CN102807544B - Method for extracting anthocyanin from elderberry fruits - Google Patents
Method for extracting anthocyanin from elderberry fruits Download PDFInfo
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- CN102807544B CN102807544B CN201210279323.XA CN201210279323A CN102807544B CN 102807544 B CN102807544 B CN 102807544B CN 201210279323 A CN201210279323 A CN 201210279323A CN 102807544 B CN102807544 B CN 102807544B
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Abstract
The invention discloses a method for extracting anthocyanin from elderberry fruits. The method comprises the following steps: (1) crushing material at low temperature after cleaning; (2) adding the crushed material into 3-6 weight/mass times of ethanol water, of which the pH value is 3-6 and the mass percent is 10-30%, carrying out countercurrent extraction for 3-6 hours at 25-50 DEG C, and (3) collecting extracting solution, and concentrating under reduced pressure to obtain the anthocyanin extract of the elderberry fruit. By adopting the countercurrent extraction method, the anthocyanin ingredient can be extracted more fully, and compared with the method in the prior art, the method has the characteristics that the material treatment is easier; the solvent is saved; the extraction efficiency is high and the cost is low, and the method is suitable for industrial production.
Description
Technical field
The present invention relates to plant efficient part extractive technique field, be specifically related to the extracting method of anthocyanidin in a kind of erberry.
Background technology
Sambucus Sambucus L. system Caprifoliaceae Caprofoliaceae plant, about 20 kinds, the whole world, be distributed in temperate zone and subtropical zone, about there are 5 kinds of Caprifoliaceae Sambucus in China, be respectively Herba Sambuci Adnatae S.adnata Wall.ex DC., rhizome or all-grass of Steward Elatostema S.chinensis Lindl., Williams Elder Twig S.williamsiiHance, siberian Williams Elder Twig S.sibirica Nakai, golden elder S.nigraLinn., north and south each province, Qu Junyou distribution.This platymiscium has expelling wind and removing dampness, the effect of relaxing the muscles and stimulate the blood circulation, and China is among the people is widely used in treatment rheumatic arthralgia, wound, fracture, jaundice, urticaria and oedema.
In present erberry, the extracting method of the natural compounds such as anthocyanidin is: get Williams Elder Twig fresh fruit after fragmentation, first remove the seed in fruit, again remaining pericarp is fully mixed in container with appropriate moisture rudimentary ethanol, after adjusting PH4 with hydrochloric acid, soak extraction 2 hours at normal temperatures, extracting solution after filtration, after concentration and recovery ethanol concentrated extract, concentrated extract obtains erberry extract after drying.
Prior art Problems existing is: 1. Feedstock treating trouble, needs de-seed just can carry out next procedure, be therefore not suitable for large-scale industrial production; 2. extracting method is coarse, and the product after extraction is the crude product without separation and purification, and product recovery rate is low, and target component content is low, count 85%, but wherein anthocyanidin content only has 4 ~ 5% with the anthocyanidin rate of recovery; 3. product color difference (brown), is unfavorable for the application of product.
Summary of the invention
Technical problem to be solved of the present invention is to provide the extracting method of anthocyanidin in a kind of erberry, the present invention adopts countercurrent extraction method, can extract Anthocyanins more fully, there is Feedstock treating compared with art methods be more prone to, save the features such as solvent, extraction efficiency is high, cost is low, be suitable for suitability for industrialized production.
The present invention solves the problems of the technologies described above adopted technical scheme: the extracting method of anthocyanidin in a kind of erberry, comprises the steps: successively
Step (1), after raw material is cleaned, pulverize at low temperature; With
Step (2), by pulverize after raw material adds 3-6 weight/volume times amount, pH value is 3-6, mass percent is the aqueous ethanolic solution of 10-30%, countercurrent extraction 3-6h at 25-50 DEG C; And
Step (3), collect extracting solution, concentrating under reduced pressure, obtains elderberry anthocyanin extract.
In above-mentioned steps (2), aqueous ethanolic solution add-on is preferably 4.5 weight/volume times amount, and pH value is preferably 4, ethanol mass percent preferably 20%; Temperature is preferably 40 DEG C; Extraction time is preferably 4h.Weight/volume times amount as herein described refers to by weight or volume computing, and aqueous ethanolic solution is relative to the multiple of raw material.
