CN110051709A - A kind of blackcurrant polyphenol extract and preparation method thereof - Google Patents
A kind of blackcurrant polyphenol extract and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of blackcurrant polyphenol extracts and preparation method thereof, belong to Secondary metabolites and isolate and purify field.It mainly includes the following steps: step 1, using blackcurrant as raw material, is freeze-dried and crushes；Step 2, ethyl alcohol extract；Step 3, centrifugation retain supernatant；Step 4, concentration.Blackcurrant polyphenol of the invention is to angiotensin converting enzyme-I (ACE) inhibitory activity studies have shown that blackcurrant polyphenol has inhibiting effect to ACE.Blackcurrant polyphenol can induce the apoptosis of gastric carcinoma cells MKN-28, and blackcurrant essence proposes the effect of polyphenol and slightly mentions polyphenol better than blackcurrant.
Description
Technical field
The present invention relates to one kind from blackcurrant extract and its polyphenol method, belongs to Secondary metabolites and isolates and purifies
Field.
Background technique
In recent years polyphenol because of it with high security, naturally, the features such as almost non-toxic side effect and molecular weight are small, and become
The hot spot of health food research.As people gradually understand and pay close attention to the special small berries in Northeast China, blackcurrant is due to itself
The higher nutritive value that contains and the concern and attention for having attracted researcher.The polyphenol that it wherein contains, which has, removes oxygen freedom
The oxidation resistance of base possesses potential medical value and nutritive value.Oneself verified polyphenol of many scientific researches is to the heart
Vascular diseases have effects that effectively to prevent.Seek from natural food it is more effective it is safer can control blood pressure it is raised at
Point, it reduces hypertension and drug for hypertension is medical investigator, nutrition expert and food to the negative effect of hypertensive patient
Product scientist's facing challenges, therefore the effectively extraction of polyphenol is particularly important.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of blackcurrant polyphenol, include the following steps:
Blackcurrant is carried out pre-freeze first using blackcurrant as raw material by step 1 under the conditions of -20 DEG C, then with freeze-drying
Equipment removed moisture for blackcurrant vacuum freeze drying 2~3 days, freeze-drying blackcurrant was obtained, finally by the blackcurrant of freeze-drying with ten thousand
Energy pulverising mill carries out being crushed to -80 mesh of 60 mesh, and powder is thinner, and recovery rate is higher, obtains freeze-drying blackcurrant powder, seals at -20 DEG C
Preservation, it is spare.
Step 2 weighs the freeze-drying blackcurrant powder that step 1 obtains, with solid-liquid ratio 1:5-7g/mL (preferably solid-liquid ratio be 1:
Ethanol solution 7g/ml) under 35-45 DEG C of water-bath (preferably 40 DEG C), extract 1.5-3h (preferably 2h), the ethanol solution be containing
(95% ethyl alcohol is 100 ethyl alcohol water by matter for 0.1% acetic acid dehydrated alcohol (100% ethyl alcohol) or 95% ethyl alcohol containing 0.1% acetic acid
Amount matches).
Step 3, the substance that step 2 is obtained, with 7000-9000r/min (preferably 8000r/min), at -8~0 DEG C
(preferably -4 DEG C) are centrifuged 10-30min (preferably 20min), retain supernatant.
Step 4 is concentrated the supernatant that step 3 obtains in vacuum rotary evaporator, when ethyl alcohol all steams
When, it the supernatant of concentration is poured into container (big ware) to be put into refrigerator is refrigerated to whole solidifications.
Solidification sample made from step 4 is carried out vacuum freeze drying, obtains blackcurrant and slightly mention polyphenol by step 5.
Preferably, preparation method further includes step 6: the blackcurrant that step 5 obtains slightly being mentioned polyphenol and purify
Polyphenol is mentioned to blackcurrant essence.
Preferably, the purification step includes:
Step a, the blackcurrant for taking step 5 to obtain slightly mention polyphenol, are dissolved in suitable distilled water, stirring in water bath, completely molten
Xie Houyong needle draws blackcurrant polyphenol leaching liquor, crosses ester phase microfiltration membranes and is cleaned in advance, obtains micro-filtration permeate.
Step b, the micro-filtration permeate obtained using step a is stoste, the ultrafiltration for being 1kDa through molecular cut off (MWCO)
Film obtains the product after ultrafiltration, and operating pressure is no more than 50Pa, and micro-filtration permeate temperature is 25 DEG C.Pay attention to sample being placed on ice
In, prevent polyphenol oxidase.
