CN105434486A - Cape gooseberry extract and application thereof in preparation of liver cell oxidative damage inhibitors - Google Patents
Cape gooseberry extract and application thereof in preparation of liver cell oxidative damage inhibitors Download PDFInfo
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- CN105434486A CN105434486A CN201510943697.0A CN201510943697A CN105434486A CN 105434486 A CN105434486 A CN 105434486A CN 201510943697 A CN201510943697 A CN 201510943697A CN 105434486 A CN105434486 A CN 105434486A
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- radix ribis
- ribis manschurici
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- 239000000284 extract Substances 0.000 title claims abstract description 50
- 230000004792 oxidative damage Effects 0.000 title claims abstract description 11
- 239000003112 inhibitor Substances 0.000 title claims abstract description 10
- 210000005229 liver cell Anatomy 0.000 title claims abstract description 9
- 238000002360 preparation method Methods 0.000 title abstract description 4
- 235000001982 Physalis edulis Nutrition 0.000 title abstract 5
- 244000064622 Physalis edulis Species 0.000 title description 2
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 9
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims abstract description 8
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 claims abstract description 4
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- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 claims abstract description 4
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Medical Informatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines Containing Plant Substances (AREA)
- Bioinformatics & Cheminformatics (AREA)
Abstract
The invention discloses a cape gooseberry extract comprising effective components by the mass percentage: 45.57%-49.73% of total phenols, 1.95%-5.32% of total flavonoids, 1.56%-1.79% of Vc, 1.02%-1.51% of caffeic acid, 0.81%-1.28% of chlorogenic acid, 0.01%-0.05% of proanthocyanidin, 41.37%-43.24% of total polysaccharides, and 0.42%-0.53% of total reducing sugar. Extraction steps comprise that fresh cape gooseberry is subjected to enzymolysis through pepsase and trypsin and then is subjected to centrifugal separation to obtain a supernatant, the supernatant is filtered by an ultrafiltration membrane, components with the molecular weight of greater than 5 kDa are intercepted, and the filtrate is subjected to vacuum freeze drying to obtain the cape gooseberry extract. The invention also discloses an application of the cape gooseberry extract in preparation of liver cell oxidative damage inhibitors.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of Radix Ribis manschurici extract and preparing the application in liver cell oxidative damage inhibitor.
Background technology
Radix Ribis manschurici (PhysalisperuvianaL.), the annual or perennial herbaceous plant of Solanaceae Physalis, various food (fruit jam, preserved fruit, canned food and salad etc.) can be eaten or be processed into Radix Ribis manschurici maturation in yellow, raw afterwards.Radix Ribis manschurici sweet and sour taste, nutritious, containing higher vitamin C, and containing a large amount of active substances, mainly comprise: Polyphenols, flavonoid and organic acid composition.Radix Ribis manschurici is a kind of herb medicinal plants, and among the people, the monkey flower comprising Radix Ribis manschurici is widely used in prevention and therapy cancer, leukemia, antibacterial, diuresis, antiinflammatory and immunomodulating etc.
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 then attack various organelle, the tissue of infringement body, and then cause chronic disease and aging effect and bring out various disease.
Acrylamide (Acrylamide, AA) be that a kind of chemical substance international cancer research institution (IARC) with neurotoxicity and potential carcinogenecity evaluates its carcinogenecity for 1994, acrylamide being classified as 2 class carcinogens (2A) the i.e. mankind may carcinogen.Acrylamide is extensively present in the many kinds of substance of daily life, and containing acrylamide in much food, especially fried, baking class height starchy food, also all contains acrylamide in drinking-water, smoking, cosmetics and packaging material.(the Zhang Xichun such as Zhang Xichun, Zhang Minai, acrylamide liver metabolism and Cytochrome P450 2E1 protein expression [J]. modern preventive medicine, 2007,34 (5): 910-915) with 1.0,2.0,4.0mmol/ml3 the AA of individual dosage and rats'liver are cut into slices and are jointly hatched, find the increase along with AA concentration, the GA that liver slice metabolism generates also increases thereupon, and the expression of CYP2E1 also increases.Liver is the vitals that human-body biological transforms and detoxifies, be metabolism place initial after AA enters human body, the AA of high concentration can be assembled, and CYP2E1 in liver cytochrome P 450 is as a kind of monooxygenase of metabolism alien material, AA can be oxidized to GA, and GA has more cytotoxicity than AA.