Compared with prior art, tool has the following advantages in the present invention:
(1) the present invention adopts countercurrent extraction method, can extract Anthocyanins more fully, has Feedstock treating and is more prone to, saves the features such as solvent, extraction efficiency is high, cost is low, be suitable for suitability for industrialized production compared with art methods.
(2) elderberry anthocyanin extract for preparing of the present invention demonstrates good effect in minimizing eosinophilic granulocyte, and namely they can effectively for various diseases that prevention and therapy is caused by too much eosinophilic granulocyte.
(3) elderberry anthocyanin extract that the present invention prepares can as cell death inducer, antianaphylaxis, anti-inflammatory and protective foods.
Accompanying drawing explanation
Fig. 1 is the mass percent (%) of ethanol and the graph of a relation of leach liquor anthocyanidin content (g).
Fig. 2 is the graph of a relation of duration of contact (h) and leach liquor anthocyanidin content (g).
Fig. 3 is the graph of a relation of temperature (DEG C) and leach liquor anthocyanidin content (g).
Fig. 4 is the schematic diagram of the device that the extracting method of anthocyanidin in a kind of erberry of the present invention uses.
Embodiment
One, extracting factor is investigated
1, leaching condition experiment
(1) alcohol concn is on the impact of anthocyanidin compound leaching effect
In industrial production, due to normal containing certain moisture in raw material, ethanol is also containing certain moisture after recycling, and alcohol concn exists very large impact to the leaching of anthocyanidin compound, therefore with different concentration ethanol liquid, leaching experiment is carried out to anthocyanidin material, with anthocyanidin content trace analysis.At alcohol concn on leaching from raw material in the impact experiment of anthocyanidin, get 5 parts of raw materials respectively, every part of 40.00g, tests, and with each 180.00ml of the aqueous ethanolic solution of different concns, adjust pH value to be 4, lixiviate 6h, compares the amount of gained anthocyanidin as shown in Figure 1.
As shown in Figure 1, under the same terms, leaching ability power is followed successively by 20%>15%>25%>10 %>30%, therefore, selects 20% aqueous ethanolic solution as leaching agent.
(2) duration of contact is on the impact of anthocyanidin compound leaching effect
Get 6 parts of raw materials respectively, every part of 40.00g, respectively add the ethanol 180.00ml that concentration is 20%, adjust pH value to be 4, leach, duration of contact is respectively 1h, 2h, 3h, 4h, 5h, 6h, and it leaches result as shown in Figure 2.
As shown in Figure 2, under room temperature (26 DEG C), extraction time is 4h, and Leaching reaction reaches balance substantially; Extend extraction time, leaching yield is substantially constant.
(3) temperature is on the impact of anthocyanidin compound leaching effect
Get 6 parts of raw material powders respectively, every part of 40.00g, respectively add the ethanol that 180.00ml concentration is 20%, adjust pH value to be 4.0 to leach at different temperature, extraction time is 2h, and sampling detects anthocyanidin content in leach liquor, and result as shown in Figure 3.
As shown in Figure 3, along with the increase of extraction temperature, the leaching yield of anthocyanidin also increases accordingly; When temperature reaches 40 DEG C, increase temperature, the leaching yield increase of anthocyanidin is tending towards slow.