It is furthermore preferred that further including step c, the product after the ultrafiltration for first obtaining step b is in a reservoir in (culture dish)
It is placed into pre-freeze in -20 DEG C of refrigerator after one layer of thin layer, then freezes 8h in -80 DEG C of refrigerator.Then solid will be frozen into
Sample freeze dryer place 48h, slough moisture, finally scrape sample in centrifuge tube, deposit in -20 DEG C of refrigerator.
The present invention also provides a kind of blackcurrant polyphenol extract product preparation method, by the blackcurrant slightly mention polyphenol and
The blackcurrant essence mentions polyphenol and is dissolved separately in phosphoric acid solution or sodium carbonate liquor, and concrete operations can are as follows: by step 5 and
The blackcurrant that step c is obtained slightly mentions polyphenol and blackcurrant essence mentions polyphenol and prepared respectively, first weighs the sample that step 5 obtains
The sample obtained with step c is configured to the mother liquor of 10mg/mL, is then diluted to different concentration, manner of formulation one by one are as follows:
It is prepared with phosphate buffer solution or sodium carbonate buffer.
The present invention extracts product using a kind of blackcurrant polyphenol that the preparation method of blackcurrant polyphenol obtains as described above,
Blackcurrant polyphenol concentration is greater than 750 μ g/mL.
It is furthermore preferred that above-mentioned blackcurrant polyphenol concentration is 1-10mg/mL.
It is furthermore preferred that above-mentioned blackcurrant polyphenol concentration is 2.5-10mg/mL.
The present invention also provides a kind of blackcurrant polyphenol to extract product, and polyphenol extracts molecular weight < 1kDa of product.
Beneficial effect
The present invention extracts preparation containing the blackcurrant compared with high polyphenolic content and antioxidant activity using organic solvent extractionprocess
Extract --- blackcurrant polyphenol, and molecular cut off < 1kDa blackcurrant polyphenol is prepared for using membrane separation process, then distinguish
ACE (angiotensin converting enzyme-I) activity suppression research and antitumor reality have been carried out to the blackcurrant polyphenol before and after UF membrane
It tests.Blackcurrant polyphenol of the invention is to angiotensin converting enzyme-I (ACE) inhibitory activity studies have shown that blackcurrant polyphenol pair
ACE has inhibiting effect.Blackcurrant polyphenol can induce the apoptosis of gastric carcinoma cells MKN-28, and blackcurrant essence proposes the effect of polyphenol
Polyphenol is slightly mentioned better than blackcurrant.
Detailed description of the invention
Fig. 1 is polyphenol concentration standard curve figure；
Fig. 2 is thick polyphenol (i.e. blackcurrant slightly mentions polyphenol) hydroxyl radical free radical clearance rate figure；
Fig. 3 is molecular cut off < 1kDa sample (i.e. blackcurrant essence mentions polyphenol) hydroxyl radical free radical clearance rate figure；
Fig. 4 is thick polyphenol DPPH clearance rate figure；
Fig. 5 is molecular cut off < 1kDa sample DPPH clearance rate figure；
Fig. 6 is Trolox canonical plotting；
Fig. 7 is inhibiting rate figure of the thick polyphenol to ACE；
Fig. 8 is the inhibiting rate figure of molecular cut off < 1kDa polyphenol (i.e. blackcurrant essence mentions polyphenol) to ACE；
Fig. 9 is influence diagram of the thick polyphenol to cell survival rate；
Figure 10 is influence diagram of molecular cut off < 1kDa polyphenol to cell survival rate.
Specific embodiment
Embodiment of the present invention is described in detail combined with specific embodiments below.It should be appreciated that reality of the invention
It applies and is not limited by the following examples, the accommodation in any form and/or change made to the present invention fall within this hair
Bright protection scope.
In a wherein example, the extraction of blackcurrant polyphenol and its membrane separating and purifying includes the following steps:
Blackcurrant pretreatment → extraction → centrifugation → concentration → freeze-drying → blackcurrant slightly mention polyphenol → ultrafiltration → black plus
Logical sequence essence mentions polyphenol；
(1) blackcurrant pre-processes
Using blackcurrant as raw material, blackcurrant is subjected to freezing 1h under conditions of -20 DEG C first, then use freeze drying equipment
By blackcurrant, vacuum freeze drying 48h removes moisture under conditions of vacuum degree is 10 pas, temperature is -20 DEG C, is lyophilized
Blackcurrant.The blackcurrant of freeze-drying is crushed with high speed Universal pulverizer (Tianjin Stettlen Instrument Ltd.), crosses 60
Mesh obtains freeze-drying blackcurrant powder, spare in -20 DEG C of sealing preservations.
