CN107412278B - Antioxidation and anti-aging application of dictyota water extract - Google Patents

Antioxidation and anti-aging application of dictyota water extract Download PDF

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CN107412278B
CN107412278B CN201710388926.6A CN201710388926A CN107412278B CN 107412278 B CN107412278 B CN 107412278B CN 201710388926 A CN201710388926 A CN 201710388926A CN 107412278 B CN107412278 B CN 107412278B
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precipitate
supernatant
water extract
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dictyococcus
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CN107412278A (en
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黄泽波
胡丽莉
张琪
郑凌凌
许怡宁
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Kelemei Guangzhou Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
    • 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
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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
    • A61K2236/30—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
    • 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
    • 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 application of a dictyococcus water extract in preparation of health-care food and medicines with antioxidant and/or anti-aging functions. The preparation method of the dictyota algae water extract comprises the steps of repeatedly freezing and thawing fresh wet dictyota algae, and centrifuging to remove supernatant; adding ethanol into the precipitate, performing ultrasonic extraction, and centrifuging to remove supernatant; washing the precipitate with water, adding water, and heating for extraction. The invention provides a new application of dictyococcus, which applies the dictyococcus water extract to the preparation of health-care food and medicine with antioxidant and/or anti-aging functions. The dictyocline water extract provided by the invention can prolong the survival time of caenorhabditis elegans under oxidative stress conditions, improve the survival rate, promote the elimination of active oxygen free radicals in vivo, reduce the level of the active oxygen free radicals in vivo, and has remarkable antioxidant and anti-aging effects.

Description

Antioxidation and anti-aging application of dictyota water extract
Technical Field
The invention belongs to the technical field of marine organisms, relates to an application of a dictyota aqueous extract, and particularly relates to an anti-oxidation and anti-aging application of the dictyota aqueous extract.
Background
Genus Glomulina (A)Dictyosphaeriumehrenbergianum) Plants are an original set population, cells are spherical, elliptical, oval, or kidney-shaped, often in groups of 4 or 2 cells, separated from each other, joined by parent cell walls that divide into 4 pieces, forming a dendritic structure. The outside of the population is always provided with a transparent and colorless coating. The individual cells of a shaped population often sporulate simultaneously and then attach to the top of the respective parent cell wall lobe to form a composite original shaped population. The chromoplasts were single, cup-shaped, with 1 protein core. Rubber netAlgae reproduce asexually like parent spores and are often found in soft lakes and ponds.
Microalgae are a kind of unicellular photoautotrophic eukaryote and mainly live in various water bodies. At present, microalgae have many applications in human and animal nutrition, cosmetics and production of high value-added products (such as pigments, fatty acids, stable isotopes and the like), but the research on the types of microalgae is narrow, the research mainly focuses on several common microalgae such as spirulina platensis, porphyridium, chlorella and the like, relatively few reports are made on other microalgae, and the development of novel functional microalgae is beneficial to promoting and accelerating the development of microalgae resources and the development of marine economy.
Through retrieval, no report about the related application of the dictyococcus laurentii, in particular the report about the anti-oxidation and anti-aging application of the dictyococcus laurentii water extract is found in the prior art.
Disclosure of Invention
The invention provides application of a dictyococcus water extract in preparing a medicine with antioxidant and/or anti-aging functions. The water extract of the dictyota algae provided by the invention is prepared by repeatedly freezing and thawing fresh wet dictyota algae and centrifuging to remove supernatant; adding ethanol into the precipitate, performing ultrasonic extraction, and centrifuging to remove supernatant; the precipitate is taken, washed by water and then heated and extracted by adding water, has obvious antioxidation and anti-aging effects, and can be applied to preparing medicines with antioxidation and/or anti-aging functions.