Therefore, develop a kind of material hepar damnification being had to protective effect, the hepar damnification especially caused by acrylamide has the material of protective effect to be necessary.
Summary of the invention
The invention discloses a kind of Radix Ribis manschurici extract, adopt the mode of simulation human gastrointestinal tract digestion to prepare.Pepsin, trypsin etc. is adopted to prepare Radix Ribis manschurici extract.Radix Ribis manschurici 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; And pass through the Radix Ribis manschurici product of traditional method for extracting, after entering human body, the active substance having part is decomposed digestion by digestion, and thus functional have unpredictability, can not represent the truth after entering human body.
A kind of Radix Ribis manschurici extract, extraction step is as follows:
(1) mass mixing such as Radix Ribis manschurici fresh fruit and water is pulled an oar, and adjusts pH to 1.5 ~ 2, add pepsin with HCl, at 37 DEG C, water-bath vibration 2 ~ 3h under 200 ~ 300rpm;
(2) mixed serum that step (1) obtains is got, adjust pH to 6 with NaOH, then add pancreatic juice, then adjust pH to 7.2 ~ 7.5 with NaHCO3,37 DEG C, under 200 ~ 300rpm after water-bath vibration 2 ~ 3h, under 5000 ~ 8000rpm condition, centrifugal 10 ~ 15min obtains supernatant;
(3) get the composition that step (2) supernatant molecular cut off after ultrafiltration membrance filter is greater than 5kDa, filter liquor, through vacuum lyophilization, obtains Radix Ribis manschurici extract;
By percentage to the quality, effective ingredient in described Radix Ribis manschurici extract: total phenol content is 45.57%-49.73%, general flavone content is 1.95%-5.32%, Vc content is 1.56%-1.79%, caffeic acid content is 1.02%-1.51%, and chlorogenic acid content is 0.81%-1.28%, and procyanidin content is 0.01%-0.05%, total polysaccharides content is 41.37%-43.24%, and total reducing sugars content is 0.42%-0.53%.
As preferably, in step (1), the concentration of described HCl is 5mol/L.
As preferably, in step (1), described pepsic addition is 2000 ~ 4000 unit pepsin/20g homogenate.
As preferably, in step (2), the concentration of described NaOH is 1mol/L.
The concentration of described NaHCO3 is 1mol/L.
As preferably, in step (2), in described pancreatic juice, the content of pancreatin is 5 ~ 6mg/mL, and the content of cholate is 20 ~ 30mg/mL;
The addition of described pancreatic juice is 5mL/18mL mixing homogenate.
The invention also discloses the application of Radix Ribis manschurici extract in the inhibitor preparing liver cell oxidative damage, expand the application of Radix Ribis manschurici.
Further disclose the application of Radix Ribis manschurici extract in the inhibitor preparing the liver cell oxidative damage of being induced by acrylamide.
As preferably, in described inhibitor, the effective dose of Radix Ribis manschurici extract is 0.5mg/mL.
Compared with prior art, the present invention has the following advantages:
The mode that the present invention simulates human gastrointestinal tract digestion prepares Radix Ribis manschurici extract.
The oxidative damage that Late Cambrian of the present invention Radix Ribis manschurici extract can effectively suppress acrylamide to be induced, has good DEVELOPMENT PROSPECT.
The present invention is that Radix Ribis manschurici extract provides new medical application, has expanded a new application.