2, four sections of adverse current percolations (process diagram is shown in Fig. 4)
Installed 6 ф 25.4mm × 1000mm glass altogether and drawn together mouth pipe, as the leacher (leaching post) of four sections of adverse current diacolation leaching experiments, wherein four operations, two are done week conversion.150.00g erberry raw material powder particle (being called for short raw material, as follows) is loaded, with volume pump by leaching agent (10-30% aqueous ethanolic solution), with about 5mlmin in every root post
-1flow squeeze in first leaching post, ethanol is with about 1cmmin
-1speed diacolation, after the first post flows out 450ml leach liquor, connects the first post and the second post.The recent filling that volume pump is squeezed into, through the first post, flows out and enters the second post; Identical with upper, after the second post flows out 450ml black leach liquor, the second post and the 3rd post are connected.The rest may be inferred, after the 4th post flows out 450ml effluent liquid by the time, the 4th post and the 5th post connected.Meanwhile, volume pump outlet pipe is moved to the second post upper end from the first post upper end and the second post is connected (the first post is withdrawn from from system).Now, new leaching agent, enters the second post through volume pump, after three, four, five posts, flows out, get 450ml leach liquor equally from the 5th post; After this, recent filling enters the 3rd post, goes out leach liquor from the 6th post.The rest may be inferred, and constantly circulation carries out four sections of adverse current diacolation leaching experiments to reaching leaching balance.In this test, the 9th post starts to stop to the 12 post, the leach liquor that these four posts flow out approximate equilibrium time leach liquor, therefore when new leaching agent enters, through ten, 11,12 posts from from the 9th post, and flow out 450ml black leach liquor from the 12 post after, stop leaching test.The leach liquor sampling of being flowed out by nine, ten, 11,12 posts, censorship, measures the content of erberry leach liquor anthocyanidin.(note: cylinder is and recycles, nine, ten, 11,12 only represent label, do not have a practical significance)
3, four sections of adverse current diacolation leaching test results
Counter-current described above is tested.Every increment product are 150.0g, and with 20% ethanol for leach liquor, adjust pH value to be lixiviate 4h at 4,40 DEG C, liquid-solid ratio is 4.5.The leach liquor getting nine, ten, 11,12 posts outflows measures, and its result is as shown in table 1.Described liquid-solid ratio refers to that aqueous ethanolic solution is relative to the quality of sample or volume multiple.
Table 1 four sections of adverse current diacolation leaching test results
As known from Table 1, in erberry, anthocyanidin content is in 0.41%, then four sections of its leaching yields of adverse current diacolation lixiviation process (in leach liquor) reach 95.77%(in anthocyanidin).
Two, embodiment: below by way of specific embodiment, the present invention is described in further detail.
Embodiment 1:
After the cleaning of 500 grams of raw materials, pulverize at low temperature, adds 3 weight/volume times amount by the raw material after pulverizing, pH value is 6, mass percent is the aqueous ethanolic solution of 10%, countercurrent extraction 6h at 50 DEG C, collect extracting solution, concentrating under reduced pressure, obtains elderberry anthocyanin extract 19.16 grams.
Embodiment 2
After the cleaning of 500 grams of raw materials, pulverize at low temperature, adds 4.5 weight/volume times amount by the raw material after pulverizing, pH value is 4, mass percent is the ethanol of 20%, countercurrent extraction 4h at 40 DEG C, collect extracting solution, concentrating under reduced pressure, obtains elderberry anthocyanin extract 19.51 grams.
Embodiment 3
After the cleaning of 500 grams of raw materials, pulverize at low temperature, adds 6 weight/volume times amount by the raw material after pulverizing, pH value is 3, mass percent is the ethanol of 30%, countercurrent extraction 3h at 25 DEG C, collect extracting solution, concentrating under reduced pressure, obtains elderberry anthocyanin extract 19.03 grams.
The test of embodiment 4 eosinophilic apoptosis-inducing and the experiment of MBP kinase activity
Eosinophilic's apoptosis-inducing is tested: the method (Journal ofImmunological Methods 184 volume 39-51 page nineteen ninety-five) of experimental evidence Vermes etc. is carried out.Make eosinophilic granulocyte sexual cell HL-60 cell suspension in RPMI1640 substratum, cell concn is 1-3 × 10
6cell/ml.Tested substance (finally collecting the erberry extract obtained in above-described embodiment 1-3) or substratum is added in this cell suspending liquid 500 μ l, the concentration of erberry extract in cell suspending liquid is made to be 100 μ g/ml, at 37 DEG C, 5%CO
2condition under cultivate 6 hours or 24 hours.Centrifugal clean after, add phosphatide phthalein associated proteins (Annexin) V damping fluid, then add 5u1 phosphatide phthalein associated proteins V-FITC.By flow cytometry method, using phosphatide phthalein associated proteins V positive cell as apoptosis-inducing cell, with the per-cent evaluation of relatively total cell count.
