CN106943437B - Ethanol extract of microcystis and preparation method and application thereof - Google Patents
Ethanol extract of microcystis and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- 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
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- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- 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
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- 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
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- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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Abstract
The invention belongs to the technical field of marine organisms, and provides a microcystis ethanol extract and a preparation method and application thereof. The microcystis ethanol extract provided by the invention is safe and nontoxic, can promote the clearance of in vivo active oxygen free Radicals (ROS), reduce the level of in vivo active oxygen free Radicals (ROS), can relieve the premature senility phenomenon caused by oxidative stress, has obvious antioxidant and anti-aging effects, and can be applied to the preparation of health-care foods and medicines with antioxidant functions.
Description
Technical Field
The invention belongs to the technical field of marine organisms, and particularly relates to a microcystis ethanol extract and a preparation method and application thereof.
Background
The human organism is composed of many elements, and the high level of oxidation resistance of the human body depends on the constituent elements and the composition thereof. For example, selenium is an essential trace element for living organisms, is also an important antioxidant in vivo, and has important effects on maintaining human health and delaying aging. Free radicals are normal metabolites of the human body, and the main free radicals in the organism are oxygen radicals. The oxygen free radicals are oxygen-containing radicals with high chemical reaction activity generated by insufficient electron acceptance during oxygen reduction, and mainly include superoxide free radicals, hydroxyl free radicals, singlet oxygen, peroxy free radicals, peroxynitrogen free radicals and the like. Under physiological conditions, the body can scavenge oxygen free radicals in the body by effective enzymatic and non-enzymatic systems, thereby placing it in physiological homeostasis. When some factor makes oxygen free radical produce too much to exceed the organism's eliminating capacity or weaken it, the oxygen free radical level in vivo is higher than the balance point, and the oxygen free radical can react with DNA, protein and unsaturated fatty acid to cause DNA oxidative damage, protein cross-linking and lipid peroxidation, thus causing inflammation, causing molecular degradation, organism aging or diseases related to aging. Therefore, the auxiliary improvement of the oxidation resistance of the organism by screening substances with oxidation resistance is one of the direct, effective and convenient important methods for improving the oxidation resistance of the organism and delaying aging.
Microalgae are unicellular microalgae which exist independently or in chains and clusters in water body environments such as oceans, lakes, rivers and the like. Microalgae have great biodiversity, about 200000 to 800000 species of microalgae, of which about 35000 species have been described, and over 15000 species of novel compounds produced from algal biomass have been chemically identified. The active ingredients produced and contained in the microalgae have important economic value and social value, and are widely applied to the aspects of medicines, health-care foods, feeds, chemical industry, environmental protection and the like. However, the types of microalgae studied at present are narrow, and most of the application studies of microalgae are mainly focused on the aspect of efficient oil production, and the application studies of microalgae in the aspects of medicine and health-care food are mainly focused on several common microalgae such as spirulina, chlorella and porphyridium, and relatively few reports are reported for other microalgae.
The cells of Chroococcaceae of Chroococcales of Cyanophyta of Microcystis are spherical, and are wrapped in colloid to form irregular population. The group of colorless, soft and soluble quilts is in a spherical shape, an oblong shape, a reticular shape with irregular shape or a window lattice shape, and can be seen microscopically or visually. The Microcystin (MC) is a hepatotoxin generated by the outbreak of microcystis, is a cyclic heptapeptide compound with biological activity, can strongly inhibit the activity of protein phosphatase, and is also a strong liver tumor promoter.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a microcystis ethanol extract. The ethanol extract of microcystis provided by the invention has obvious antioxidation, and can be applied to preparation of health-care food and medicines with antioxidation function.
The technical solution of the present invention will be further apparent and explained by the following detailed description.