(2) organic solvent extracts
The freeze-drying blackcurrant powder that 100g step (1) obtains is weighed, is added to the ethanol solution of 700mL and (contains 0.1% second
Acid) in, at 40 DEG C of water-bath, extract 2h.
(3) it is centrifuged
The liquid that step (2) is obtained is centrifuged 20min with 8000r/min at -4 DEG C, retains supernatant.
(4) it is concentrated
Supernatant obtained in step (3) is concentrated in vacuum rotary evaporator, revolving 1h to no liquid is steamed,
When ethyl alcohol all steams, the supernatant of concentration is poured into big ware and is put into -20 DEG C of refrigerator freezings for 24 hours to whole solidifications.
(5) it is freeze-dried
It is cold that consolidated article obtained in step (4) is subjected to vacuum under conditions of vacuum degree is 8 pas, temperature is -25 DEG C
It dry 36h is lyophilized obtains blackcurrant and slightly mention polyphenol.
(6) ultrafiltration
A. micro-filtration cleans
It takes blackcurrant slightly to mention polyphenol, is dissolved in suitable distilled water, stirring in water bath, draw blackcurrant with needle after being completely dissolved
Polyphenol leaching liquor crosses organic phase microfiltration membranes (ester phase microfiltration membranes) (Nanjing Shou De experiment equipment Co., Ltd-Jin Long pin filtering
Device, aperture are 0.45 μm) it is cleaned in advance.
B. ultrafiltration
Using micro-filtration permeate as stoste, ultrafiltration is carried out through the ultrafiltration membrane that molecular cut off (MWCO) is 1kDa and obtains ultrafiltration
Product afterwards, operating pressure are no more than 50Pa, 25 DEG C of micro-filtration permeate temperature.Pay attention to for the product after ultrafiltration being placed in ice, prevent
Only polyphenol oxidase.
C. it is lyophilized
Product after ultrafiltration falls first in multiple culture dishes and is placed into pre-freeze 2h in -20 DEG C of refrigerator after one layer of thin layer,
Then in -80 DEG C of refrigerator freezing 8h, then the sample for being frozen into solid is placed in -20 DEG C of freeze dryer and is freeze-dried 48h, is sloughed
Moisture finally scrapes sample in centrifuge tube, puts that -20 DEG C of refrigerators are spare to mention polyphenol to get to blackcurrant essence.
In other examples, change different extraction conditions, obtain different recovery rates:
One of example is, the above-mentioned example the step of in (2), change solid-liquid ratio is 1:5, and other conditions are constant, i.e.,
The freeze-drying blackcurrant powder that 100g step (1) obtains is weighed, is added to the ethanol solution of 500mL, polyphenol recovery rate is
58.83%.
In another example, the above-mentioned example the step of in (2), change solid-liquid ratio is 1:6, and other conditions are constant, i.e.,
The freeze-drying blackcurrant powder that 100g step 1 obtains is weighed, is added to the ethanol solution of 600mL, polyphenol recovery rate is
62.56%.
In another example, the above-mentioned example the step of in (2), changing temperature is water-bath (25 DEG C) under normal temperature condition,
Other conditions are constant, polyphenol recovery rate 63.36%.
In another example, the above-mentioned example the step of in (2), change extraction time is 1h, and other conditions are constant, more
Phenol extraction rate is 60.47%.
The measuring method of blackcurrant polyphenol content is as follows:
(1) drafting of polyphenol concentration standard curve
It is as follows to draw standard curve method:
Prepare the gallic acid titer of 0.1mg/mL；7 50mL volumetric flasks are taken, 0,1,2,3,4,5 and 6mL is sequentially added
Gallic acid titer simultaneously marks；Then the forint phenol (room temperature reaction 5min) that 5mL concentration is 10wt% is added, adds 4mL
Concentration is the saturated sodium carbonate solution of 7.5wt%, and constant volume simultaneously shakes up, and is protected from light 30 DEG C of water-bath 1h；Use No. 0 sample as blank pair
According to the absorbance (each sample is measured in parallel three times) of measurement solution, the polyphenol concentration standard curve drawn at 765nm
As shown in Figure 1；
The calculating of blackcurrant polyphenol recovery rate is indicated with the percentage that the polyphenol extracted accounts for total polyphenols in pomace.
