from Fig. 1 and Fig. 2; Finally carry out separation and purification by Semipreparative chromatography method and just can obtain 3 kinds of compounds, gained target compound purity is high, and foreign matter content is extremely low, this point can from Fig. 4 to Fig. 6 find out.In addition, also there is following advantage:
(1), after Chinese water extracts of propolis naturally cools to room temperature, beeswax can be separated out automatically, and the beeswax solidified is easy to removing, repeatedly processes several times, can remove beeswaxs most in aqueous extract.Polysaccharide can remove by aqueous extract after alcohol precipitation, at utmost can reduce the contaminated degree of macroporous adsorbent resin.
(2) carry out roughing out with macroporous adsorbent resin to Chinese Water extracts of propolis, use water as moving phase and can remove most strong polar impurity, mixture can be made to realize grouping with the ethanol-water solution gradient elution of different concns, composition is simplified.
(3) carry out separation and purification by Semipreparative chromatography method to compound, can obtain 3 kinds of high-purity monomer compounds, the method is simple to operate, and efficiency is high, and process cycle is short, saves reagent, reduces production cost.
(4) only use water, ethanol and methyl alcohol in extracting and developing, purge process, do not use environment and the large organic solvent such as chloroform, benzene of harm, alcohol-water, methanol-water eluent can be reused repeatedly after underpressure distillation is reclaimed, environmental protection.
(5) optimize the separation condition (composition of elutriant and flow velocity) of Semipreparative chromatography method, the purity of compound and efficiency are all greatly improved.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of Chinese Water extracts of propolis.
Fig. 2 is the high-efficient liquid phase chromatogram of sample 1.
Fig. 3 is the Semipreparative chromatography figure of sample 1.
Fig. 4 is caffeinic high-efficient liquid phase chromatogram and ultraviolet spectrogram.
Fig. 5 is high-efficient liquid phase chromatogram and the ultraviolet spectrogram of forulic acid.
Fig. 6 is high-efficient liquid phase chromatogram and the ultraviolet spectrogram of isoferulic acid.
In figs. 1-6, I: coffic acid; II: forulic acid; III: isoferulic acid.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with embodiment and accompanying drawing, but protection domain is not by this restriction.In embodiment, equipment used or raw material all can obtain from market.Agents useful for same is all purchased from Jinan reagent head factory, and water used is deionized water.
embodiment:from Chinese Water extracts of propolis, the method for separation and purification coffic acid, forulic acid and isoferulic acid, the steps include:
(1) preparation of Chinese Water extracts of propolis: by freezing for Chinese propolis, pulverizes, heats extraction 4 times with 8 times amount distilled water in 80 DEG C of water-baths, each 2h, filtering, Aqueous extracts naturally cooling, is about 1.2 through being evaporated to relative density after the beeswax that solidifies of removing upper strata.
(2) alcohol precipitation: adding about 2 times amount 95% ethanol to ethanol contents in above-mentioned concentrated solution is about 70%, and hold over night, gets supernatant concentration and obtain medicinal extract.
(3) macroporous adsorbent resin roughing out: upper HPD826 type macroporous adsorptive resins after above-mentioned medicinal extract is dissolved with suitable quantity of water, first wash with water, then be eluent gradient wash-out with 20%, 40%, 60%, 80%, 95% ethanol-water solution successively, collect 20% ethanol elution part, concentrated, obtain sample 1.
(4) Semipreparative chromatography separation and purification: by sample 1 Semipreparative chromatography separation and purification, chromatographic column is C
18sMB100 post (400mm × 25.4mmI.D., 10 μm), moving phase is methanol-water (15:85, V/V), and determined wavelength is 280nm, collects target components cut, by the cut concentrating under reduced pressure obtained, namely obtains the monomeric compound that will be separated.
Contriver makes moving phase by using the methyl alcohol of different concns, adopt different types of elution, the flow velocity controlling methanol-water eluent is the preferred 25mL/min of 20-30mL/min(), optimized the purification condition realizing the object of the invention, regarding assay result is as follows:
The preparative high performance liquid chromatography separation condition of table one sample 1
In embodiment 1, adopt 20% methanol-water to be elutriant isocratic elution, each composition elution time is shorter, but separating effect is not ideal enough each other, and gained material is lower.Adopt 15% methanol-water to be elutriant isocratic elution in embodiment 2, be separated good between each composition, disengaging time is also comparatively suitable.Adopt 10% methanol-water to be elutriant isocratic elution in embodiment 3, each component separating is good, but disengaging time is oversize.Embodiment 4 adopts methanol-water gradient elution, also can obtain good separating effect within the suitable time, but causes being difficult to realize recycling because the concentration of elutriant constantly changes.
Fig. 3 is the Semipreparative chromatography figure when selecting embodiment 2 system, and as seen from Figure 3, each component separating is good, and disengaging time is also comparatively suitable.According to color atlas manual collection each peak component, after recycling design, corresponding high-purity compound can be obtained.Through high performance liquid chromatography area normalization method analytical test, purity higher than 98%, this point can from Fig. 4 to Fig. 6 find out.