A microcapsule algae ethanol extract is prepared by the following steps:
s1, taking fresh microcystis wet algae, freezing for 12 hours at the temperature of-20 to-40 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 3-5 times, centrifuging for 5-15 minutes at the rotating speed of 5000-10000 r/min, separating supernatant from precipitate, and taking the precipitate for later use; the supernatant is the intracellular fluid of the microcystis;
s2, adding ethanol with the volume fraction of 70% -99% into the precipitate obtained in the step S1, performing ultrasonic extraction for 2-4 times, centrifuging for 5-15 min under the condition that the rotating speed is 5000-10000 r/min, separating the supernatant from the precipitate, taking the supernatant for vacuum drying, and dissolving the product after vacuum drying with primary water to obtain the microcystis ethanol extract.
Preferably, ethanol is added in step S2 according to the solid-to-liquid ratio of the wet algae to the ethanol of 1g/mL for ultrasonic extraction.
Preferably, the frequency of ultrasonic extraction in step S2 is 35-40 kHz, and the temperature is 4 ℃.
Preferably, the time of each ultrasonic extraction in the step S2 is 10-30 min.
The Microcystis (Microcystis sp.) is provided by aquatic organism research institute of Chinese academy of sciences, and is numbered as FACHB-1349 in freshwater algae seed bank of Chinese academy of sciences.
The inventor conducts a great deal of experiments in the process of studying microalgae, and unexpectedly finds that the microcapsule algae intracellular fluid and the ethanol extract of the microcapsule algae obtained by ethanol ultrasonic extraction of the residue left after extracting the microcapsule algae intracellular fluid both have obvious antioxidant effect.
The test is carried out on the whole animal level by using an oxidative stress model and an aging related model, and the test result shows that the ethanol extract of the microcystis provided by the invention can obviously prolong the survival time of the caenorhabditis elegans under the oxidative stress condition and improve the survival rate of the caenorhabditis elegans; meanwhile, the composition can promote the clearance of active oxygen free Radicals (ROS) in vivo and obviously reduce the level of the active oxygen free Radicals (ROS) in vivo. The ethanol extract of the microcystis provided by the invention has obvious antioxidation, and the test process does not find that the ethanol extract of the microcystis has toxic action on caenorhabditis elegans, so that the ethanol extract of the microcystis provided by the invention is safe and nontoxic, and can be applied to preparation of health-care food and medicines with antioxidation function.
The ethanol extract of the microcystis can be used alone or together with other active ingredients, and is used as the only or main active ingredient for preparing health-care food and medicines with antioxidant function, and the dosage forms of the health-care food and the medicines are preferably tablets, capsules, injections, oral liquid, granules, pills, powder, dripping pills and the like.
The antioxidant effect of the present invention is a preventive and therapeutic effect on aging and aging-related diseases which are characterized mainly by changes due to oxidative stress under pathological or physiological conditions.
Compared with the prior art, the invention has the following beneficial effects:
(1) the microcystis ethanol extract provided by the invention can promote the clearance of in vivo active oxygen free Radicals (ROS), obviously reduce the level of in vivo active oxygen free Radicals (ROS), and has obvious antioxidation effect.
(2) The ethanol extract of the microcystis provided by the invention can prolong the survival time of caenorhabditis elegans under oxidative stress conditions, improve the survival rate of the caenorhabditis elegans, and has obvious anti-aging and anti-oxidation effects.
(3) The preparation method of the microcystis ethanol extract provided by the invention has the characteristics of short period, low organic solvent consumption, simple process, safety, no toxicity, stability, high efficiency and the like, and is suitable for industrial production.
Drawings
FIG. 1 Effect of ethanol extract of microcystis on the survival rate of caenorhabditis elegans under oxidative stress conditions.
FIG. 2 Effect of ethanol extract of Microcystis on the level of ROS in C.elegans.
FIG. 3 Effect of microcystis intracellular fluid on the survival rate of C.elegans under oxidative stress conditions.
FIG. 4 Effect of microcystis intracellular fluid on the ROS levels in C.elegans.
FIG. 5 Effect of intracellular fluid of Microcystis on SOD activity in C.elegans.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The test methods used in the examples of the present invention are all conventional methods unless otherwise specified; the materials and reagents used, unless otherwise specified, are all commercially available reagents and materials.