Total phenol yield w (%)=m/M × 100 (1)
In formula, total phenol quality in m-extracting solution, unit g；
M-sample quality, unit g；
(2) measurement of blackcurrant polyphenol content
Folin-Ciocalteu colorimetric method is used to carry out the measurement of polyphenol content: using gallic acid as standard items, in room
Under the conditions of temperature, wavelength is that absorbance is measured at 765nm, and the content of polyphenol is calculated according to standard curve.According to GB/T 8313-
2008 method: take supernatant obtained in 1mL step (3) in 100mL volumetric flask, addition 5.0mL concentration is 10wt%'s
Forint phenol reagent reacts 5min, and the saturated sodium carbonate solution that 4mL concentration is 7.5wt% is added, distilled water is added to be settled to scale simultaneously
And it shakes up.30 DEG C are protected from light water-bath 1h, with fluorescence microplate reader (the full-automatic Bio-Tek company, the microplate reader U.S. of ELX800) in 765nm
The absorbance of place's measurement solution.Polyphenol content is calculated with gallic acid standard curve, formula:
Note: in formula: C --- gallic acid mass concentration, μ g/mL；V --- extract supernatant volume, mL；N --- dilution
Multiple；M --- sample volume, mL；W --- polyphenol content, μ g/mL.
The active measuring method of blackcurrant polyphenol ACE is as follows:
The preparation (totally 7 concentration) of blackcurrant polyphenol test fluid: being respectively 1mg/mL, 750 μ g/, 500 μ g/mL, 250 μ g/
ML, 100 μ g/mL, 50 μ g/mL, 10 μ g/mL, are directly prepared with phosphate buffer solution or sodium carbonate buffer.
ACE inhibiting rate is measured using 96 orifice plates: 40uL test fluid, the FAPGG of 50uL1.0mM being successively added in 96 orifice plates
(0.0012g FAPGG+3mL HEPES, which is vortexed, to be shaken), 10uL ACE.Control group replaces test with the HEPES buffer solution of 40uL
Liquid.5min is preheated at 37 DEG C, the change rate of 30min internal absorbance is measured under 340nm wavelength, every 30s reads primary.According to
Following equation calculates ACE activity suppression.
Note: Δ A in formulaSampleWith Δ ABlankIt is to represent blackcurrant sample and blank sample ACE activity change per minute respectively
Rate.
Mtt assay is as follows to the determination step of blackcurrant extract induction stomach cancer cell MKN-28 apoptosis:
(1) recovery of cell
The stomach cancer cell MKN-28 that freezen protective is taken out from liquid nitrogen container is then immersed in 37 DEG C of warm water (speed is melted), the phase
Between rock and enable its thawing several times, 2min or so, taking-up when there remains the big ice cube of mung bean in pipe is melted using residual temperature；Open lid
Son draws cell suspension, is transferred to new EP pipe, adds the fresh culture medium of 8-10mL, and FBS10% is added in RPMI-1640,
1% dual anti-(playing the role of diluting DMSO, washing away serum etc. freezing substance), 3min centrifugation, 1000r/min；Liquid is discarded supernatant,
Cell is resuspended in the fresh medium that 1mL rewarming is added, and 6 orifice plates have added culture medium, and every plate spreads the above-mentioned resuspension cell liquid of 500 μ L, writes
Upper cell category, operator, date mix.(or fresh medium is added, cell is resuspended, it counts, adjusts cell density, inoculation
Into deep bid)；Containing 5%CO2Incubator in 37 DEG C of constant temperature cultures, culture solution replace after 4~6h, is removed extremely thin
Born of the same parents continue to cultivate.+ 2,4 holes (every 90 μ L of pore capacities) 60mm (diameter) ware that a general cryopreservation tube can spread 6 orifice plates is (every
300 μ L of ware capacity).
