CN105920046B - A kind of polar glycolipids extract and its application - Google Patents
A kind of polar glycolipids extract and its application Download PDFInfo
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Classifications
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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
- A61K35/56—Materials from animals other than mammals
- A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- 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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- General Health & Medical Sciences (AREA)
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- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
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- Pharmacology & Pharmacy (AREA)
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Abstract
The technical problem to be solved by the present invention is to be to provide extract and its application of echinoderm polar glycolipids, it is used to prepare the food and medicine that can be improved, improve Neural Differentiation, has the effect of promoting the development of nervous system or prevention neurogenic disease.Extraction purification of the present invention sea cucumber, sea urchin, starfish polar glycolipids, and acted on nerve cell, the results showed that three kinds of echinoderm polar glycolipids extracts can significantly improve neural cellular differentiation rate, to promoting, improve Neural Differentiation to have significant effect.Therefore, product can be made using echinoderm polar glycolipids extract as a kind of component or auxiliary material cooperation.The preparation method of echinoderm polar glycolipids extract is at low cost in the present invention, operating method is relatively simple, gained echinoderm polar glycolipids extract has the effect of well promoting neurodevelopment through cell experiment confirmation, it can be used for developing safely and effectively to improve the development of prematurity somatic nervous system and the food or drug of prevention of neurodegenerative diseases.
Description
Technical field
The invention belongs to screening bioactive compounds technical fields, and in particular to a kind of polar glycolipids extract and its application.
Background technique
Nerve growth and differentiation be people a kind of important physiological phenomenon, it is not only related with infancy intellectual development, and with it is old
The neurodegenerative disease of Nian Yifa such as Alzheimer syndrome (AD), parkinsonism (PD) etc. is closely related.Nerve
Differentiation is formed as main feature, including neural process (including dendron and aixs cylinder) starting, extension, branch and neural network with aixs cylinder
Formation, this is most important for regenerating after the development of nervous system and neure damage.If breaking down in the process,
It can give rise to diseases and illness, including feeblemindedness, neurodegenerative disease etc..Therefore, normal nerve growth, differentiation not
The only key factor of human health, the social development pushed simultaneously for the mankind are also of great significance.
In mammalian nervous system growth course, the variation of content and type and the nerve fiber form of polar glycolipids are sent out
It educates with correlation.By taking gangliosides (gangliosides, GLS) as an example, mesoderm growing early stage nervous system is mainly expressed
The better simply gangliosides of structure, such as monosaccharide groups Ganglioside, GD3 and GM3.With the growth of protrusion and cynapse, four glycosyls
Ganglioside GM1 and GD1 content increase sharply, and finally internal in maturation, the type and content of gangliosides reach stable.
In primary hippocampal, Dorsal Root Ganglion Neurons (DRG) originally culture or the PC12 cell no matter cultivated in vitro, through in vitro through inducing
The phenomenon that differentiation, presentation ganglioside content significantly increases.Furthermore it studies have found that, is added into PC12 cell exogenous
Gangliosides can significantly increase nerve growth factor (NGF) function, promote nerve regneration.This is not only predictive of exogenous polarity
Glycolipid can promote, improve a possibility that Neural Differentiation and regeneration, also drive scholars to be constantly dedicated to research Neural Differentiation logical
Road, the mechanism of action of neuronal inhibition factor are conducive to food, drug that preferably exploitation has neurotrophic activity.
Echinoderm (Echinodermata) be it is a kind of live in seabed, body be in radial symmetrical type invertebrate
5 guiding principles such as general designation, including Ophiuroidea, Crinoidea, Echinoidea, Asteroidea and Holothuroidea.Wherein sea cucumber, sea urchin are not only to be good for
The food of Kang Youyi, and several physiological active substances are rich in, so being taken as rare nourishing food or medicinal material always；And starfish
Substantially without edible value, but in vivo containing there are many tool antibacterials, antitumor, decompression active material.Further, since the office of region
Sex-limited, the development of marine active substance is slower, though in recent years constantly studies have reported that isolated out of echinoderm body
Active material have the effects that improve immunity, suppression cancer, reducing blood lipid, blood pressure lowering, neuroprotection, and about its nerve growth,
The effect of Neural Differentiation, has not been reported.
