CN104547112A - Red raspberry extract and application thereof in preparation of intestinal cell oxidative damage inhibitor - Google Patents
Red raspberry extract and application thereof in preparation of intestinal cell oxidative damage inhibitor Download PDFInfo
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- CN104547112A CN104547112A CN201510019573.3A CN201510019573A CN104547112A CN 104547112 A CN104547112 A CN 104547112A CN 201510019573 A CN201510019573 A CN 201510019573A CN 104547112 A CN104547112 A CN 104547112A
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Abstract
The invention discloses a red raspberry extract. The red raspberry extract consists of the following components in percentage by mass: 15.6-22.3% of cyanidin-3-glucoside, 3.5-6.4% of cyanidin-3-rutinoside, 40.8-49.2% of cyanidin-3-sophoroside, 4.7-8.1% of cyanidin-3-glucose rutinoside, 11.3-16.8% of pelargonidin-3-glucoside and 5.1-8.5% of pelargonidin-3-rutinoside. An extraction method comprises the following steps: sequentially performing enzymatic hydrolysis on fresh red raspberry fruits by using pepsin and trypsin, then performing centrifugal separation to obtain supernatant liquid, filtering the supernatant liquid by virtue of an ultra-filtration membrane, intercepting components of which the molecular weight is more than 10kDa, and performing vacuum freeze drying on a filtrate to obtain the red raspberry extract. The invention also discloses an application of the red raspberry extract in preparation of an intestinal cell oxidative damage inhibitor.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of red raspberry extract and preparing the application in intestinal cell oxidative damage inhibitor.
Background technology
Free radical is considered to one of reason causing human senility and some chronic disease to occur, when the free radical in human body produces too much or when removing slow, will transfer attack various organelle and make it to sustain damage, thus accelerate the aging course of body and bring out various disease.
Small intestinal is a part for digestive system, is the main place that human body absorbs.Intestinal health has material impact to health, if intestinal cell damaged, human nutrition absorbs and will be affected.
Urethanes (Ethyl carbonate, EC) be a kind of material that may be carcinogenic, be the material of food natural generation in fermentation or storage process, be prevalent in fermented food and beverage, after absorption of human body, potential threat existed to health.Intestinal is owing to being the main place that human consumption absorbs, and intestinal cell easily sustains damage after contact urethanes, and human nutrition absorbs and will be affected.
Therefore, develop a kind of material intestinal injury being had to protective effect, the intestinal injury especially caused by urethanes has the material of protective effect to be necessary.
Red raspberry (Red Raspberry) is Rosaceae (Rasaceae) rubus (Rubus.L) Berry plant, has another name called rasp berry, pallet, Malin, Fructus Rubi etc.1993, FAO (Food and Agriculture Organization of the United Nation) (FAO) recommended Fructus Rubi to be in the world " third generation fruit ".Red raspberry fruits is cerise time ripe, the tender succulence of fruit, color and luster are pleasant, delicate mouthfeel, sweet and sour taste, local flavor fragrance, containing mineral elements such as abundant Vc, Ve, Va, Vb, protein, edible fiber and K, Fe, Zn, Ca, be especially rich in Polyphenols, flavonoid, SOD, the sour isoreactivity material of tanning.Active component in fruit can remove Superoxide anion free radical in human body, has anticancer, health care and to wait for a long time effect, also have strengthening immune system function simultaneously, improve blood circulation, and the effect such as reduce blood pressure.But at present, not for the research of red raspberry extract in protection intestinal cell oxidative damage.
Summary of the invention
The invention discloses a kind of red raspberry extract, adopt the mode of simulation human gastrointestinal tract digestion to prepare.Red raspberry extract of the present invention contains the effective ingredient that human body finally absorbs, and owing to adopting in-vitro simulated mode, remains effective ingredient to greatest extent, and it is functional has practicality; Traditional method adopts 80% acetone extraction, and after its composition enters human body, wherein have part active substance and decomposed digestion by digestion, thus functional have unpredictability, can not represent the truth after entering human body.
