CN102772468B - Elderberry anthocyanin extract - Google Patents
Elderberry anthocyanin extract Download PDFInfo
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- CN102772468B CN102772468B CN201210278160.3A CN201210278160A CN102772468B CN 102772468 B CN102772468 B CN 102772468B CN 201210278160 A CN201210278160 A CN 201210278160A CN 102772468 B CN102772468 B CN 102772468B
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Abstract
The invention discloses an elderberry anthocyanin extract, which is obtained by using a counterflow extraction method. The counterflow extraction method comprises the following steps of: step (1), crashing at low temperature after cleaning raw materials; step (2), adding the crashed raw materials into ethanol water, and carrying out counterflow extraction for 3-6h at the temperature of 25-50 DEG C, wherein the amount of the added ethanol water is 3-6 times of the crashed raw materials in weight/volume, the pH of the ethanol water is 3-6, and the mass percent is 10-30%; and step (3), collecting extracting solution, and decompressing for compression so as to obtain the elderberry anthocyanin extract. The elderberry anthocyanin extract disclosed by the invention uses the counterflow extraction method, and can more completely extract the active ingredients such as anthocyanin; compared with the existing technical method, the counterflow extraction method disclosed by the invention has the advantages that the elderberry anthocyanin extract is more easy to process raw materials, the solvent is saved, the extraction efficiency is high, the cost is low, and the like, and thus the counterflow extraction method is suitable for industrial production.
Description
Technical field
The present invention relates to the extract that plant effective site extractive technique prepares, be specifically related to a kind of erberry extract.
Background technology
Sambucus Sambucus L. is Caprifoliaceae Caprofoliaceae plant, approximately 20 kinds, the whole world, be distributed in temperate zone and subtropical zone, approximately there are 5 kinds of Caprifoliaceae Sambucus in China, be respectively Herba Sambuci Adnatae S.adnata Wall.ex DC., Rhizoma seu Herba Elatostematis stewardii S.chinensis Lindl., Ramulus Sambuci Williamsii S.williamsii Hance, Siberia Ramulus Sambuci Williamsii S.sibirica Nakai, Sambucus nigra L. S.nigraLinn., each province, north and south, Qu Junyou distribute.This platymiscium has expelling wind and removing dampness, the effect of relaxing muscles and tendons to promote blood circulation, China's treatment rheumatic arthralgia, traumatic injury, fracture, jaundice, urticaria and edema of being widely used among the people.
The complex chemical composition that Sambucus plant is contained, has multiple biological activity.In recent years, Chinese scholars has been done comparatively deep research to its pharmacological action, biological activity and clinicing aspect respectively from different perspectives, for its clinical efficacy provides foundation.But do not find yet that so far it has the erberry extract of cell death inducing, antiallergic and antiinflammatory action.
In erberry, the preparation method of cyanidin extract is now: get Ramulus Sambuci Williamsii fresh fruit after fragmentation, first remove the seed in fruit, again remaining peel is fully mixed homogeneously in container with appropriate moisture rudimentary ethanol, with hydrochloric acid, adjust after PH4, soak and extract 2 hours at normal temperatures, extracting solution after filtration, after concentration and recovery ethanol concentrated extract, concentrated extract obtains erberry extract after drying.
Summary of the invention
Technical problem to be solved of the present invention is to provide a kind of erberry extract, extract of the present invention is used counter-current to prepare, in the erberry extract that this method is extracted, can extract more fully anthocyanidin isoreactivity composition, compare with art methods and there is the feature such as save solvent, extraction efficiency is high, cost is low, be suitable for suitability for industrialized production; And in erberry extract of the present invention, there is induction eosinophil apoptosis and the kinase whose material of activation MBP.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of erberry extract, and adopt countercurrent extraction method to extract and obtain, described countercurrent extraction method comprises the following steps:
Step (1), after raw material is cleaned, pulverize at low temperature; With
Step (2), adds by the raw material after pulverizing the ethanol water that 3-6 weight/volume is doubly measured, pH value is 3-6, mass percent is 10-30%, countercurrent extraction 3-6h at 25-50 ℃; And
Step (3), collects extracting solution, and concentrating under reduced pressure, obtains described erberry extract.
In above-mentioned steps (2), ethanol water addition is preferably 4.5 weight/volume and doubly measures, and pH value is preferably 4, ethanol mass percent preferably 20%; Temperature is preferably 40 ℃; Extraction time is preferably 4h.Weight/volume as herein described is doubly measured and is referred to by weight or volume calculating, and ethanol water is with respect to the multiple of raw material.
Further, described erberry extract has the activity of cell death inducing.
Further, described erberry extract has the activity of induction eosinophil apoptosis.
Further, described erberry extract can activate MBP kinases.
Compared with prior art, tool has the following advantages in the present invention:
(1) erberry extract of the present invention adopts countercurrent extraction method, can to anthocyanidin isoreactivity composition, extract more fully, compare with art methods and there is raw material and process and to be more prone to, to save the features such as solvent, extraction efficiency is high, cost is low, be suitable for suitability for industrialized production.