In the embodiment of the invention, the microcystis is provided by institute of aquatic organisms of Chinese academy of sciences, and the microcystis is numbered as FACHB-1349 in freshwater algae seed bank of Chinese academy of sciences.
S basic solution: weigh 5.8g NaCl, 1.3g K2HPO4·3H2O and 6.0g KH2PO4Placing in a reagent bottle, adding 750mL deionized water, dissolving completely, diluting to a constant volume of 1L, sterilizing for 30min, and cooling to 40-50 deg.C for use.
S Medium solution: adding 1L sterilized S Basal solution dropwise into 1mL 5.0mg/mL cholesterol ethanol solution while shaking to dissolve completely (the solution changes from colorless transparency to microemulsion transparency without obvious precipitation), and sequentially adding 3mL 1M CaCl2Solution, 3mL 1M MgSO410mL of 1M potassium citrate solution (pH 6)0) and 10mL of trace element solution, and standing at normal temperature for later use.
Example 1 preparation of ethanol extract of microcystis
S1, taking fresh microcystis wet algae, freezing for 12 hours at-30 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 4 times, centrifuging for 10 minutes at the rotation speed of 8000r/min, separating the supernatant from the precipitate, and taking the precipitate for later use;
s2, adding ethanol with volume fraction of 85% into the precipitate in the step S1 according to the solid-liquid ratio of the wet algae to the ethanol of 1g/mL, carrying out ultrasonic extraction for 3 times, wherein the frequency of ultrasonic extraction is 40kHz, the temperature is 4 ℃, the time of each ultrasonic extraction is 20min, centrifuging for 10min at the rotation speed of 8000r/min, separating the supernatant from the precipitate, taking the supernatant for vacuum drying, dissolving the product after vacuum drying with primary water, and adjusting the concentration of the product relative to the weight of microcystis (wet algae) to be 1gA/mL (A represents microcystis wet algae), thus obtaining the microcystis ethanol extract.
And (4) adding primary water into the supernatant obtained by centrifuging and separating in the step S1 to adjust the concentration of the supernatant relative to the weight of the microcystis (wet algae) to be 2gA/mL (A represents the microcystis wet algae), thus obtaining the microcystis intracellular fluid.
Example 2 preparation of ethanol extract of microcystis
S1, taking fresh microcystis wet algae, freezing for 12 hours at the temperature of-20 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 5 times, centrifuging for 8 minutes at the rotation speed of 10000r/min, separating supernatant from precipitate, and taking the precipitate for later use;
s2, adding ethanol with volume fraction of 70% into the precipitate in the step S1 according to the solid-liquid ratio of the wet algae to the ethanol of 1g/mL, carrying out ultrasonic extraction for 3 times, wherein the frequency of ultrasonic extraction is 35kHz, the temperature is 4 ℃, the time of each ultrasonic extraction is 30min, centrifuging for 10min at the rotation speed of 8000r/min, separating the supernatant from the precipitate, taking the supernatant for vacuum drying, dissolving the product after vacuum drying with primary water, and adjusting the concentration of the product relative to the weight of microcystis (wet algae) to be 1gA/mL (A represents microcystis wet algae), thus obtaining the microcystis ethanol extract.
Example 3 preparation of ethanol extract of microcystis
S1, taking fresh microcystis wet algae, freezing for 12 hours at-40 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 3 times, centrifuging for 15 minutes at the rotation speed of 8000r/min, separating the supernatant from the precipitate, and taking the precipitate for later use;
s2, adding 90% ethanol by volume fraction into the precipitate in the step S1 according to the solid-liquid ratio of the wet algae to the ethanol of 1g/mL, performing ultrasonic extraction for 4 times, wherein the ultrasonic extraction frequency is 40kHz, the temperature is 4 ℃, the ultrasonic time is 15min each time, centrifuging for 15min under the condition that the rotating speed is 5000r/min, separating the supernatant from the precipitate, taking the supernatant for vacuum drying, dissolving the product after vacuum drying with primary water, and adjusting the concentration of the product relative to the weight of microcystis (wet algae) to be 1gA/mL (A represents microcystis wet algae), so as to obtain the microcystis ethanol extract.