A kind of red raspberry extract, extraction step is as follows:
(1) mass mixing such as red raspberry fresh fruit and water is pulled an oar, and adds HCl and regulates pH=1.5 ~ 2 of homogenate, then add pepsin, 37 DEG C, water-bath vibration 1.5 ~ 3h under 100 ~ 200rpm;
(2) get the mixed serum that step (1) obtains, add NaOH and regulate pH=6 ~ 7, then add pancreatic juice, then use NaHCO
3regulate pH to 7.4,37 DEG C, after 100 ~ 200rpm water-bath vibration, 2 ~ 3h, more centrifugal 5 ~ 10min obtains supernatant under 5000 ~ 8000rpm;
(3) get the supernatant of step (2), after ultrafiltration membrance filter, molecular cut off is greater than the composition of 10kDa, and filter liquor, through vacuum lyophilization, obtains described red raspberry extract;
The composition of described red raspberry extract: Cy-3-G (cyanidin-3-glucoside), cyanidin-3-rutinoside (cyanidin 3-rutinoside), cyanidin-3-sophoroside (cyanidin-3-sophoroside), cyanidin-3-glucose rutinoside (cyanidin 3-glucosylrutinoside), pelargonin-3-glucoside (Pelargonidin-3-O-glucoside) and pelargonin-3-rutinoside (Pelargonidin-3-O-rutinoside), mass percentage is as follows:
As preferably, in step (1), the concentration of described HCl is 5mol/L.
As preferably, in step (1), described pepsic addition is 5000 ~ 7000 unit pepsin/20g homogenate.
As preferably, in step (2), the concentration of described NaOH is 1mol/L;
Described NaHCO
3concentration be 1mol/L.
As preferably, in step (2), in described pancreatic juice, the content of pancreatin is 4 ~ 5mg/mL, and the content of cholate is 25 ~ 30mg/mL;
The addition of described pancreatic juice is 5mL/18mL mixed serum.
The invention also discloses the novelty teabag of described red raspberry extract in the inhibitor preparing intestinal cell oxidative damage, expand the application of blackberry.
Further disclose the application of described red raspberry extract in the inhibitor preparing the intestinal cell oxidative damage of being induced by urethanes.
As preferably, in described inhibitor, the effective dose of red raspberry extract is 1mg/mL.
Compared with prior art, the present invention has the following advantages:
The mode that the present invention simulates human gastrointestinal tract digestion prepares blackberry extract.
The oxidative damage that Late Cambrian of the present invention red raspberry extract can effectively suppress urethanes to be induced is a material with good DEVELOPMENT PROSPECT;
The present invention is that red raspberry extract provides new purposes, has expanded a new application.
Accompanying drawing explanation
Fig. 1 is that the red raspberry extract of embodiment 1 preparation is to the protective effect of the Caco-2 cell oxidative damage that urethanes is induced;
Fig. 2 is that the red raspberry extract of embodiment 1 preparation is to the inhibitory action of the Caco-2 cytoactive oxygen-derived free radicals ROS (DCF fluorescence) that urethanes is induced;
Fig. 3 is that the red raspberry extract of embodiment 2 preparation is to the protective effect of the Caco-2 mitochondrial membrane potential in anoxic (MMP) that urethanes is induced;
Fig. 4 is the inhibitory action that the red raspberry extract of embodiment 2 preparation reduces the Caco-2 cellular glutathione content that urethanes is induced;
Fig. 5 is that the red raspberry extract of embodiment 3 preparation is to the inhibitory action of the Caco-2 nucleus damage that urethanes is induced.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, and what below enumerate is only specific embodiments of the invention, but protection scope of the present invention is not limited in this:
Embodiment 1
Red raspberry fresh fruit and water mixed in equal amounts are pulled an oar, with 5M HCl, red raspberry homogenate is acidified to pH=1.5, add pepsin (7000 unit pepsin/20g homogenate), at 37 DEG C, 100rpm water-bath vibration 1.5h, after simulation peptic digestion, continue to adjust pH to 6 with 1M NaOH, every 18mL diabetes involving middle-JIAO compound adds 5mL pancreatic juice (in pancreatic juice, pancreatin content is 4mg/mL, and cholate content is 25mg/mL), then uses 1M NaHCO
3adjust pH to 7.4, at 37 DEG C, centrifugal 6min under 100rpm water-bath vibration 2.5h, 5000rpm condition, supernatant is red raspberry crude extract.Utilizing MilliporePellicon ultrafiltration system, is the regenerated cellulose ultrafiltration membrane surface of 10kDa by said extracted liquid pump to molecular cut off, and collect filter liquor, vacuum lyophilization, obtains red raspberry extract.