The purpose of the invention is realized by the following technical scheme: provides the application of the dictyococcus water extract in preparing the medicines with the functions of antioxidation and/or anti-aging. Further, the preparation method of the dictyococcus water extract comprises the following steps:
s1, taking fresh wet algae of the dictyota, freezing for 10-12 hours at the temperature of-20 to-40 ℃, thawing for 10-12 hours at room temperature, repeatedly freezing and thawing for 3-5 times, centrifuging for 5-15 minutes at the rotation speed of 5000 to 10000r/min, separating supernatant from precipitate, and taking the precipitate for later use; the supernatant is the intracellular fluid of the dictyococcus;
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, and taking the precipitate for later use;
s3, washing the precipitate for 1-3 times by primary water in the step S2, centrifuging for 5-15 min under the condition that the rotating speed is 5000-10000 r/min, separating the supernatant from the precipitate, removing the supernatant, adding the primary water into the precipitate, heating and extracting for 3-5 times, 30-60 min each time, centrifuging for 5-15 min under the condition that the rotating speed is 5000-10000 r/min, separating the supernatant from the precipitate, and taking the supernatant to obtain the dictyotis water extract.
Further, in 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.
Further, the frequency of ultrasonic extraction in the step S2 is 35-40 kHz, and the temperature is 4 ℃.
Further, the time of each ultrasonic extraction in the step S2 is 10-30 min.
Further, in step S3, primary water is added according to the solid-to-liquid ratio of the wet algae to the primary water of 1g/mL for heating extraction.
Further, the heating and extracting temperature in the step S3 is 70-100 ℃.
The Dictyosphaeriumehrenbergianum is provided by aquatic organism research institute of Chinese academy of sciences, and is numbered as FACHB-1273 in freshwater algae seed bank of Chinese academy of sciences.
The primary water of the invention meets the standard of primary water in GB/T6682-2008.
The antioxidant and anti-aging effects of the invention refer to the prevention and treatment effects on aging and aging-related diseases which are mainly characterized by changes caused by oxidative stress under pathological or physiological conditions.
The dictyota dichotoma water extract can be used alone or in combination with other active ingredients as the only or main active ingredient for preparing a medicine with an antioxidant function, and the dosage form of the medicine is preferably tablets, capsules, injections, oral liquids, granules, pills, powder, dropping pills and the like.
In further research, the inventor of the invention unexpectedly finds that the intracellular fluid of the dictyococcus has obvious antioxidant and anti-aging effects, and can be applied to preparation of medicines with antioxidant and/or anti-aging functions.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a new application of dictyococcus, the dictyococcus water extract and intracellular fluid have obvious antioxidation and anti-aging effects, and can be applied to preparing medicines with antioxidation and/or anti-aging functions.
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FIG. 1 Effect of aqueous extracts of Nemacystus Alternifolius on the survival rate of C.elegans under oxidative stress conditions.
FIG. 2 Effect of aqueous extracts of Nemacystus glenoides on the levels of ROS in C.elegans.
FIG. 3 Effect of Nemacystus intracellular fluid on the survival rate of C.elegans under oxidative stress conditions.
FIG. 4 Effect of Nemacystus intracellular fluid on the mean life span of C.elegans under natural conditions.
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 dictyococcus is provided by aquatic organism research institute of Chinese academy of sciences, and the number of the dictyococcus is FACHB-1273 in freshwater algae seed bank of Chinese academy of sciences.
S basic solution: weighing 5.8g NaCl, 1.3g K2HPO4·3H2O and 6.0gKH2PO4Placing 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 of 5.0mg/mL cholesterol ethanol solution, shaking to dissolve completely (the solution changes from colorless transparency to microemulsion transparency without obvious precipitation), and sequentially adding 3mL of 1MCaCl2Solution, 3mL1MMgSO410mL of 1M Potassium citrate solution (p)H = 6.0) and 10mL of trace element solution, and left to stand at normal temperature for use.
Example 1 preparation of aqueous extract of Nemacystus
S1, taking fresh wet algae of the dictyota, freezing for 12 hours at the temperature of minus 20 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 5 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 ethanol with the volume fraction of 80% 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 3 times, wherein the ultrasonic extraction frequency is 40kHz, the temperature is 4 ℃, the ultrasonic time is 30min each time, centrifuging for 15min under the condition that the rotating speed is 8000r/min, separating the supernatant from the precipitate, and taking the precipitate for later use;
s3, washing the precipitate for 2 times by primary water in the step S2, centrifuging for 15min at the rotation speed of 8000r/min, separating the supernatant from the precipitate, removing the supernatant, adding the primary water into the precipitate according to the solid-to-liquid ratio of 1g/mL of wet algae to the primary water, extracting for 4 times at the temperature of 100 ℃ for 45min each time, centrifuging for 15min at the rotation speed of 8000r/min, separating the supernatant from the precipitate, taking the supernatant, adding the primary water to adjust the concentration of the supernatant to be 1gA/mL (A represents wet algae of the dictyozoa) relative to the weight of the dictyota (wet algae), and obtaining the water extract of the dictyota.