(2) passage of cell
Inverted microscope shows that, when cell will cover ware bottom, cell starts to pass on；Draw old culture medium, it is added 1~
2mLPBS is cleaned 1~2 time, exhausts PBS (adding against wall, not break up cell)；1~2mL, 0.25% trypsin solution is added, inclines
Tiltedly shaking makes solution not have ware bottom, and cell dissociation situation is observed under inverted microscope, until until 90% attached cell is rounded
(general 20~30s)；About 3~5 times of volumes of the culture medium containing FBS are added and terminate digestion；It is gently blown and beaten with 1mL pipette tips, using up can
Cell more than energy is suspended in culture solution.The cell suspension of preparation is gone in the centrifuge tube of a new 15mL, is centrifuged 3min,
1000r/min discards supernatant liquid；2~3mL fresh medium resuspension cell is added (if cell is too many, to discard after can blowing and beating uniformly
A part, then plus culture solution dilution), it is transferred in new culture dish with volume ratio 1:2 or 1:3 ratio, adds fresh medium, write
Upper cell category, operator, date, up and down or eight words mix.(100mm ware adds 7~8mL, and 60mm ware adds 3~4mL)；37
DEG C, volumetric concentration 5%CO2Continue to cultivate.
(3) MTT experiment method and process are as follows:
The density of cell is 5000~10000 cells/wells, is increased using 0.25% trypsin digestion logarithm of mass percent
Long-term MKN-28 stomach cancer cell is resuspended in 1640 complete culture solution of RPMI of 10% calf serum containing mass percent, root
It is adjusted to the single cell suspension of various concentration according to drug treating time difference, is inoculated in 96 orifice plates, every 200 μ L of hole.At 37 DEG C,
Add 5% CO2Humidified incubator in cultivate for 24 hours, after cell is adherent, discard culture solution, sample (complete culture solution be added.
Then for 24 hours by the culture solution (DOX) and polypeptide co-incubation of cell and various concentration, make in hole sample concentration (respectively
0.625,1.25,2.5,5,2.5,5,7.5,10mg/mL.The complete culture solution control group and solvent control that drug is not added are set simultaneously
Group, every group of 3 multiple holes.After being put into incubator for 24 hours, culture medium is removed, rinses cell with preheating PBS.By 120 μ L- methyl
Azo azoles salting liquid (MTT) (1mg/mL is dissolved in culture medium) is added to 4h on every mouthful of plate.After discarding culture medium, 50 μ are added
L dimethyl sulfoxide (DMSO), micro-mixer shake 20min, after making to crystallize sufficiently dissolution, mixing in Microplate Reader
The absorbance value on plate is read at 540nm.
Note: A --- absorbance
Measurement result is as follows:
1. the extraction of blackcurrant polyphenol
Measuring blackcurrant under the wavelength of 765nm and slightly mentioning the light absorption value of polyphenol is 0.0842, according to the standard curve in Fig. 1
It is about 3.8 μ g/mL that available blackcurrant, which slightly mentions polyphenol content in polyphenol,；Blackcurrant essence mentions the light absorption value in polyphenol
0.0910, slightly mentioning polyphenol polyphenol content according to the available blackcurrant of standard curve in Fig. 1 is about 4.14 μ g/mL.Explanation mentions
After pure, blackcurrant slightly proposes the increase of the polyphenol purity in polyphenol.
Blackcurrant is extracted repeatedly to colourless, is calculated according to formula (1), 10g freeze-drying has been shared in this experimentation
Blackcurrant powder is extracted 5 times altogether, it can thus be concluded that blackcurrant when once being extracted to blackcurrant polyphenol slightly proposes polyphenol yield is
67.85%.
2. hydroxyl radical free radical (OH) Scavenging activity of blackcurrant polyphenol
From Fig. 2 and Fig. 3 it is found that whether blackcurrant slightly mentions polyphenol or blackcurrant essence mentions polyphenol, hydroxyl radical free radical is clear
Except rate rises with the raising of polyphenol concentration.When its concentration is less than 250 μ g/mL, Scavenging activity improves unobvious；When dense
When degree is in 250-750 μ g/mL, as polyphenol concentration increases, Scavenging activity obviously gets a promotion；When concentration is greater than 750 μ g/mL
When, Scavenging activity has certain promotion.And under same concentrations, the sample (i.e. essence mentions polyphenol) of molecular weight < 1kDa is compared
Polyphenol free radical scavenging activity, which is slightly mentioned, in blackcurrant about improves 5%-7%.Blackcurrant polyphenol in low concentration, due to its content compared with
It is few effectively to inhibit or block free chain reaction quickly by great amount of hydroxy group free-radical oxidation, pass through chain reaction freedom
Base is lived again quickly, therefore its hydroxyl radical free radical clearance rate is lower when concentration is less than 250 μ g/mL；When blackcurrant polyphenol is further
Rise, as its concentration increases, hydroxyl radical free radical can not be regenerated by free chain reaction in time after being removed, therefore more
In 250-750 μ g/mL, hydroxyl radical free radical clearance rate significantly improves phenol concentration；When blackcurrant polyphenol concentration continues to increase,
Due to can not be thoroughly fully erased by hydroxyl radical free radical, free chain reaction be still had, therefore hydroxyl radical free radical clearance rate
It tends to be steady.Under a certain concentration, product all has higher hydroxyl radical free radical Scavenging activity, namely shows it with good
Antioxidant effect.