Known through liquid chromatograph test, the main component of this red raspberry extract is as shown in the table:
Table 1
| Composition | Content |
| Cy-3-G | 20.4％ |
| Cyanidin-3-rutinoside | 4.6％ |
| Cyanidin-3-sophoroside | 47.7％ |
| Cyanidin-3-glucose rutinoside | 5.8％ |
| Pelargonin-3-glucoside | 14.9％ |
| Pelargonin-3-rutinoside | 6.6％ |
Embodiment 2
Red raspberry fresh fruit and water mixed in equal amounts are pulled an oar, with 5M HCl, red raspberry homogenate is acidified to pH=1.7, add pepsin (6000 unit pepsin/20g homogenate), at 37 DEG C, 150rpm water-bath vibration 2h, after simulation peptic digestion, continue to adjust pH to 6.5 with 1M NaOH, every 18mL diabetes involving middle-JIAO compound adds 5mL pancreatic juice (in pancreatic juice, pancreatin content is 4mg/mL, and cholate content is 30mg/mL), then uses 1M NaHCO
3adjust pH to 7.4, at 37 DEG C, centrifugal 8min under 150rpm water-bath vibration 2h, 6000rpm condition, supernatant is red raspberry crude extract.Utilizing MilliporePellicon ultrafiltration system, is the regenerated cellulose ultrafiltration membrane surface of 10kDa by said extracted liquid pump to molecular cut off, and collect filter liquor, vacuum lyophilization, obtains red raspberry extract.
Embodiment 3
Red raspberry fresh fruit and water mixed in equal amounts are pulled an oar, with 5M HCl, red raspberry homogenate is acidified to pH=2, add pepsin (5000 unit pepsin/20g homogenate), at 37 DEG C, 200rpm water-bath vibration 3h, after simulation peptic digestion, continue to adjust pH to 7 with 1M NaOH, every 18mL diabetes involving middle-JIAO compound adds 5mL pancreatic juice (in pancreatic juice, pancreatin content is 5mg/mL, and cholate content is 30mg/mL), then uses 1M NaHCO
3adjust pH to 7.4, at 37 DEG C, centrifugal 10min under 200rpm water-bath vibration 3h, 8000rpm condition, supernatant is red raspberry crude extract.Utilizing Millipore Pellicon ultrafiltration system, is the regenerated cellulose ultrafiltration membrane surface of 10kDa by said extracted liquid pump to molecular cut off, and collect filter liquor, vacuum lyophilization, obtains red raspberry extract.
Performance test
The red raspberry extract prepared with embodiment 1 ~ 3, for sample, tests its protective effect of intestinal cell oxidative damage to urethanes induction
(1) cell survival rate measures (mtt assay)
Mtt assay is adopted to detect the survival rate of cell.Select the cell of exponential phase, with 0.25% pancreatin extraction cell monolayer, be made into individual cells suspension by the RPIM1640 culture medium containing 10% new Ox blood serum, seed cells in 96 porocyte culture plates that (concentration is 5 × 10
3individual cells/well), after hatching 24h; Red raspberry extract (1mg/mL) pretreatment 24h prepared by embodiment 1, then use urethanes (62.5mM) to process Caco-2 cell, then use MTT (0.5mg/mL) to hatch 4h.The precipitate dissolves generated, in the DMSO of 200 μ L, detects the absorbance at 490nm place by microplate reader.
