CN104522459A - Use of rhodiola root and spine date seed compound polysaccharide in preparation of anti neurodegenerative disease health food - Google Patents
Use of rhodiola root and spine date seed compound polysaccharide in preparation of anti neurodegenerative disease health food Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
-
- 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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- Food Science & Technology (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract
The present invention discloses use of rhodiola root and spine date seed compound polysaccharide in preparation of anti neurodegenerative disease health food, the compound polysaccharide is a composition of rhodiola root polysaccharide and spine date seed polysaccharide, the compound polysaccharide is prepared from rhodiola root and /or spine date seed as raw materials by water extraction, concentration and alcohol precipitation process, the preparation method is simple, stable, efficient, and suitable for industrial production. By caenorhabditis elegans model experiment verification, the rhodiola root and spine date seed compound polysaccharide can effectively inhibit neurotoxicity of amyloid beta-protein and polyglutamine, the action mode is related with toxic protein aggregation inhibition, heat shock protein expression increase and reactive oxygen free radical level reduction, and neuroprotective effect can be shown.
Description
Technical field
The invention belongs to healthy food/technical field of health care food, be specifically related to a kind of rhodiola root and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation.
Background technology
Along with the aggravation of social population's aging degree, nerve degenerative diseases has become the major disease of serious harm people life and health and quality of life, although alleviate the medicine listing of patients symptomatic existing, these medicines lack the concern to disease generation in early stage and development.Therefore, active development has great people's livelihood meaning undoubtedly for the healthy food/health food of nerve degenerative diseases.Multiple nerve degenerative diseases known at present all relates to the abnormal aggregation of protein, and such as, the major pathologic features of Alzheimer's disease shows as extracellular amyloid-beta (amyloid β protein; A β) assemble the precipitation formed, the major pathologic features of Huntington's chorea shows as in cell by poly glumine (polyglutamine; PolyQ) inclusion bodies of protein caused extremely is extended.The product formed after these toxic protein abnormal aggregation has strong neurotoxicity, can inducing neural retrogression pathological changes.Therefore, by suppressing disease protein matter abnormal aggregation and alleviating the Critical policies that the toxicity stress damage caused by different polyprotein is control Neurodegenerative conditions.
C. Elegans Automatic Screening a kind ofly studies the conventional model animal of nervous function and relevant disease, has the outstanding advantages such as life cycle is short, experimental implementation is simple, genetic resources is abundant.The genome of C. Elegans Automatic Screening contains the homologous gene of the study of human disease-related gene position of 2/3, and easily build transgenic disease model, such as utilize GFP labelling technique, in individual level, can carry out the complete lifelong follow-up study of system to retrogression pathological changes and potential drug, this seems particularly important to the correlative study of the nervous system disease.Therefore, C. Elegans Automatic Screening is adopted to be conducive to promoting the screening of neuroprotective activity compound and the research of mechanism of action thereof.
The Chinese herbal medicine kind of China's abundant is that control nerve degenerative diseases provides huge resources bank, and wherein integration of drinking and medicinal herbs kind has the medicinal and healthy nutritive value of huge medical treatment undoubtedly.Such as, rhodiola root is the traditional blood-enriching qi-invigorating class Chinese medicine of China, have tonifying Qi disappear tired, intelligence development is nourished heart, the effect such as controlling of bleeding with astringents and eliminating stasis to subdue swelling; Spina date seed, as famous intelligence development class Chinese medicine of calming the nerves, has nourishing the liver, Ning Xin, calms the nerves and the effect such as arrest sweating.Modern study shows, the polysaccharide of these resources of Chinese medicinal herb also has the potential source biomolecule activity of control nerve degenerative diseases.But, generation development due to nerve degenerative diseases has many because of property and complexity, may be there is the problems such as action target spot is limited, active function is not enough in single polysaccharide, therefore expand and strengthen active polysaccharide to be absolutely necessary for deep exploitation and improvement polysaccharide product.Although the report at present about complex polysaccharide activity research is few, there are some researches show recently, astragalus polyose and sEPS coupling can improve chicken immune systemic-function, thus alleviation endoxan causes immunosupress.This shows compared to single polysaccharide, and the mutual coupling of herbal polysaccharide has the potentiality strengthening therapeutic action.Therefore, strengthen the research of complex polysaccharide in anti-nerve degenerative diseases and be not only conducive to promoting its deep application in medicines and health protection field, widen the range of application of polyose, novel active can also be provided for the control of disease.