And (4) adding primary water into the supernatant obtained by centrifugation in the step S1 to adjust the concentration of the supernatant relative to the weight of the dictyococcus zoffii (wet algae) to be 2gA/mL (A represents the wet dictyococcus zoffii), thus obtaining the dictyococcus zoffii intracellular fluid.
Example 2 preparation of aqueous extracts of Nemacystus
S1, taking fresh wet algae of the dictyota, freezing for 10 hours at the temperature of minus 30 ℃, thawing for 10 hours at room temperature, repeatedly freezing and thawing for 4 times, centrifuging for 5 minutes at the rotation speed of 10000r/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 3 times, wherein the ultrasonic extraction frequency is 35kHz, the temperature is 4 ℃, the ultrasonic time is 20min each time, centrifuging for 10min under the condition that the rotation speed is 10000r/min, separating the supernatant from the precipitate, and taking the precipitate for later use;
s3, washing the precipitate for 3 times by primary water in the step S2, centrifuging for 15min at the rotation speed of 10000r/min, separating the supernatant from the precipitate, removing the supernatant, adding the primary water into the precipitate according to the solid-to-liquid ratio of 1g/mL of wet algae to the primary water, extracting for 3 times at the temperature of 90 ℃ for 30min each time, centrifuging for 15min at the rotation speed of 10000r/min, separating the supernatant from the precipitate, taking the supernatant, adding the primary water to adjust the concentration of the primary water to be 1gA/mL (A represents wet algae of the dictyozoa) relative to the weight of the dictyota (wet algae), and obtaining the water extract of the dictyota.
Example 3 preparation of aqueous extract of Nemacystus
S1, taking fresh wet algae of the dictyota, freezing for 10 hours at the temperature of minus 40 ℃, thawing for 12 hours at room temperature, repeatedly freezing and thawing for 3 times, centrifuging for 15 minutes at the rotating speed of 5000r/min, separating the supernatant from the precipitate, and taking the precipitate for later use;
s2, adding ethanol with the 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, performing ultrasonic extraction for 4 times, wherein the ultrasonic extraction frequency is 40kHz, the temperature is 4 ℃, the ultrasonic time is 20min each time, centrifuging for 15min under the condition that the rotating speed is 5000r/min, separating the supernatant from the precipitate, and taking the precipitate for later use;
s3, washing the precipitate for 2 times by primary water in the step S2, centrifuging for 15min at the rotation speed of 10000r/min, separating the supernatant from the precipitate, removing the supernatant, adding the primary water into the precipitate according to the solid-to-liquid ratio of 1g/mL of wet algae to the primary water, extracting for 5 times at the temperature of 70 ℃ for 60min each time, centrifuging for 10min at the rotation speed of 10000r/min, separating the supernatant from the precipitate, taking the supernatant, adding the primary water to adjust the concentration of the primary water to be 1gA/mL (A represents wet algae of the dictyozoa) relative to the weight of the dictyota (wet algae), and obtaining the water extract of the dictyota.
Example 4 Effect of Dictyophora Water extract on the survival Rate 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 aqueous extract of C.galbana prepared in example 1 was added to adult wild type C.elegans (N2) (provided by the genetic information collection of C.elegans, university of Minnesota) at a final concentration of 300mgA/mL, to a control group, an equal volume of S Medium solution was added, the group was incubated at 20 ℃ for 24 hours, paraquat at a final concentration of 50mM was added to the group for oxidative stress molding, and the survival rate of C.elegans was counted every 12 hours until all of the species died. 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 the dictyocaulus water extract can significantly prolong the survival time of C.elegans under oxidative stress conditions and increase the survival rate thereof. Test results show that the dictyococcus water extract 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 Dictyophora Water extract on ROS levels in C.elegans
Taking a 24-hole 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 synchronized L4-stage wild type C.elegans (N2) and the dictyozoa aqueous extract prepared in example 1, the final concentration of the dictyococcus aqueous extract is 100mgA/mL, the control group is added with synchronized L4-stage wild type C.elegans (N2) and the S Medium solution with the same volume as the water extract of the dictyococcus 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 the C.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 μ M is added into the supernatant, fluorescence intensity was measured with a fluorescence microplate reader at 485nm excitation wavelength and 538nm emission wavelength for 2h at room temperature, once every 10min, and the results are shown in FIG. 2.