Cell survival rate (%)=[A
sample/ A
blank] × 100%;
As shown in Figure 1, red raspberry extract (1mg/mL) prepared by embodiment 1 acts on Caco-2 cell, and compared with urethanes group, cell quantity showed increased, cell survival rate adds 23.01%.Above-mentioned experimental result confirms that red raspberry extract has protective action to the intestinal cell oxidative damage that urethanes causes.
(2) intracellular ROS level detects (DCF fluorescence)
Utilize fluorescent probe DCFH-DA to carry out active oxygen detection, DCFH-DA itself does not have fluorescence, can pass freely through cell membrane, after entering cell, can be generated DCFH by intracellular esterase hydrolyzed.And DCFH can not permeabilized cells film, thus probe is made to be easy to be loaded onto in cell.Intracellular active oxygen can be oxidized non-blooming DCFH and generate the DCF having fluorescence.The fluorescence intensity detecting DCF just can know the level of reactive oxygen species, and DCF fluorescence is stronger, and ROS is more.
Select the cell of exponential phase, with 0.25% pancreatin extraction cell monolayer, be made into individual cells suspension, with 5x10 by the RPIM1640 culture medium containing 10% new Ox blood serum
5the density in individual/hole is inoculated in 6 orifice plates, put in incubator and cultivate sucking-off culture medium after 24h, add the red raspberry extract effect 24h that whole dosage is embodiment 1 preparation of 1mg/mL, urethanes (62.5mM) is used to process intestinal cell again, 2 times are washed with PBS, the DCFH-DA adding 10 μMs dyes 30min, and dyestuff is taken pictures with fluorescence microscope after cleaning, and calculates average optical density value.
As shown in Figure 2, urethanes can cause Caco-2 cell and produces a large amount of reactive oxygen free radical ROS (compared with Normal group, effect 48h fluorescence intensity is 392.76%); Dosage is that the red raspberry extract of 1mg/mL significantly can reduce intracellular ROS level, and cell ROS fluorescence intensity reduces 142.40%.The above results shows that red raspberry extract can reduce ROS in born of the same parents and generate and accumulation, thus intervenes the oxidative damage of intestinal cell.
(3) mitochondrial membrane potential in anoxic MMP horizontal detection
Select the cell of exponential phase, with 0.25% pancreatin extraction cell monolayer, be made into individual cells suspension, with 5 × 10 by the RPIM1640 culture medium containing 10% new Ox blood serum
5the density in individual/hole is inoculated in 6 orifice plates, put in incubator and cultivate sucking-off culture medium after 24h, add the red raspberry extract effect 24h that whole dosage is embodiment 2 preparation of 1mg/mL, urethanes (62.5mM) is used to process intestinal cell again, 2 times are washed with PBS, then mitochondrial membrane potential probe Rhodamine 123 (RH123) fluorescent probe is adopted to dye, hatch 30min for 37 DEG C, take pictures with fluorescence microscope after dyestuff is cleaned, then analyze its fluorescence intensity.
Some researchs show that mitochondrial membrane potential change is associated with the generation of reactive oxygen free radical (ROS).Mitochondrion is the structure manufacturing energy in cell, and the vigor of it and cell is closely related, and mitochondrial membrane potential effectively can reflect the functional status of intracellular mitochondrial.
As shown in Figure 3, compared with matched group (control), Caco-2 cell is acted on through urethanes (62.5mM), cell membrane potential significantly reduces (fluorescence intensity drops to 34.39%), the reduction of the cell membrane potential that red raspberry extract 1mg/mL dosage can significantly suppress urethanes to be induced, cell membrane potential fluorescence intensity is elevated to 79.30%.The above results shows that the cell membrane potential that red raspberry extract can effectively suppress urethanes to be induced reduces.