Accompanying drawing explanation
Fig. 1 is Radix Ribis manschurici extract A BTS
+radical scavenging activity;
Fig. 2 is Radix Ribis manschurici extract in vitro anti-oxidation (FRAP method);
Fig. 3 is the protective effect of Radix Ribis manschurici extract to the HepG2 cell oxidative damage that acrylamide is induced;
Fig. 4 is the inhibitory action of Radix Ribis manschurici extract to the HepG2 cytoactive oxygen-derived free radicals ROS (DCF fluorescence) that acrylamide 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
Radix Ribis manschurici fresh fruit and water mixed in equal amounts are pulled an oar, pH to 2 is adjusted with 5M hydrochloric acid, add pepsin (4000 units/20g homogenate), at 37 DEG C, 250rpm water-bath vibration 2.5h, the product continuation 1MNaOH of in-vitro simulated peptic digestion adjusts pH to 6, and (in pancreatic juice, pancreatin content is 6mg/mL to add 5mL pancreatic juice, cholate content is 20mg/mL), then use 1MNaHCO
3adjust pH to 7.2, at 37 DEG C, 200rpm water-bath vibration 3h, after simulation intestinal digestion terminates in vitro, centrifugal 10min under 8000rpm condition, supernatant is Radix Ribis manschurici crude extract.Utilizing MilliporePellicon ultrafiltration system, is the regenerated cellulose ultrafiltration membrane surface of 5kDa by said extracted liquid pump to molecular cut off, and collect filtrate, vacuum lyophilization, obtains Radix Ribis manschurici extract.
By percentage to the quality, effective ingredient in described Radix Ribis manschurici extract: total phenol content is 48.73%, general flavone content is 3.11%, Vc content is 1.79%, caffeic acid content is 1.76%, and chlorogenic acid content is 0.81%, and procyanidin content is 0.03%, total polysaccharides content is 43.24%, and total reducing sugars content is 0.53%.
Embodiment 2
Radix Ribis manschurici fresh fruit and water mixed in equal amounts are pulled an oar, pH to 1.5 is adjusted with 5M hydrochloric acid, add pepsin (6000 units/20g homogenate), at 37 DEG C, 200rpm water-bath vibration 3h, the product continuation 1MNaOH of in-vitro simulated peptic digestion adjusts pH to 6, and (in pancreatic juice, pancreatin content is 5mg/mL to add 5mL pancreatic juice, cholate content is 25mg/mL), then use 1MNaHCO
3adjust pH to 7.4, at 37 DEG C, 250rpm water-bath vibration 2.5h, after simulation intestinal digestion terminates in vitro, centrifugal 12min under 6000rpm condition, supernatant is Radix Ribis manschurici crude extract.Utilizing MilliporePellicon ultrafiltration system, is the regenerated cellulose ultrafiltration membrane surface of 5kDa by said extracted liquid pump to molecular cut off, and collect filtrate, vacuum lyophilization, obtains Radix Ribis manschurici extract.
Embodiment 3
Radix Ribis manschurici fresh fruit and water mixed in equal amounts are pulled an oar, pH to 2 is adjusted with 5M hydrochloric acid, add pepsin (6000 units/20g homogenate), at 37 DEG C, 300rpm water-bath vibration 2h, the product continuation 1MNaOH of in-vitro simulated peptic digestion adjusts pH to 6, and (in pancreatic juice, pancreatin content is 6mg/mL to add 5mL pancreatic juice, cholate content is 25mg/mL), then use 1MNaHCO
3adjust pH to 7.5, at 37 DEG C, 300rpm water-bath vibration 2h, after simulation intestinal digestion terminates in vitro, centrifugal 15min under 5000rpm condition, supernatant is Radix Ribis manschurici crude extract.Utilizing MilliporePellicon ultrafiltration system, is the regenerated cellulose ultrafiltration membrane surface of 5kDa by said extracted liquid pump to molecular cut off, and collect filtrate, vacuum lyophilization, obtains Radix Ribis manschurici extract.
Performance test
Radix Ribis manschurici extract is to the protective effect of the liver cell oxidative damage that acrylamide is induced
(1) antioxidation in vitro measures (ABTS method)
ABTS [2,2-azino-two (3-ethyl-benzothiazole-6-sulfonic acid)] method can be used for the oxidation resistance measuring biological sample.ABTS generates stable aeruginous radical cation ABTS after active oxygen oxidizes
+if, tested material energy and ABTS
+react and reaction system is faded, then pointing out tested material to have reduction ABTS
+the activity of free radical.