Summary of the invention:
The technical problem to be solved by the present invention is to be to provide a kind of polar glycolipids extraction obtained from echinoderm
Object and its application can be used for preparing promotion, improve Neural Differentiation, prevent the food or drug of neural class disease.
Polar glycolipids extract is provided first in the present invention, it is specific the preparation method is as follows:
1) echinoderm edible part is taken, screening obtains dry powder after freeze-drying grinding；
2) sample dry powder is taken, chloroform and methyl alcohol mixed liquor extraction (1:1~5, v/v) is added, centrifugation obtains supernatant；
3) add water vortex oscillation to extract through water leaching liquor, be collected by centrifugation upper layer methanol-water phase, and to lower layer's chloroformic solution
Middle addition KCl solution and methanol (1:1~6, v/v) extract again, merge upper solution.By its activated C8 solid-phase extraction column
Extraction, methanol elution collect eluent and obtain echinoderm lipid crude extract.
Eluent is subjected to thin-layer silicon offset plate chromatography, solvent is chloroform-methanol-water (10~20:2~6:1, v/v), is received
Collect RfThe band that value is 0.8~0.9, is concentrated to get sample powder for band；
4) sample powder for obtaining step 3) is dissolved in chloroform-methanol-water (6~15:3~10:1, v/v), carries out ion
Exchange column chromatography；Then chloroform-methanol-water (1:1~2:3~10, v/v) solution containing 0.1~1.0mol/L ammonium acetate is passed through
Linear elution obtains final echinoderm polar glycolipids ingredient after eluent concentration.
The echinoderm polar glycolipids extract of offer of the invention is used to prepare for promoting, improving Neural Differentiation, in advance
The product of anti-neural class disease.
The product, is food or drug, and the drug is oral administration preparation or parenteral formulations, including powder
Injection, granule, tablet, electuary, powder, oral solution, enteric coated tablet, capsule etc..
The cell experiment of echinoderm polar glycolipids extract of the invention the result shows that, echinoderm polar glycolipids extract
Object can significantly improve neural cellular differentiation rate, increase the expression of Neural Differentiation GAP-associated protein GAP, i.e., to promotion, improvement Neural Differentiation tool
There is significant effect.Product can be made using echinoderm lipid-soluble extract as a kind of component or auxiliary material cooperation.Spine in the present invention
The preparation method of skin animal lipid extract is at low cost, and operating method is simple, and gained echinoderm lipid-soluble extract is through cell reality
Verifying is real to be had the effect of good promotion, improves Neural Differentiation, and can be used for developing safely, effectively improves child's neurodevelopment,
The food or drug for preventing old degenerative disease of easily going crazy.
Detailed description of the invention
Fig. 1: shadow of ocean of the embodiment of the present invention echinoderm polar glycolipids extract to the NGF PC12 cell differentiation mediated
It rings.Normal group cell is almost undifferentiated, is added to the model control group differentiation rate of 10ng/mL NGF close to 50%, adds
50 μ g/mL sea cucumbers, sea urchin, starfish polar glycolipids extract experimental group compared to the control group, PC12 cell differentiation rate mentions respectively
It is high by 41%, 36.3%, 30.3%, have extremely significant sex differernce (P < 0.01), and compared with bovine brain GM1, has preferably living
Property effect, and not purified ocean echinoderm lipid crude extract fails to significantly improve differentiation rate (P > 0.05).Show to extract
The lipid crude extract of Yu Haiyang echinoderm, the polar glycolipids extract obtained after purified, is remarkably improved experimental group PC12
The differentiation rate of cell.
Specific embodiment
Applicant has finally been determined that it has and has been promoted, improves mind by the detection to echinoderm polar glycolipids extract
Through breaking up the preparation method of effective component, interference of the other materials to active effect in echinoderm is eliminated, and then facilitate
The present invention.
The present invention will be described in detail below with reference to the drawings of preferred embodiments, whereby to the present invention how applied technology method
Technical problem is solved, and the realization process for reaching technical effect can fully understand and implement.
Embodiment 1: the preparation of sea cucumber lipid crude extract
The first step takes the sea cucumber of gutting, and -40 DEG C of freeze-dryings, screening obtains dry powder after grinding.