The results in FIG. 2 show that there was a significant decrease in the ROS levels in C.elegans after feeding with the aqueous extract of Nemacystus galbana compared to the control group. Test results show that the dictyococcus water extract provided by the invention can promote the removal of ROS in vivo and reduce the ROS level in vivo, and has remarkable antioxidant capacity.
Example 6 Effect of Nemacystus Alternifolia intracellular fluid on the survival of C.elegans under oxidative stress conditions
The intracellular fluid of the dictyococcus laurentii prepared in example 1 is added into wild type 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 24 hours, 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 12 hours 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 the intracellular fluid of Nemacystus polygama can prolong the survival time and increase the survival rate of C.elegans under oxidative stress conditions. Test results show that the dictyota intracellular fluid 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 Nemacystus Alternifolia intracellular fluid on the mean Life time of caenorhabditis elegans under Natural conditions
The synechocystis intracellular fluid prepared in example 1 was added to the synchronized wild type caenorhabditis elegans (N2) at L4 stage to a final concentration of 100mgA/mL, an equal volume of S Medium solution was added to the control group, the cells were incubated at 20 ℃ and the survival of caenorhabditis elegans was counted every 1 day until all of them died. The life span of C.elegans is expressed as a survival curve, and the results are analyzed by the Kaplan-Meier method. The results are shown in FIG. 4.
FIG. 4 shows that the intracellular fluid of Nemacystus decipiens is capable of prolonging the life expectancy of C.elegans under natural conditions, as compared to the control. Test results show that the dictyota intracellular fluid provided by the invention has the effect of delaying the aging process, namely, has a remarkable anti-aging effect.
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 (1)

1. The application of the dictyococcus water extract in preparing the medicines with the functions of antioxidation and anti-aging is characterized in that the preparation method of the dictyococcus water extract comprises the following steps:
s1, taking fresh wet algae of the dictyota, freezing for 10-12 hours at the temperature of-20 ℃ to-40 ℃, thawing for 10-12 hours at room temperature, repeatedly freezing and thawing for 3-5 times, centrifuging for 5-15 minutes at the rotation speed of 5000r/min to 10000r/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, and taking the precipitate for later use;
step S2, adding ethanol according to the solid-to-liquid ratio of the wet algae to the ethanol of 1g/mL for ultrasonic extraction, wherein the frequency of ultrasonic extraction is 35-40 kHz, the temperature is 4 ℃, and the time of each ultrasonic extraction is 10-30 min;
s3, washing the precipitate in the step S2 with primary water for 1-3 times, centrifuging for 5-15 min under the condition that the rotating speed is 5000-10000 r/min, separating the supernatant from the precipitate, removing the supernatant, adding the primary water into the precipitate, heating and extracting for 3-5 times, 30-60 min each time, centrifuging for 5-15 min under the condition that the rotating speed is 5000-10000 r/min, separating the supernatant from the precipitate, and taking the supernatant to obtain the dictyotis water extract;
in step S3, primary water is added according to the solid-to-liquid ratio of the wet algae to the primary water of 1g/mL for heating extraction, and the heating extraction temperature is 70-100 ℃.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
产多糖海洋微藻筛选、培养及其多糖提取与抗氧化性研究;付婷婷;《广西大学》;20170315;第2,11,38页 *
付婷婷.产多糖海洋微藻筛选、培养及其多糖提取与抗氧化性研究.《广西大学》.2017,第2,11,38页. *
蛋白核小球藻冻融破壁条件优化及对其抗氧化活性的影响;陈艺煊等;《食品科技》;20160520;第41卷(第5期);第75-80页 *

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