MBP Kinase activity assays: the method (Blood 99 volume 3432-3438 page 2002) of experimental evidence De Souza etc. is carried out.By eosinophilic granulocyte sexual cell HL-60 cell suspension in RPMI 1640 substratum, cell concn is 1-3 × 10
6cell/ml.Add tested substance or substratum in this cell suspending liquid 500 μ l, make concentration be 100 μ g/ml, at 37 DEG C, 5%CO
2condition under cultivate 6 hours or 24 hours.Centrifugal clean after, add solubilized damping fluid, dissolved cell, use the gel containing MBP 5mg/ml to carry out electrophoresis.Gel, after de-sex change, again denaturing treatment, is used
32p-ATP marks, and implements the phosphorylation reaction of 3 hours.After gel drying, Quantimet is used to resolve radioactivity.When 36kDa grows indicia band, be denoted as the MBP kinase activation positive (+).
Elderberry anthocyanin extract observed the apoptosis-inducing of concentration dependent in HL-60 cell.When carrying out gel MBP kinase assays (In-gel MBP kinase assay) in addition, elderberry anthocyanin extract activates 36kDa MBP kinases.Above result prompting.Elderberry anthocyanin extract is to eosinophilic granulocyte energy cell death inducing, and this apoptosis is relevant with 36kDa MBP kinases.Infer in elderberry anthocyanin extract of the present invention containing the composition that may be used for anti-eosinophilic's inflammatory effect medicine thus.
Acquired results is as shown in table 2 below:
Confirm that embodiment 2 and 3 has strong apoptosis-inducing activity, and with the kinase whose activation of MBP.
Table 2
As mentioned above, just the present invention can be realized preferably.
Claims (1)
1. the extracting method of anthocyanidin in erberry, is characterized in that, comprise the following steps:
Step (1), after raw material is cleaned, pulverize at low temperature; With
Step (2), by after pulverizing raw material adds 4.5 weight/volume times amount, pH value is 4, mass percent is the aqueous ethanolic solution of 20%, countercurrent extraction 4h at 40 DEG C; And
Step (3), collect extracting solution, concentrating under reduced pressure, obtains elderberry anthocyanin extract.
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| CN105294631B (en) * | 2015-11-13 | 2018-08-28 | 大兴安岭林格贝寒带生物科技股份有限公司 | A kind of industrial method preparing anthocyanidin from erberry |
| CN113694561A (en) * | 2021-08-27 | 2021-11-26 | 湖南华康生物科技股份有限公司 | Extraction and separation method of elderberry polyphenol |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070248700A1 (en) * | 2006-03-17 | 2007-10-25 | Alberte Randall S | Extractions and Methods Comprising Elder Species |
| CN101415332A (en) * | 2006-03-17 | 2009-04-22 | 草药科学新加坡私人有限公司 | Extractions and methods comprising elder species |
| CN101463027A (en) * | 2009-01-08 | 2009-06-24 | 陕西明锐天然产物有限责任公司 | Method for extracting and separating flavone and anthocyanidin from elder fruit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070248700A1 (en) * | 2006-03-17 | 2007-10-25 | Alberte Randall S | Extractions and Methods Comprising Elder Species |
| CN101415332A (en) * | 2006-03-17 | 2009-04-22 | 草药科学新加坡私人有限公司 | Extractions and methods comprising elder species |
| CN101463027A (en) * | 2009-01-08 | 2009-06-24 | 陕西明锐天然产物有限责任公司 | Method for extracting and separating flavone and anthocyanidin from elder fruit |
Non-Patent Citations (3)
| Title |
|---|
| Michael Schwarz,等.Application of high-speed countercurrent chromatography to the large-scale isolation of anthocyanins.《Biochemical Engineering Journal》.2003,第14卷(第3期), * |
| 天然色素的提取和研究进展;姚成立,等;《安徽化工》;20070630;第33卷(第3期);第1-3页 * |
| 李希,等.动态逆流提取技术在中药提取中的应用.《实用中医药杂志》.2008,第24卷(第12期), * |
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