(2) erberry extract of the present invention is demonstrating good effect aspect minimizing eosinophilic granulocyte, and it can be effectively for preventing and treat the various diseases being caused by too much eosinophilic granulocyte.
(3) erberry extract of the present invention can be used as cell death inducer, antiallergic, antiinflammatory and health food.
Accompanying drawing explanation
Fig. 1 is the mass percent (%) of ethanol and the graph of a relation of leachate anthocyanidin content (mg).
Fig. 2 is the graph of a relation of time of contact (h) and leachate anthocyanidin content (mg).
Fig. 3 be temperature (℃) with the graph of a relation of leachate anthocyanidin content (mg).
Fig. 4 is the schematic diagram of the device that uses of the extracting method of anthocyanidin in a kind of erberry of the present invention.
The specific embodiment
One, extraction process condition is investigated
1, leaching condition experiment
(1) impact of concentration of alcohol on anthocyanidin compound leaching effect
In commercial production, owing to often containing certain moisture in raw material, ethanol also contains certain moisture after recycling, on the leaching of anthocyanidin compound, there is very large impact in concentration of alcohol, therefore anthocyanidin material is carried out to leaching experiment with different concentration ethanol aqueous solution, with anthocyanidin content trace analysis.At concentration of alcohol, on leaching in the impact experiment of anthocyanidin, get respectively 5 parts of raw materials, every part of 40.00g from raw material, test, with each 180.00ml of ethanol water of variable concentrations, adjusting pH value is 4, lixiviate 6h, the amount of comparison gained anthocyanidin, its result is 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% ethanol water as leaching agent.
(2) impact of time of contact on anthocyanidin compound leaching effect
Get respectively 6 parts of raw materials, every part of 40.00g, respectively adding concentration is 20% ethanol 180.00ml, adjusting pH value is 4, leaches, and is respectively 1h time of contact, 2h, 3h, 4h, 5h, 6h, it leaches result as shown in Figure 2.
As shown in Figure 2, under room temperature (26 ℃), extraction time is 4h, and Leaching reaction reaches balance substantially; Extend extraction time, leaching rate is substantially constant.
(3) impact of temperature on anthocyanidin compound leaching effect
Get respectively 6 parts of raw material powders, every part of 40.00g, respectively adding 180.00ml concentration is 20% ethanol, and adjusting pH value is 4.0 at different temperature, to leach, and extraction time is 2h, and sampling detects anthocyanidin content in leachate, and result is as shown in Figure 3.
As shown in Figure 3, along with the increase of extraction temperature, the leaching rate of anthocyanidin also increases accordingly; When temperature reaches 40 ℃, increase temperature, the leaching rate increase of anthocyanidin is tending towards slow.
2, four sections of adverse current percolations (process diagram is shown in Fig. 4)
6 ф 25.4mm * 1000mm glass have been installed altogether and have been drawn together a mouthful pipe, as the infuser (leaching post) of four sections of adverse current percolation leaching experiments, wherein four operations, do week conversion for two.In every post, pack 150.00g erberry raw material powder particle (be called for short raw material, as follows) into, with dosing pump by leaching agent (10-30% ethanol water), with about 5mlmin
-1flow squeeze in first leaching post, ethanol is with about 1cmmin
-1speed percolation, when the first post flows out after 450ml leachate, connects the first post and the second post.The new leaching agent that dosing pump is squeezed into, through the first post, flows out and enters the second post; With upper identical, when the second post flows out after 450ml black leachate, the second post and the 3rd post are connected.The rest may be inferred, and the 4th post flows out after 450ml effluent by the time, and the 4th post and the 5th post are connected.Meanwhile, dosing pump outlet is moved to the second post upper end and the second post from the first post upper end and connect (the first post is withdrawn from from system).Now, new leaching agent, enters the second post through dosing pump, after three, four, five posts, from the 5th post, flows out, and gets equally 450ml leachate; After this, new leaching agent enters the 3rd post, from the 6th post, goes out leachate.The rest may be inferred, constantly loops four sections of adverse current percolation leaching experiments to reaching leaching balance.In this test, the 9th post starts to stop to the 12 post, the leachate that these four posts flow out is leachate during approximate equilibrium, therefore when new leaching agent is from entering from the 9th post, through ten, 11,12 posts, and from the 12 post, flow out after 450ml black leachate, stop leaching test.The leachate sampling that nine, ten, 11,12 posts are flowed out, censorship, the content of mensuration erberry leachate 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 percolation leaching test results
Counter-current described above is tested.Every duplicate samples is 150.0g, take 20% ethanol as leachate, and adjusting pH value is lixiviate 4h at 4,40 ℃, and liquid-solid ratio is 4.5.Get the leachate of nine, ten, 11,12 posts outflows and measure, its result is as shown in table 1.Described liquid-solid ratio refers to that ethanol water is with respect to quality or the volume multiple of sample.
Four sections of adverse current percolation leaching test results of table 1
As known from Table 1, in erberry, anthocyanidin content is in 0.41%, and its anthocyanidin leaching rate of four sections of adverse current percolation solvent extraction method (in leachate) reaches 95.77%(in anthocyanidin).