According to the method for list of references, preparation ABTS
+working solution, at appropriate ABTS
+add appropriate sample liquid in working solution, fully mix, under room temperature condition, react 6min (lucifuge), measure the absorbance A under 734nm
i, replace sample liquid to do blank with the distilled water of equivalent, remember that its absorbance is A
0, using ascorbic acid as positive control.ABTS
+free radical scavenging activity (%)=[(A
0-A
i)/A
0] × 100%.As Fig. 1 display, Radix Ribis manschurici extract has good antioxidant activity in vitro, as calculated, and the IC of blank group
50for 12.97mg/ml, the IC of experimental group
50for 8.06mg/ml.
(2) antioxidation in vitro measures (FRAP method)
FRAP method (iron ion reducing process) is usually used in the evaluation of natural product antioxidant activity.Ferric ion through reduction after can with TPTZ (2,4,6-TPTZ) reaction generation blue complex, the reducing power of its growing amount and tested material is proportional, if tested material can make system produce this blue complex with its reaction, then tested material is pointed out to have the ability of reduction ferric ion.
Method according to list of references: preparation FRAP working solution, i.e. the acetate buffer (pH3.5) of the 300mM of 100ml, the 20mM of the 10mMTPTZ of 10ml, 10ml but iron chloride are with 10:1:1 volume mixture.In appropriate FRAP working solution, add appropriate sample liquid, fully mix, under 37 DEG C of water-baths, lucifuge reaction 30min, measures the absorbance under 593nm.The oxidation resistance of sample is represented with Vc equivalent.As Fig. 2 display, Radix Ribis manschurici has antioxidant activity in vitro preferably, and as calculated, blank group is 0.3315 μ gVc/mg, and experimental group is 0.3386 μ gVc/mg.
(3) cell survival rate measures (mtt assay)
According to the method for list of references, mtt assay is adopted to detect the survival rate of cell.The HepG2 cell of logarithmic (log) phase is inoculated in 96 porocyte culture plates that (concentration is 3.5 × 10
3individual cells/well), after hatching 24h; Under the condition containing 0.5mg/mL concentration Radix Ribis manschurici extract, process HepG2 cell 24h with acrylamide (5mM), then use MTT (0.5mg/mL) to hatch 3.5h.Generate precipitate dissolves in the DMSO of 150 μ L, with the absorbance at microplate reader check fee 490nm place.
Cell survival rate (%)=[A
sample/ A
blank] × 100%
As Fig. 3 display, HepG2 cell damages after 2h through 5mMAA, and cell density obviously reduces, and compared with normal group, cell survival rate is 54.99%, has statistical significance (p<0.05).When Radix Ribis manschurici extract is under 5mMAA mass action; good protective effect is had to HepG2 cell; compared with matched group; there is significant difference; under the effect of 0.5mg/mL Radix Ribis manschurici extract, (cell survival rate reaches 84.23%; p<0.05), above results demonstrate that Radix Ribis manschurici extract has the protective effect of the cell injury of intervening acrylamide induction.
(4) intracellular ROS level detects (DCF fluorescence)
Collect the HepG2 cell of logarithmic (log) phase, seed cells in 12 porocyte culture plates that (concentration is 3.5 × 10
4individual cells/well), after hatching 24h; Under the condition containing 0.5mg/mL concentration Radix Ribis manschurici extract, use acrylamide (5mM) to process HepG2 cell 24h respectively, then with DCFH-DA dyeing, hatch 30min for 37 DEG C, take pictures with fluorescence microscope after dyestuff is cleaned, then analyze its fluorescence intensity.
As Fig. 4 display, compared with untreated matched group (control), through 5mMAA effect hepatocyte HepG22h, fluorescence intensity obviously strengthens (fluorescence intensity 198.9%).Radix Ribis manschurici extract significantly can reduce the ROS fluorescence intensity that AA induction produces, and has statistical significance, 0.25mg/mL Radix Ribis manschurici extract best results (fluorescence intensity 132.6%).The above results shows that Radix Ribis manschurici extract effectively can reduce the cell ROS level of AA induction generation.