Second step weighs sample powder 20g, and chloroform/methanol (1:2, v/v) extraction is primary, stands after vortex oscillation 1min
Upper layer extracting solution is collected by centrifugation in 10min；Residue repeats extraction 2~3 times with chloroform-methanol (3:1, v/v) afterwards, merges on twice
Clear liquid obtains leaching liquor through filtering.
Upper layer methanol-water phase is collected by centrifugation sufficiently after extraction in leaching liquor plus water vortex oscillation by third step；To lower layer's chlorine
0.01mol/L KCl solution is added in imitative solution and methanol (1:3, v/v) extracts again, merges upper solution.Then it is splined on
The C8 solid-phase extraction column of activation, methanol elution rotate eluent to dry, acquisition sea cucumber lipid crude extract.
Embodiment 2: the preparation of sea cucumber polar glycolipids extract
The first step takes sea cucumber edible part, and -40 DEG C of freeze-dryings, screening obtains dry powder after grinding；
Second step weighs sample powder 10g, and chloroform/methanol (1:1, v/v) extraction is primary, stands after vortex oscillation 1min
10min；Upper layer extracting solution is collected by centrifugation, residue repeats extraction 2~3 times with chloroform-methanol (1:1, v/v) afterwards, merges on twice
Clear liquid obtains leaching liquor through filtering；
Upper layer methanol-water phase is collected by centrifugation sufficiently after extraction in leaching liquor plus water vortex oscillation by third step；To lower layer's chlorine
0.01mol/L KCl solution is added in imitative solution and methanol (1:1, v/v) extracts again, merges upper solution.Then it is splined on
The C8 solid-phase extraction column of activation, methanol elution, by eluent carry out thin-layer silicon offset plate chromatography (TLC), wherein solvent be chloroform/
Methanol/water (10:2:1, v/v).By RfValue obtains sample powder for 0.8~0.9 Tape samples concentration.
Sample powder is dissolved in appropriate chloroform-methanol-water (6:3:1, v/v) and dissolved, carries out anion-exchange column by the 4th step
Chromatography；Then through chloroform-methanol-water (1:1:3, v/v) linear elution, eluent is collected, final sea cucumber pole can be obtained in concentration
Property glycolipid 10.13mg.
Embodiment 3: the preparation of sea urchin polar glycolipids extract
The first step takes the edible part of sea urchin, and -40 DEG C of freeze-dryings, screening obtains dry powder after grinding；
Second step weighs sample powder 40g, and chloroform/methanol (1:2, v/v) extraction is primary, stands after vortex oscillation 1min
10min；Upper layer extracting solution is collected by centrifugation, residue repeats extraction 2~3 times with chloroform-methanol (3:1, v/v) afterwards, merges on twice
Clear liquid obtains leaching liquor through filtering；
Upper layer methanol-water phase is collected by centrifugation sufficiently after extraction in leaching liquor plus water vortex oscillation by third step；To lower layer's chlorine
0.01mol/L KCl solution is added in imitative solution and methanol (1:3, v/v) extracts again, merges upper solution.Then it is splined on
The C8 solid-phase extraction column of activation, methanol elution, eluent carry out thin-layer silicon offset plate chromatography (TLC), and wherein solvent is chloroform/first
Alcohol/water (15:3:1, v/v).By RfValue obtains sample powder for 0.8~0.9 Tape samples concentration.
4th step will be dissolved in appropriate chloroform-methanol-water (10:5:1, v/v) dissolution, carry out anion friendship in sample powder
Change column chromatography；Then through chloroform-methanol-water (1:2:8, v/v) linear elution, eluent is collected, final sea can be obtained in concentration
Gallbladder polar glycolipids 38.02mg.