Example 4 Effect of ethanol extract of Microcystis on the survival of caenorhabditis elegans under oxidative stress conditions
Paraquat, known as methyl viologen, is a strong oxidant and can induce a large amount of Reactive Oxygen Species (ROS) to be generated in vivo, so that the organism is in an oxidative stress environment and further has the premature senility phenomenon.
The ethanol extract of microcystis prepared in example 1, added to wild type caenorhabditis elegans (N2) (provided by the genetic information collection of caenorhabditis elegans, Minnesota university, USA) at a final concentration of 100mgA/mL, added to a control group with an equal volume of S Medium solution, cultured at 20 deg.C for 24h, added with paraquat at a final concentration of 50mM for oxidative stress molding, and counted every 12h until all caenorhabditis elegans die. The survival rate of C.elegans was expressed as a survival curve, and the results were analyzed by the Kaplan-Meier method. The results are shown in FIG. 1.
The results in FIG. 1 show that ethanol extract of microcystis can prolong the survival time of C.elegans under oxidative stress and increase the survival rate. Test results show that the ethanol extract of microcystis provided by the invention can relieve the premature senility phenomenon caused by oxidative stress and has obvious antioxidant and anti-aging effects.
Example 5 Effect of ethanol extract of Microcystis on ROS levels in C.elegans
Taking a 24-well culture plate, arranging a test group and a control group, wherein each group is provided with two holes, the final volume of each hole is 1mL, the test group is added with a synchronized L4-stage wild type caenorhabditis elegans (N2) and the microcapsule algae ethanol extract prepared in example 1, the final concentration of the microcapsule algae ethanol extract is 100mgA/mL, the control group is added with a synchronized L4-stage wild type caenorhabditis elegans (N2) and an S Medium solution with the same volume as the microcapsule algae ethanol extract, culturing is carried out for 24h at 20 ℃, paraquat is added into each hole until the final concentration is 2mM, after culturing is carried out for 48h, each group of caenorhabditis elegans is collected and homogenized under the ice bath condition respectively, the homogenate is centrifuged for 5min at 4 ℃ and 10000g, the supernatant is collected, a DCFH-DA probe (purchased from Sigma company, CAS number: 2044-85-1) solution with the final concentration of 50 muM is added into the supernatant, and detecting the fluorescence intensity by using a fluorescence microplate reader under the excitation wavelength of 485nm and the emission wavelength of 538nm for 2 hours at room temperature, and detecting once every 10 min. The results were analyzed by the F-test, and the results are shown in FIG. 2.
The results in FIG. 2 show that the ROS levels in C.elegans were significantly reduced after feeding with ethanol extract of microcystis, compared to the control group. Test results show that the microcystis ethanol extract provided by the invention can promote the removal of ROS in vivo, reduce the ROS level in vivo and has remarkable oxidation resistance.
Example 6 Effect of Microcystis intracellular fluid on the survival of caenorhabditis elegans under oxidative stress conditions
The intracellular fluid of the microcystis prepared in example 1 is added into wild caenorhabditis elegans (N2) at the early stage of imago according to the final concentration of 300mgA/mL, an equal volume of S Medium solution is added into a control group, after the control group is placed at 20 ℃ for culturing for 24h, paraquat with the final concentration of 50mM is added for oxidative stress molding, and then the survival rate of the caenorhabditis elegans is counted every 12h until all caenorhabditis elegans die. The survival rate of C.elegans was expressed as a survival curve, and the results were analyzed by the Kaplan-Meier method. The results are shown in FIG. 3.
The results in FIG. 3 show that intracellular fluid of microcystis can prolong the survival time of caenorhabditis elegans under oxidative stress conditions and improve the survival rate. The test result shows that the intracellular fluid of the microcystis provided by the invention can relieve the premature senility phenomenon caused by oxidative stress and has obvious antioxidant and anti-aging effects.