(4) glutathion inside cell GSH horizontal detection
Collect the Caco-2 cell of logarithmic (log) phase, seed cells in 6 porocyte culture plates that (concentration is 5 × 10
5individual cells/well), after hatching 24h; 24h is acted under the condition of the red raspberry extract of embodiment 2 preparation containing 1mg/mL dosage, with urethanes process Caco-2 cell, then with the dyeing of GSH fluorescent probe, hatch 30min for 37 DEG C, take pictures with fluorescence microscope after dyestuff is cleaned, then analyze its fluorescence intensity.
GSH is as a kind of regulator of good cellular redox state, and energy scavenging activated oxygen, has protective effect to the oxidative damage caused by poisonous substance.As shown in Figure 4, compared with matched group (control), through urethanes effect Caco-2 cell, cellular glutathione content significantly reduces (fluorescence intensity 30.35%).The cellular glutathione content that red raspberry extract 1mg/mL dosage all can suppress urethanes to be induced effectively reduces, and glutathione content is elevated to 70.81%.The above results shows the reduction of the cellular glutathione content that red raspberry extract can suppress urethanes to be induced.
(5) Hoechst nucleus detects
Hoechst 33258 be a kind of can the blue fluorescent dyes of permeates cell membranes, lower to the toxicity of cell, can be used for nuclear targeting.Hoechst33258 is specific DNA dyestuff, closes with A-T bond, can dye immediately to dead cell or through the cell that 70% cold ethanol is fixing.Dyeing for living cells is then gradual, reaches capacity in 10min.Observe under fluorescence microscope, living cells core be the even fluorescence of disperse, when there is apoptosis, and the particulate mass fluorescence of visible dense dye densification in nucleus or Cytoplasm.
Select the cell of exponential phase, with 0.25% pancreatin extraction cell monolayer, be made into individual cells suspension, with 5 × 10 by the RPIM1640 culture medium containing 10% new Ox blood serum
5the density in individual/hole is inoculated in 6 orifice plates, put in incubator and cultivate sucking-off culture medium after 24h, add the red raspberry extract pretreatment 24h that whole dosage is embodiment 3 preparation of 1mg/mL, again with urethanes process intestinal cell, 2 times are washed with PBS, the Hoechst 33258 adding 10 μMs dyes 30min, and dyestuff is taken pictures with fluorescence microscope after cleaning. and calculate average optical density value.
Can be observed by Fig. 5, normal Caco-2 cell (control) nucleus volume is larger, chromatin is homogeneous, through urethanes effect Caco-2 cell, the chromatin of part cell obviously there occurs concentrated, and occurs apoptotic body, and after adding red raspberry extract (1mg/mL) pretreatment 24h, chromatin is slightly unfolded, and karyomorphism recovers to some extent, shows the nucleus damage that red raspberry extract can suppress urethanes to be induced.
Finally, the present invention can summarize with other the concrete form without prejudice to spirit of the present invention and principal character.Therefore, no matter from that, above-mentioned embodiment of the present invention all can only be thought explanation of the present invention and can not limit the present invention, claims indicate scope of the present invention, and scope of the present invention is not pointed out in above-mentioned explanation, therefore, any change in the implication suitable with claims of the present invention and scope, all should think to be included in the scope of claims.