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 (9)
1. a Radix Ribis manschurici extract, is characterized in that, extraction step is as follows:
Radix Ribis manschurici extract, according to this after pepsin, trypsin digestion, centrifugally obtains supernatant; Described supernatant molecular cut off after ultrafiltration membrance filter is greater than the composition of 5kDa, and filter liquor, through vacuum lyophilization, obtains Radix Ribis manschurici extract;
By percentage to the quality, effective ingredient in described Radix Ribis manschurici extract: total phenol content is 45.57%-49.73%, general flavone content is 1.95%-5.32%, Vc content is 1.56%-1.79%, caffeic acid content is 1.02%-1.51%, and chlorogenic acid content is 0.81%-1.28%, and procyanidin content is 0.01%-0.05%, total polysaccharides content is 41.37%-43.24%, and total reducing sugars content is 0.42%-0.53%.
2. Radix Ribis manschurici extract according to claim 1, it is characterized in that, described Radix Ribis manschurici extract through the concrete steps of pepsin enzymolysis is:
The mass mixings such as Radix Ribis manschurici fresh fruit and water are pulled an oar, and adjust pH to 1.5 ~ 2, add pepsin with HCl, at 37 DEG C, water-bath vibration 2 ~ 3h under 200 ~ 300rpm, obtain mixed serum.
3. Radix Ribis manschurici extract according to claim 2, it is characterized in that, in step (1), the concentration of described HCl is 5mol/L, and described pepsic addition is 2000 ~ 4000 unit pepsin/20g homogenate.
4. Radix Ribis manschurici extract according to claim 1, it is characterized in that, described Radix Ribis manschurici extract through the concrete steps of trypsin digestion is:
To learn from else's experience the mixed serum after pepsin enzymolysis, adjust pH to 6 with NaOH, then add pancreatic juice, then use NaHCO
3adjust pH to 7.2 ~ 7.5,37 DEG C, under 200 ~ 300rpm after water-bath vibration 2 ~ 3h, under 5000 ~ 8000rpm condition, centrifugal 10 ~ 15min obtains supernatant.
5. Radix Ribis manschurici extract according to claim 4, it is characterized in that, in step (2), the concentration of described NaOH is 1mol/L, described NaHCO
3concentration be 1mol/L.
6. Radix Ribis manschurici extract according to claim 4, it is characterized in that, in step (2), in described pancreatic juice, the content of pancreatin is 5 ~ 6mg/mL, and the content of cholate is 20 ~ 30mg/mL;
The addition of described pancreatic juice is 5mL/18mL mixing homogenate.
7. one kind according to the application of described Radix Ribis manschurici extract in the inhibitor preparing liver cell oxidative damage.
8. preparing the application in the inhibitor of liver cell oxidative damage of being induced by acrylamide according to described Radix Ribis manschurici extract for one kind.
9. the application according to claim 7 or 8, is characterized in that, in described inhibitor, the effective dose of Radix Ribis manschurici extract is 0.5mg/mL.
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|---|---|---|---|---|
| CN107266602A (en) * | 2017-07-27 | 2017-10-20 | 李欣瑜 | Extraction of Polysaccharides in Fruit of Physalis and preparation method thereof |
| CN111721848A (en) * | 2019-03-21 | 2020-09-29 | 仙乐健康科技股份有限公司 | Method for measuring content of phosphatidylserine |
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| CN103805661B (en) * | 2012-11-15 | 2015-12-02 | 中国科学院大连化学物理研究所 | A kind of preparation method being rich in the Franchet Groundcherry Calyx and Fruit peptide of zeaxanthin |
| CN104606306B (en) * | 2015-01-14 | 2017-07-07 | 浙江大学 | A kind of blackberry extract and its application in liver cell oxidative damage inhibitor is prepared |
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| CN107266602A (en) * | 2017-07-27 | 2017-10-20 | 李欣瑜 | Extraction of Polysaccharides in Fruit of Physalis and preparation method thereof |
| CN111721848A (en) * | 2019-03-21 | 2020-09-29 | 仙乐健康科技股份有限公司 | Method for measuring content of phosphatidylserine |
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