Embodiment 4: the preparation of starfish polar glycolipids extract
The first step takes the edible part of starfish, and -40 DEG C of freeze-dryings, screening obtains dry powder after grinding；
Second step weighs sample powder 40g, and chloroform/methanol (1:5, v/v) extraction is primary, stands after vortex oscillation 1min
10min；Upper layer extracting solution is collected by centrifugation, residue repeats extraction 2~3 times with chloroform-methanol (5:1, v/v) afterwards, merges on twice
Clear liquid obtains leaching liquor through filtering；
Upper layer methanol-water phase is collected by centrifugation sufficiently after extraction in leaching liquor plus water vortex oscillation by third step；To lower layer's chlorine
0.01mol/L KCl solution is added in imitative solution and methanol (1:6, v/v) extracts again, merges upper solution.Then it is splined on
The C8 solid-phase extraction column of activation, methanol elution, eluent carry out thin-layer silicon offset plate chromatography (TLC), and wherein solvent is chloroform/first
Alcohol/water (20:6:1, v/v).By RfValue obtains sample powder for 0.8~0.9 Tape samples concentration.
4th step will be dissolved in appropriate chloroform-methanol-water (15:10:1, v/v) dissolution, carry out anion friendship in sample powder
Change column chromatography；Then through chloroform-methanol-water (1:4:10, v/v) linear elution, eluent is collected, final sea can be obtained in concentration
Star polar glycolipids 31.13mg.
Embodiment 5: the effect of echinoderm polar glycolipids extract promotion neural cellular differentiation
One, material and method
(1) foundation and experimental group of cell model
To logarithmic growth phase, Suitable Density is inoculated in through poly-D-lysine coated 6 hole culture routine culture PC12 cell
Plate.The 2%FBS-RPMI culture medium of NGF containing various concentration (0,5,10 and 20ng/L) is added after cell for 24 hours is adherent, persistently incubates
144h is educated, every the differentiation rate for taking pictures and counting cell for 24 hours.Cell can normally be survived, NGF concentration when differentiation rate is up to 50%
And action time is as model group condition.
(2) cell differentiation rate measures
Cell culture condition and fabric swatch method same (1) are divided into model control group (10ng/mL after cell for 24 hours is adherent
NGF), experimental group.In the sea cucumber lipid crude extract and embodiment 2 that the embodiment 1 that experimental group adds 50 μ g/mL respectively obtains
Starfish polar glycolipids in sea urchin polar glycolipids, embodiment 4 in sea cucumber polar glycolipids, embodiment 3 (contain 10ng/mL
NGF), take pictures after being persistently incubated for 6 days, and cell differentiation rate, axon length, aixs cylinder number are counted.
(3) expression of immunofluorescence assay synaptophysin and growth Protein G AP-43
Cell fabric swatch and cultural method are the same as (1).After comparison model group and experimental group are incubated for for 24 hours, through conventional Tri-labeling method
Method processing, wherein corresponding primary antibody solution (Synaptophysin and GAP-43), fluorescence secondary antibody is added according to specification in antibody
After incubation, PBS buffer solution is sufficiently washed, and DAPI redyes 20min.Last confocal laser scanning microscope is taken pictures.
(4) expression of RT-qPCR method measurement synaptophysin and growth Protein G AP-43
Cell fabric swatch and cultural method after addition tested material is incubated for for 24 hours, extract PC12 cell using Trizol method with (1)
Total serum IgE.The purity and content of RNA are calculated, and detects the integrality of RNA.Take 1 μ g RNA in the reverse transcription reaction system of 25 μ L
In, it is catalyzed through M-MLV reverse transcriptase and generates cDNA, saved at 4 DEG C stand-by.Then according to SYBR Green I Master Mix
Specification detects the expression quantity of each target gene mRNA with RT-qPCR.The mrna expression amount of each gene is with β-actin mRNA
Expression quantity is corrected as internal reference, and comparison model group is set to 100%.
Two, experimental result
NGF can maintain the survival of PC12 cell under low serum state, and Cell differentiation inducing activity is at the thin of neural characteristic
Born of the same parents.Determine that NGF induces noble cells model are as follows: 10ng/mL NGF, be incubated for 6 days.Spine skin obtained is purified in embodiment 2,3,4
Animal polar glycolipids can be obviously promoted the PC12 cell differentiation of NGF induction, and sea cucumber, sea urchin, starfish polar glycolipids can be respectively increased
Cell differentiation rate 61%, 47%, 30%, cell of the axon length greater than 4 times of cell space dramatically increases (P < 0.05), and embodiment
Sea cucumber lipid crude extract in 1, which is not apparent from, promotes cell differentiation (P > 0.05).In addition, being added to the reality of echinoderm polar glycolipids
Group is tested compared with model group, the mRNA level in-site of synaptophysin and growth Protein G AP-43 significantly rise (P < 0.01).It is comprehensive apparent
Index shows purified echinoderm polar glycolipids extract (embodiment 2,3,4) compared to echinoderm lipid crude extract
(embodiment 1), model group have more significant effect to promotion, improvement Neural Differentiation.