The chemical structural formula of 3 compounds obtained through Modern spectroscopy data acknowledgement institute extraction purification is as follows:
Caffeic Acid And Ferulic Acid In Doca isoferulic acid
The qualification result of 3 compounds is as follows:
Coffic acid:
1h-NMR (400MHz, DMSO-d6): δ ppm:12.01 (1H, br ,-COOH), 8.90 ~ 9.90 (2H, br, 4-OH, 3-OH), 7.42 (1H, d,
j=16Hz, 7-H), 7.02 (1H, d,
j=2.0Hz, 2-H), 6.96 (1H, dd,
j=2.0,8.0Hz, 6-H), 6.76 (1H, d,
j=8.0Hz, 5-H), 6.17 (1H, d,
j=15.9Hz, 8-H).
13c-NMR (100MHz, DMSO-d6): δ ppm:167.80 (9-C), 148.07 (4-C), 145.50 (3-C), 144.49 (7-C), 125.67 (1-C), 121.07 (8-C), 115.71 (2-C), 115.10 (5-C), 114.59 (6-C).
Forulic acid:
1h-NMR (400MHz, DMSO-d6): δ ppm:7.45 (1H, d,
j=16.0Hz, 7-H), 7.09 (1H, s, 2-H), 7.07 (1H, s, 6-H), 6.94 (1H, d,
j=8.0Hz, 5-H), 6.24 (1H, d,
j=15.6Hz, 8-H), 3.81 (3H, s ,-OCH
3).
13c-NMR (100MHz, DMSO-d6): δ ppm:167.66 (9-C), 149.79 (3-C), 146.63 (4-C), 144.10 (7-C), 127.05 (1-C), 120.89 (6-C), 116.22 (2-C), 114.05 (5-C), 111.99 (8-C), 55.59 (OCH
3).
Isoferulic acid:
1h-NMR (400MHz, DMSO-d6): δ ppm:7.45 (1H, d,
j=15.6Hz, 7-H), 7.09 (1H, d,
j=2.0Hz, 5-H), 7.07 (1H, s, 2-H), 6.95 (1H, d,
j=8.0Hz, 6-H), 6.24 (1H, d,
j=16.0Hz, 8-H), 3.81 (3H, s ,-OCH
3).
13c-NMR (100MHz, DMSO-d6): δ ppm:167.76 (9-C), 149.82 (4-C), 146.64 (3-C), 144.14 (7-C), 127.06 (1-C), 120.97 (8-C), 116.25 (6-C), 114.06 (2-C), 111.95 (5-C), 55.60 (OCH
3).
It should be pointed out that embodiment is the more representational example of the present invention, obvious technical scheme of the present invention is not limited to above-described embodiment.A lot of distortion can also be had.Those of ordinary skill in the art, mentions or associates disclosed in from then in file, all should think the claimed scope of this patent.
Claims (6)
1. the method for separation and purification coffic acid, forulic acid and isoferulic acid from Chinese Water extracts of propolis, it is characterized in that, step is:
(1) preparation of Chinese Water extracts of propolis: by freezing for Chinese propolis, pulverizes, and extract with distilled water heating, filter, Aqueous extracts naturally cools to room temperature, the beeswax removing of being solidified on upper strata, and lower aqueous solution is 1.2 through being evaporated to relative density;
(2) alcohol precipitation: adding 95% ethanol to ethanol content in above-mentioned concentrated solution is 70%, and hold over night, gets supernatant concentration and obtain medicinal extract;
(3) macroporous adsorbent resin roughing out: upper macroporous adsorptive resins after above-mentioned medicinal extract suitable quantity of water is dissolved, first wash with water, then be eluent gradient wash-out with 20%, 40%, 60%, 80%, 95% ethanol-water solution successively, collect 20% ethanol elution part, concentrated, obtain sample 1;
(4) Semipreparative chromatography separation and purification: by sample 1 Semipreparative chromatography separation and purification, chromatographic column is C
18sMB100 post, parameter is 400mm × 25.4mmI.D., 10 μm, and moving phase is methanol-water, ratio is 15:85, V/V, and determined wavelength is 280nm, collect target components cut, by the cut concentrating under reduced pressure obtained, namely obtain monomeric compound coffic acid, forulic acid and the isoferulic acid that will be separated.
2. method according to claim 1, is characterized in that, step (1) heating extraction time is 0.5-2.5 hour.
3. method according to claim 1, is characterized in that, step (1) extraction time is 2-6 time.
4. method according to claim 1, is characterized in that, when step (1) heating is extracted, the consumption of distilled water is 3 times amount-10 times amount.
5. method according to claim 1, is characterized in that, temperature when step (1) heating is extracted is 60 DEG C-90 DEG C.
6. method according to claim 1, is characterized in that, step (3) big pore adsorption resin is the macroporous adsorbent resin of HPD series, and model is 100,400,500,600,722 or 826.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1555814A (en) * | 2004-01-08 | 2004-12-22 | 兰州大学 | Extracting method of propolis effective component and its use |
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| CN1555814A (en) * | 2004-01-08 | 2004-12-22 | 兰州大学 | Extracting method of propolis effective component and its use |
Non-Patent Citations (2)
| Title |
|---|
| "Preparation and antioxidant properties of water extract of propolis";T. Nagai et al.;《Food Chemistry》;20031231;第80卷;29-33 * |
| 不同萃取方式对蜂胶化学成分的影响以及抗油脂氧化作用的研究;余平兰 等;《海峡两岸第六届蜜蜂与蜂产品研讨会论文》;20070831;71-74 * |
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