3. the DPPH Scavenging activity of blackcurrant polyphenol
DPPH can capture electronics, can be used as effective monitoring agent of radical reaction, can be seen that from Fig. 4 and Fig. 5
DPPH clearance rate has significant rising with the increase of sample concentration, but when concentration is greater than 600 μ g/mL, the promotion of clearance rate
Slightly slow down.The DPPH clearance rate of molecular weight < 1kDa polyphenol is promoted than slightly mentioning polyphenol under same concentrations, but is compared
For overall DPPH clearance rate, promoted it is not fairly obvious, only 5% or so.Blackcurrant polyphenol is in low concentration, since it contains
Measure it is less can not effectively inhibit or block free chain reaction quickly by a large amount of free-radical oxidations, it is free to pass through chain reaction
Base is lived again quickly, therefore its free radical scavenging activity is lower when concentration is less than 150 μ g/mL；When blackcurrant polyphenol is further up,
As its concentration increases, free radical can not be regenerated by free chain reaction in time after being removed, therefore be existed in polyphenol concentration
When 300-600 μ g/mL, free radical scavenging activity is significantly improved；When blackcurrant concentration continues to increase, due to can not be thoroughly by hydroxyl
Base free radical is fully erased, and free chain reaction still has, therefore the rising of hydroxyl radical free radical clearance rate is slowed down.Certain dense
Under degree, product all has higher DPPH Scavenging activity, namely shows it with good antioxidant effect.
4. the ORAC value (total antioxidant capacity index) of blackcurrant polyphenol
Resulting Trolox standard curve is drawn by above-mentioned experimental program, such as Fig. 6:
After converting blackcurrant slightly mention polyphenol and blackcurrant essence mention polyphenol ORAC value it is as follows:
The ORAC value that blackcurrant slightly mentions polyphenol is 467.69 μm of ol/mg；
The ORAC value that blackcurrant essence mentions polyphenol is 492.74 μm of ol/mg.
Carrying out analysis to data can be seen that blackcurrant polyphenol with outstanding ORAC value, be 3 times of left sides of typical apple
The right side, is the fruit with high concentration Natural Antioxidants rare in nature, and blackcurrant slightly mentions polyphenol and tentatively mentioned
After pure, ORAC value has promotion by a small margin, there is the potentiality further refined.
5. blackcurrant polyphenol is to the activity suppression of ACE
From the figure we can see that the increase that the inhibiting rate of ACE proposes polyphenol concentration with blackcurrant essence is positively correlated, explanation
Blackcurrant blackcurrant essence, which mentions polyphenol, can inhibit the activity of angiotensin converting enzyme-I.Fig. 7 is compared with Fig. 8, when dense
When degree is all 1120 μ g/mL, it is 73.17% to the inhibiting rate of ACE that blackcurrant, which slightly mentions polyphenol, and blackcurrant essence mentions polyphenol to ACE's
Inhibiting rate is 84.25%, so blackcurrant essence, which mentions polyphenol, slightly mentions polyphenol higher than blackcurrant to the inhibiting rate of ACE.Pass through what is measured
Experimental data can obtain, and blackcurrant slightly mentions the half-inhibitory concentration (IC of polyphenol50) it is about 310 μ g/mL, blackcurrant essence proposes polyphenol group
IC50Value is 210 μ g/mL, is illustrated after proposing (purification) by membrane separation technique essence, the polyphenol purity in extract also has to be mentioned greatly very much
It rises.
6. blackcurrant extract induces tumour cell (MKN-28 stomach cancer cell) apoptosis
Polyphenol substance abundant is rich in blackcurrant, blackcurrant polyphenol has outstanding antioxygenic property, hydroxyl free
Base clearance rate reaches 3 times of left sides of typical apple using 25%, DPPH Scavenging activity is reached after purification close to 90%, ORAC value
It is right --- therefore 492.74 μm of ol/mg are worth with huge deep processing, such as antioxidant is produced in exploitation from blackcurrant
Technology and anti-aging type health product.