Example 7 Effect of Microcystis intracellular fluid on the level of ROS in C.elegans
Taking a 24-hole culture plate, setting a test group and a control group, wherein each group is provided with two holes, the final volume of each hole is 1mL, the test group is added with the synchronized L4-stage wild type caenorhabditis elegans (N2) and the microcapsule algae intracellular fluid prepared in example 1, the final concentration of the microcapsule algae intracellular fluid is 100mgA/mL, the control group is added with the synchronized L4-stage wild type caenorhabditis elegans (N2) and the S Medium solution which has the same volume with the microcapsule algae intracellular fluid of the test group, after culturing for 24h at 20 ℃, paraquat is added into each hole until the final concentration is 2mM, after culturing for 48h, each group of caenorhabditis elegans is collected, homogenized under the ice bath condition respectively, the homogenate is centrifuged for 5min at 4 ℃ and 10000g, the supernatant is collected, DCFH-DA probe solution with the final concentration of 50 MuM is added into the supernatant, a fluorescence intensity is detected by using a fluorescence microplate reader under the excitation wavelength of 485nm and the emission wavelength of 538nm, the detection is carried out at room temperature for 2h, and every 10 min. The results were analyzed by the F-test and are shown in FIG. 4.
The results in FIG. 4 show that the ROS levels in C.elegans were significantly reduced after feeding with intracellular fluid from microcystis as compared to the control group. Test results show that the intracellular fluid of the microcystis provided by the invention can promote the removal of ROS in vivo and reduce the ROS level in vivo, and has remarkable oxidation resistance.
Example 8 Effect of Microcystis intracellular fluid on the Activity of SOD in C.elegans
A24-well culture plate is taken, a test group and a control group are arranged, each group is provided with two holes, the final volume of each hole is 1mL, the test group is added with synchronized L4-stage wild type caenorhabditis elegans (N2) and the microcystis intracellular fluid prepared in example 1, the final concentration of the microcystis intracellular fluid is 100mgA/mL, the control group is added with synchronized L4-stage wild type caenorhabditis elegans (N2) and S Medium solution which has the same volume with the microcystis intracellular fluid of the test group, after culturing for 72h at 20 ℃, each group of caenorhabditis elegans are collected and homogenized under ice bath conditions respectively, the homogenate is centrifuged for 5min under 4 ℃ and 10000g, the supernatant is collected, and the SOD activity in the homogenate supernatant is determined by using a total activity detection kit (purchased from Biyunnan biotechnology research make internal disorder or usurp, product number: 010S 1). The results are shown in FIG. 5.
FIG. 5 shows that the intracellular fluid of microcystis significantly enhances the activity of SOD in C.elegans compared to the control. Test results show that the intracellular fluid of the microcystis provided by the invention can obviously improve the activity of SOD in vivo and has obvious antioxidant capacity.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (4)
1. The microcapsule algae ethanol extract is characterized in that the preparation method of the microcapsule algae ethanol extract comprises the following steps:
s1, taking fresh microcystis wet algae, freezing for 12 hours at the temperature of-20 to-40 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 3-5 times, centrifuging for 5-15 minutes at the rotating speed of 5000-10000 r/min, separating supernatant from precipitate, and taking the precipitate for later use;
s2, adding ethanol with the volume fraction of 70% -99% into the precipitate obtained in the step S1, performing ultrasonic extraction for 2-4 times, centrifuging for 5-15 min under the condition that the rotating speed is 5000-10000 r/min, separating the supernatant from the precipitate, taking the supernatant for vacuum drying, and dissolving the product after vacuum drying with primary water to obtain the microcystis ethanol extract;
and in the step S2, ethanol is added according to the solid-to-liquid ratio of the wet algae to the ethanol of 1g/mL for ultrasonic extraction.
2. The ethanol extract of microcystis as claimed in claim 1, wherein the ultrasonic extraction frequency in step S2 is 35-40 kHz and the temperature is 4 ℃.
3. The ethanol extract of microcystis according to claim 1, wherein the time of each ultrasonic extraction in step S2 is 10-30 min.
4. The use of the ethanol extract of microcystis according to claim 1 for the preparation of health food and pharmaceutical products with antioxidant function.
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