Two, embodiment: by specific embodiment, the present invention is described in further detail below.
Embodiment 1:
After 1000 grams of raw materials are cleaned, pulverize at low temperature, by the ethanol water that the raw material after pulverizing adds that 3 weight/volume are doubly measured, pH value is 6, mass percent is 10%, countercurrent extraction 6h at 50 ℃, collect extracting solution, concentrating under reduced pressure, obtains 38.32 grams of erberry extracts.In the erberry extract of mensuration gained, anthocyanidin purity is 9.8%, and anthocyanidin leaching rate (in erberry, anthocyanidin content take 0.41%) is 91.59%.
Embodiment 2
After 1000 grams of raw materials are cleaned, pulverize at low temperature, by the ethanol that the raw material after pulverizing adds that 4.5 weight/volume are doubly measured, pH value is 4, mass percent is 20%, countercurrent extraction 4h at 40 ℃, collects extracting solution, and concentrating under reduced pressure, obtains 39.02 grams of erberry extracts.In the erberry extract of mensuration gained, anthocyanidin purity is 10.2%, and anthocyanidin leaching rate (in erberry, anthocyanidin content take 0.41%) is 97.07%.
Embodiment 3
After 1000 grams of raw materials are cleaned, pulverize at low temperature, by the ethanol that the raw material after pulverizing adds that 6 weight/volume are doubly measured, pH value is 3, mass percent is 30%, countercurrent extraction 3h at 25 ℃, collects extracting solution, and concentrating under reduced pressure, obtains 38.06 grams of erberry extracts.In the erberry extract of mensuration gained, anthocyanidin purity is 9.7%, and anthocyanidin leaching rate (in erberry, anthocyanidin content take 0.41%) is 90.04%.
Embodiment 4 eosinophilic granulocyte sexual cell apoptosis induction tests and the experiment of MBP kinase activity
Eosinophilic granulocyte sexual cell apoptosis induction test: the method for experimental evidence Vermes etc. (Journal of Immunological Methods 184 volume 39-51 page nineteen ninety-five) is carried out.Make eosinophilic granulocyte sexual cell HL-60 cell suspension in RPMI1640 culture medium, cell concentration is 1-3 * 10
6cell/ml.In this cell suspending liquid 500 μ l, add detected material (the last erberry extract obtaining of collecting in above-described embodiment 1-3) or culture medium, making the concentration of erberry extract in cell suspending liquid is 100 μ g/ml, at 37 ℃, 5%CO
2condition under cultivate 6 hours or 24 hours.After centrifugal cleaning, add phospholipid phthalein in conjunction with albumen (Annexin) V buffer, then add 5u1 phospholipid phthalein in conjunction with albumen V-FITC.By flow cytometry method, using phospholipid phthalein in conjunction with albumen V positive cell as apoptosis-inducing cell, with the percentage ratio evaluation of relatively total cell number.
MBP kinase activity test: 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 culture medium, cell concentration is 1-3 * 10
6cell/ml.In this cell suspending liquid 500 μ l, add detected material or culture medium, making concentration is 100 μ g/ml, at 37 ℃, 5%CO
2condition under cultivate 6 hours or 24 hours.After centrifugal cleaning, add solubilized buffer, dissolved cell, is used the gel that contains MBP 5mg/ml to carry out electrophoresis.Gel, through de-degeneration, again after degenerative treatments, is used
32p-ATP labelling, implements the phosphorylation reaction of 3 hours.After gel drying, use image analyzer to resolve radioactivity.When 36kDa grows indicia band, note is done MBP kinase activation positive (+).
Erberry extract has been observed the apoptosis-inducing of concentration dependent in HL-60 cell.While carrying out in addition gel MBP kinases analysis (In-gel MBP kinase assay), erberry extract activates 36kDa MBP kinases.Above results suggest.Erberry extract is to eosinophilic granulocyte energy cell death inducing, and this apoptosis is relevant with 36kDa MBP kinases.Infer that thus in erberry extract, contain can be for the composition of anti-eosinophilic granulocyte inflammatory effect medicine.
Acquired results is as shown in table 2 below:
Confirm that embodiment 2 and 3 has strong apoptosis-inducing active, and with the kinase whose activation of MBP.
Table 2
As mentioned above, just can realize preferably the present invention.
Claims (4)
1. an erberry extract, is characterized in that, adopts countercurrent extraction method to extract and obtains, and described countercurrent extraction method comprises the following steps:
Step (1), after raw material is cleaned, pulverize at low temperature; With
Step (2), by the ethanol water that the raw material after pulverizing adds that 4.5 weight/volume are doubly measured, pH value is 4, mass percent is 20%, countercurrent extraction 4h at 40 ℃; And
Step (3), collects extracting solution, and concentrating under reduced pressure, obtains described erberry extract.
2. a kind of erberry extract as claimed in claim 1, is characterized in that, described erberry extract has the activity of cell death inducing.
3. a kind of erberry extract as claimed in claim 1, is characterized in that, described erberry extract has the activity of induction eosinophil apoptosis.
4. a kind of erberry extract as claimed in claim 1, is characterized in that, described erberry extract can activate MBP kinases.
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