Using mtt assay measurement blackcurrant polyphenol to the inhibiting effect of stomach cancer cell MKN-28, Fig. 9 and Figure 10 are as a result seen.It can
To find out, blackcurrant polyphenol can induce the apoptosis of stomach cancer cell MKN-28.When incubation time is consistent, 0.625,1.25,
2.5,5,7.5,10mg/mL blackcurrant slightly mention the cell survival rate of polyphenol be respectively 85.09%, 53.89%, 23.56%,
18.72%, 13.85%, 7.86%, blackcurrant essence mention polyphenol cell survival rate be 59.55%, 32.38%, 10.41%,
7.4%, 0.45%, 0.11%.Illustrate that blackcurrant polyphenol concentration is higher, cell survival rate is lower.When blackcurrant slightly mention polyphenol with
Blackcurrant essence mention polyphenol concentration it is identical when, blackcurrant essence mentions the cell survival rate in polyphenol group significantly lower than thick polyphenol group, explanation
Blackcurrant essence through membrane separation mentions polyphenol for inducing the effect of tumour cell (MKN-28 stomach cancer cell) apoptosis to add better than black
Logical sequence slightly mentions polyphenol.And when blackcurrant essence proposes polyphenol concentration and reaches 7.5mg/mL, cell survival rate is already close to 0.Fig. 9 and 10
Middle discovery slightly mentions polyphenol from the blackcurrant within this concentration range of 2.5~10mg/mL and blackcurrant essence proposes polyphenol group, they it
Between cell survival rate difference be not very big, and the cell survival rate between 0.625~2.5mg/mL this concentration range
Difference is apparent.
Since blackcurrant essence mentions polyphenol to the remarkable effect of induction MKN-28 apoptosis in gastric cancer, illustrate the two of same concentration
In group solution, it is higher that blackcurrant essence proposes the polyphenol purity in polyphenol.
The present invention studies the stability of blackcurrant extract, contains in 60 DEG C of heating to polyphenol in blackcurrant polyphenol
The influence of amount is not significant (P>0.05), and 80 DEG C of heating cause content to significantly reduce (P<0.05), and 100 DEG C of heating can then make polyphenol
The amplitude that content reduces greatly increases, and heating temperature is higher, the time spent is longer, and polyphenol content is lower.This experimentation
In temperature be no more than 55 DEG C, reduce temperature and time to the greatest extent influences caused by polyphenol content.
From the above experimental results, we know that:
1, the measurement of polyphenol content is carried out using Folin-Ciocalteu colorimetric method: slightly mentioning polyphenol in freeze-drying blackcurrant powder
Content is 3.796 μ g/mL, and it is about 4.14 μ g/mL that essence, which proposes the content of polyphenol, is in addition also surveyed to the total phenol content of blackcurrant
It is fixed, and the recovery rate that blackcurrant polyphenol once extracts is calculated with this and reaches 67.85%.
2, pass through blackcurrant polyphenol to angiotensin converting enzyme-I (ACE) inhibitory activity research shows that blackcurrant polyphenol
There is inhibiting effect to ACE.Blackcurrant polyphenol can be obtained to activity suppression the mentioning with thick polyphenol sample concentration of ACE by Fig. 2, Fig. 3
It is high and improve.When thick polyphenol sample concentration is 112,280,560 μ g/mL, ACE inhibiting rate is changed greatly, respectively
21.05%, 45.28%, 69.40%, and the polyphenol group through membrane separation is also to change greatly between these concentration.Blackcurrant
It slightly mentions polyphenol and blackcurrant essence mentions the IC of polyphenol50Value is respectively 310 μ g/mL and 210 μ g/mL.
3, blackcurrant polyphenol can induce the apoptosis of gastric carcinoma cells MKN-28, and blackcurrant essence proposes the effect of polyphenol and is better than
Blackcurrant slightly mentions polyphenol.When two kinds of sample concentrations are 0.625mg/ml, blackcurrant essence mentions the cell survival in polyphenol sample
Rate is 59.55%, and it is 85.09% that blackcurrant, which slightly proposes polyphenol cells in sample survival rate, when two kinds of sample concentrations are 2.5mg/
When ml, the cell survival rate in two kinds of samples is respectively 10.4% and 23.6%, when two kinds of sample concentrations are 10mg/ml,
Cell survival rate in two kinds of samples is respectively 0.11% and 7.86%, it can be seen that essence mentions the exhausted big portion in polyphenol sample at this time
Divide tumour cell apoptosis.