Embodiment 6: echinoderm polar glycolipids extract active principle preparation
Sea cucumber in Example 2,3,4, sea urchin, starfish polar glycolipids extract 15mg, superfine silica gel powder 20g respectively, paste
Smart 400g, starch 580g are filled with 1g/ capsule, can be made into capsule after mixing is sufficiently stirred in the agitated machine of mentioned component
1000.Wherein every capsule echinoderm containing effective component polar glycolipids extract 0.015mg.
Embodiment 7: echinoderm polar glycolipids extract active principle preparation
Echinoderm polar glycolipids extract 20mg, the medical starch 1000g in Example 2,3,4, mixing are equal respectively
It after even, is pelletized with ethanol in proper amount, whole grain, tabletting obtains tablet, every 0.2g.Wherein every polar glycolipids containing echinoderm extract
Object 0.004mg.
The above described is only a preferred embodiment of the present invention, being not that the invention has other forms of limitations, appoint
What those skilled in the art changed or be modified as possibly also with the technology contents of the disclosure above equivalent variations etc.
Imitate embodiment.But without departing from the technical solutions of the present invention, according to the technical essence of the invention to above embodiments institute
Any simple modification, equivalent variations and the remodeling made, still fall within the protection scope of technical solution of the present invention.
Claims (6)
1. a kind of polar glycolipids extract, which is characterized in that the preparation method of the echinoderm polar glycolipids extract is such as
Under:
1) echinoderm edible part is taken, smashed sample is obtained after grinding；
2) smashed sample is taken, chloroform is added and methyl alcohol mixed liquor is extracted, is centrifugated residue and supernatant, supernatant
Leaching liquor is obtained through filtering；
The chloroform and methyl alcohol mixed liquor, wherein the volume ratio of chloroform and methanol is 1:1~5；
3) leaching liquor is extracted through water, is centrifugated upper layer methanol-water phase solution and lower layer's chloroformic solution；Methanol-water is mixed
The activated C8 solid-phase extraction column of liquid is extracted, and is eluted with methanol, and eluent is collected；
Eluent is subjected to thin-layer silicon offset plate chromatography, solvent is chloroform-methanol-aqueous solution, collects RfThe item that value is 0.8~0.9
Band is concentrated to get sample powder by band；
Chloroform in the solvent, methanol, water volume ratio be 10~20:2~6:1；
4) sample powder for obtaining step 3) is dissolved in chloroform-methanol-aqueous solution, carries out ion-exchange chromatography；Then through containing
There is chloroform-methanol-aqueous solution of ammonium acetate to carry out linear elution, obtains final echinoderm polar glycolipids after eluent concentration
Ingredient；
Chloroform-methanol-the aqueous solution containing ammonium acetate, wherein acetic acid ammonium concentration is 0.1~1.0mol/L, chloroform, first
Alcohol, water volume ratio be 1:1~4:3~10.
2. echinoderm polar glycolipids extract as described in claim 1, which is characterized in that the supernatant in the step 2)
Liquid also includes the leaching liquor that useful chloroform and methanol solution extract acquisition to residue again.
3. echinoderm polar glycolipids extract as described in claim 1, which is characterized in that the first in the step 3)
Alcohol-aqueous phase solution also include into lower layer's chloroformic solution be added 0.01mol/L KCl solution and methanol carry out extraction acquisition
Upper solution.
4. echinoderm polar glycolipids extract as claimed in claim 3, which is characterized in that the KCl solution and methanol
Volume ratio be 1:1~6.
5. echinoderm polar glycolipids extract described in claim 1 is preparing the product for promoting or improving Neural Differentiation
In application.
6. a kind of for promoting or improving the product of Neural Differentiation, which is characterized in that the product includes claim 5 institute
The echinoderm polar glycolipids extract stated.
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