Summary of the invention
The object of the present invention is to provide a kind of rhodiola root and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation.
For reaching foregoing invention object, the present invention proposes rhodiola root and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation.
In rhodiola root of the present invention and spina date seed complex polysaccharide, the quality proportioning of Radix Phodiolae Polyose and spina date seed polysaccharide is preferably 1:4 ~ 4:1.
The quality proportioning more preferably 2:3 ~ 3:2 of Radix Phodiolae Polyose and spina date seed polysaccharide in rhodiola root of the present invention and spina date seed complex polysaccharide.
As one of the present invention preferred embodiment: described rhodiola root and spina date seed complex polysaccharide prepare by the following method: with rhodiola root and spina date seed for raw material; through preparing complex polysaccharide containing water extraction, concentrated and alcohol precipitation operation; described complex polysaccharide has neuroprotection, can be used in preparing anti-nerve degenerative diseases health food.
In the preparation method of above-mentioned rhodiola root and spina date seed complex polysaccharide:
The quality proportioning of described rhodiola root and spina date seed is preferably 1:4 ~ 4:1.
The quality proportioning more preferably 2:3 ~ 3:2 of described rhodiola root and spina date seed.
During water extraction, the mass volume ratio of rhodiola root and spina date seed medicinal material and water is 1:2 ~ 1:20, and Extracting temperature is 50 ~ 100 DEG C, and extraction time is 1 ~ 10h; Time concentrated, thickening temperature is 40 ~ 100 DEG C; During alcohol precipitation, the volume ratio of ethanol and concentrate is 2:1 ~ 9:1.
As another preferred embodiment of the present invention: described rhodiola root and spina date seed complex polysaccharide prepare by the following method: with rhodiola root or spina date seed for raw material; through preparing Radix Phodiolae Polyose or spina date seed polysaccharide containing water extraction, concentrated and alcohol precipitation operation; again the two is mixed by metering ratio; prepare rhodiola root and spina date seed complex polysaccharide; described complex polysaccharide has neuroprotection, can be used in preparing anti-nerve degenerative diseases health food.
In the preparation method of above-mentioned rhodiola root and spina date seed complex polysaccharide:
During water extraction, the mass volume ratio of rhodiola root or spina date seed medicinal material and water is 1:2 ~ 1:20, and Extracting temperature is 50 ~ 100 DEG C, and extraction time is 1 ~ 10h; Time concentrated, thickening temperature is 40 ~ 100 DEG C; During alcohol precipitation, the volume ratio of ethanol and concentrate is 2:1 ~ 9:1.
In described rhodiola root and spina date seed complex polysaccharide, the quality proportioning of Radix Phodiolae Polyose and spina date seed polysaccharide is preferably 1:4 ~ 4:1.
The quality proportioning more preferably 2:3 ~ 3:2 of Radix Phodiolae Polyose and spina date seed polysaccharide in described rhodiola root and spina date seed complex polysaccharide.
Nerve degenerative diseases of the present invention refers to and comprises the nervous system disease that Alzheimer's disease and Huntington's chorea etc. are major pathologic features with toxic protein abnormal aggregation.
Rhodiola root provided by the invention and spina date seed complex polysaccharide have neuroprotective efficacy specifically can be embodied in its gathering toxicity that can suppress amyloid-beta and poly glumine in whole animal level; its mode of action with suppress toxic protein abnormal aggregation, improve heat shock protein expression and reduce active oxygen radical level closely related, therefore can be used for the nerve degenerative diseases that control is principle pathological mechanism with protein abnormal aggregation.
The formulation of health food of the present invention can be capsule, pulvis, granule, tablet or oral liquid etc.Such as above-mentioned complex polysaccharide is added other raw material if lactalbumin, auxiliary material are as maltodextrin, can be prepared as anti-nerve degenerative diseases healthy food or health food.