Claims (8)
1. a kind of preparation method of blackcurrant polyphenol extract, includes the following steps:
Blackcurrant is carried out pre-freeze first by step 1, then with freeze drying equipment by blackcurrant vacuum freeze drying 2-3 days,
Moisture is removed, the blackcurrant being lyophilized finally crushes the blackcurrant of freeze-drying, obtains freeze-drying blackcurrant powder, freezes close
Preservation is sealed, it is spare；
Step 2 weighs the freeze-drying blackcurrant powder that step 1 obtains, and is added after ethanol solution by the solid-liquid ratio of 1:5-7g/ml in temperature
1.5-3h is extracted in the water-bath that degree is 35-45 DEG C；
Step 3, the substance that step 2 is obtained, with the revolving speed of 7000-9000r/min, under the conditions of -8~0 DEG C of temperature from
Heart 10-30min retains supernatant；
Step 4 is concentrated the supernatant that step 3 obtains in vacuum rotary evaporator, will when ethyl alcohol all steams
The supernatant of concentration, which pours into container to be put into refrigerator, is refrigerated to whole solidifications；
Solidification sample made from step 4 is carried out vacuum freeze drying, obtains blackcurrant and slightly mention polyphenol by step 5.
2. a kind of preparation method of blackcurrant polyphenol extract according to claim 1, which is characterized in that the crushing is
Refer to and the blackcurrant of freeze-drying is crushed to -80 mesh of 60 mesh.
3. a kind of preparation method of blackcurrant polyphenol extract according to claim 1, which is characterized in that the step
In two: solid-liquid ratio 1:7g/ml, bath temperature are 40 DEG C, extraction time 2h.
4. a kind of preparation method of blackcurrant polyphenol extract according to claim 1, which is characterized in that the step
In three: revolving speed 8000r/min, temperature are -4 DEG C, centrifugation time 20min.
5. a kind of preparation method of blackcurrant polyphenol extract according to claim 1, which is characterized in that further include step
Six: the blackcurrant that step 5 obtains slightly being mentioned into polyphenol purified to obtain blackcurrant essence and mention polyphenol.
6. a kind of preparation method of blackcurrant polyphenol extract according to claim 5, which is characterized in that the purification
Step includes:
Step a, the blackcurrant for taking step 5 to obtain slightly mention polyphenol, are dissolved in suitable distilled water, stirring in water bath, after being completely dissolved
Blackcurrant polyphenol leaching liquor is drawn with needle, ester phase microfiltration membranes is crossed and is cleaned in advance, obtain micro-filtration permeate；
Step b, the micro-filtration permeate obtained using step a are carried out as stoste using through the ultrafiltration membrane that molecular cut off is 1kDa
Ultrafiltration obtains the product after ultrafiltration, and operating pressure is no more than 50Pa, and micro-filtration permeate temperature is 25 DEG C.
7. a kind of preparation method of blackcurrant polyphenol extract according to claim 6, which is characterized in that the purification
Step further includes step c: falling the product after ultrafiltration that step b is obtained in a reservoir be placed into -20 DEG C after one layer of thin layer first
Pre-freeze in refrigerator then freezes 8h in -80 DEG C of refrigerator, is then freeze-dried the sample for being frozen into solid in freeze dryer
48h is finally scraped in centrifuge tube, is deposited in -20 DEG C of refrigerator.
8. using blackcurrant polyphenol extract made from preparation method as claimed in claims 6 or 7, it is characterized in that: blackcurrant
Molecular weight < 1kDa of polyphenol extract.
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| CN111249321A (en) * | 2020-03-27 | 2020-06-09 | 东北农业大学 | Method for simultaneously preparing black currant polyphenol and polysaccharide |
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| CN111035693A (en) * | 2020-01-10 | 2020-04-21 | 中国科学院西北高原生物研究所 | Anthocyanin composition with good stability |
| CN111249321A (en) * | 2020-03-27 | 2020-06-09 | 东北农业大学 | Method for simultaneously preparing black currant polyphenol and polysaccharide |
| CN111249321B (en) * | 2020-03-27 | 2022-01-18 | 东北农业大学 | Method for simultaneously preparing black currant polyphenol and polysaccharide |
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