Claims (8)
1. a red raspberry extract, is characterized in that, extraction step is as follows:
(1) mass mixing such as red raspberry fresh fruit and water is pulled an oar, and adds HCl and regulates pH=1.5 ~ 2 of homogenate, then add pepsin, 37 DEG C, water-bath vibration 1.5 ~ 3h under 100 ~ 200rpm;
(2) get the mixed serum that step (1) obtains, add NaOH and regulate pH=6 ~ 7, then add pancreatic juice, then use NaHCO
3regulate pH to 7.4,37 DEG C, after 100 ~ 200rpm water-bath vibration, 2 ~ 3h, more centrifugal 5 ~ 10min obtains supernatant under 5000 ~ 8000rpm;
(3) get the supernatant of step (2), after ultrafiltration membrance filter, molecular cut off is greater than the composition of 10kDa, and filter liquor, through vacuum lyophilization, obtains described red raspberry extract;
The composition of described red raspberry extract: Cy-3-G, cyanidin-3-rutinoside, cyanidin-3-sophoroside, cyanidin-3-glucose rutinoside, pelargonin-3-glucoside and pelargonin-3-rutinoside, mass percentage is as follows:
2. red raspberry extract according to claim 1, is characterized in that, in step (1), the concentration of described HCl is 5mol/L.
3. red raspberry extract according to claim 1, is characterized in that, in step (1), described pepsic addition is 5000 ~ 7000 unit pepsin/20g homogenate.
4. red raspberry extract according to claim 1, is characterized in that, in step (2), the concentration of described NaOH is 1mol/L;
Described NaHCO
3concentration be 1mol/L.
5. red raspberry extract according to claim 1, is characterized in that, in step (2), in described pancreatic juice, the content of pancreatin is 4 ~ 5mg/mL, and the content of cholate is 25 ~ 30mg/mL;
The addition of described pancreatic juice is 5mL/18mL mixed serum.
6. the application of red raspberry extract in the inhibitor preparing intestinal cell oxidative damage described in a basis.
7. the application of the red raspberry extract described in a basis in the inhibitor preparing the intestinal cell oxidative damage of being induced by urethanes.
8. the application according to claim 6 or 7, is characterized in that, in described inhibitor, the effective dose of red raspberry extract is 1mg/mL.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104840523A (en) * | 2015-05-04 | 2015-08-19 | 浙江大学 | Mulberry extract and application of mulberry extract in preparing liver cell damage inhibitor |
| CN104922189A (en) * | 2015-06-15 | 2015-09-23 | 浙江大学 | Lonicera caerulea extract and application of lonicera caerulea extract in liver cell oxidative damage inhibitor preparation |
| CN109651463A (en) * | 2019-01-25 | 2019-04-19 | 辽宁大学 | A kind of method that high speed adverse current chromatogram separates Cyanidin in red raspberry fruit |
| CN110711404A (en) * | 2019-06-13 | 2020-01-21 | 青海大学 | Method for extracting anthocyanin from raspberry |
| CN116138248A (en) * | 2023-02-22 | 2023-05-23 | 西北农林科技大学 | Preparation method and application of diluent for freezing preservation of semen of dairy sheep |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104840523A (en) * | 2015-05-04 | 2015-08-19 | 浙江大学 | Mulberry extract and application of mulberry extract in preparing liver cell damage inhibitor |
| CN104922189A (en) * | 2015-06-15 | 2015-09-23 | 浙江大学 | Lonicera caerulea extract and application of lonicera caerulea extract in liver cell oxidative damage inhibitor preparation |
| CN104922189B (en) * | 2015-06-15 | 2018-06-26 | 浙江大学 | A kind of indigo berry extract and its application in liver cell oxidative damage inhibitor is prepared |
| CN109651463A (en) * | 2019-01-25 | 2019-04-19 | 辽宁大学 | A kind of method that high speed adverse current chromatogram separates Cyanidin in red raspberry fruit |
| CN109651463B (en) * | 2019-01-25 | 2022-04-05 | 辽宁大学 | Method for separating cyanidin from red raspberry fruits by high-speed counter-current chromatography |
| CN110711404A (en) * | 2019-06-13 | 2020-01-21 | 青海大学 | Method for extracting anthocyanin from raspberry |
| CN116138248A (en) * | 2023-02-22 | 2023-05-23 | 西北农林科技大学 | Preparation method and application of diluent for freezing preservation of semen of dairy sheep |
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