Tool of the present invention has the following advantages:
(1) rhodiola root in the present invention and spina date seed complex polysaccharide, its preparation technology is simply efficient, is applicable to large-scale production;
(2) rhodiola root that prepared by the present invention CKIs matter can assemble relevant Neurodegenerative conditions with verified its of spina date seed complex polysaccharide in C. Elegans Automatic Screening whole animal level, show in its solid-liquid preparation healthy food that can be applicable to anti-nerve degenerative diseases or health food, for the control of relevant disease and successful aging provide New Scheme, and deep application and the modernization of Chinese medicine development of polyose can be promoted.
Accompanying drawing explanation
Below by detailed description of the invention and pharmacodynamic study result, the invention will be further described.
Fig. 1 adopts rhodiola root and spina date seed complex polysaccharide and single polysaccharide in embodiment 1 to suppress the data characterization figure of amyloid-beta toxicity in C. Elegans Automatic Screening CL2355 body in embodiment 6, wherein Figure 1A characterizes the schematic diagram of 2mg/mL complex polysaccharide to the behavioral implications of C. Elegans Automatic Screening CL2355 chemotactic, Figure 1B characterizes the chemotactic ability that 2mg/mL complex polysaccharide, Radix Phodiolae Polyose and spina date seed polysaccharide improve C. Elegans Automatic Screening CL2355, and wherein * represents p<0.05;
Fig. 2 adopts rhodiola root and spina date seed complex polysaccharide and single polysaccharide in embodiment 2 to suppress the data characterization figure of poly glumine toxicity in C. Elegans Automatic Screening HA759 body in embodiment 7, wherein Fig. 2 A characterizes unprocessed and through the HA759 C. Elegans Automatic Screening ASH neuron schematic diagram of 2mg/mL complex polysaccharide process, Fig. 2 B characterizes 2mg/mL complex polysaccharide, Radix Phodiolae Polyose and spina date seed polysaccharide suppress poly glumine in HA759 C. Elegans Automatic Screening body to assemble toxicity, Fig. 2 C characterizes 2mg/mL complex polysaccharide, Radix Phodiolae Polyose and spina date seed polysaccharide improve the behaviouristics defect that poly glumine gathering toxicity causes, wherein * represents p<0.05,
Fig. 3 adopts rhodiola root and spina date seed complex polysaccharide in embodiment 3 to suppress the data characterization figure of nerve degenerative diseases related protein aggregation in vitro in embodiment 8, wherein Fig. 3 A characterizes the aggregation in vitro that 2mg/mL complex polysaccharide suppresses A β 42, and Fig. 3 B characterizes the aggregation in vitro that 2mg/mL complex polysaccharide suppresses polyQ40;
Fig. 4 to adopt in embodiment 4 rhodiola root and spina date seed complex polysaccharide to the data characterization figure of heat shock protein regulating and controlling effect in C. Elegans Automatic Screening CL2070 body in embodiment 9, wherein Fig. 4 A characterizes 2mg/mL complex polysaccharide expresses impact schematic diagram on HSP16.2 in C. Elegans Automatic Screening CL2070 body, Fig. 4 B characterizes the expression that 1mg/mL, 2mg/mL and 4mg/mL complex polysaccharide promotes HSP16.2 in C. Elegans Automatic Screening CL2070 body, and wherein * represents p<0.05;
Fig. 5 adopts rhodiola root and spina date seed complex polysaccharide in embodiment 5 to reduce the data characterization figure of C. Elegans Automatic Screening activity in vivo oxygen level in embodiment 10, wherein Fig. 5 A characterizes 1mg/mL, 2mg/mL and 4mg/mL complex polysaccharide reduction C. Elegans Automatic Screening CL2355 activity in vivo oxygen free radicals, Fig. 5 B characterizes 1mg/mL, 2mg/mL and 4mg/mL complex polysaccharide and reduces C. Elegans Automatic Screening HA759 activity in vivo oxygen free radicals, and wherein * represents p<0.05.
Detailed description of the invention
The present invention will be described to enumerate a part of specific embodiment below, be necessary to herein means out be following specific embodiment only for the invention will be further described, do not represent limiting the scope of the invention.Some nonessential amendments that other people make according to the present invention and adjustment still belong to protection scope of the present invention.
Embodiment 1
A kind of rhodiola root that the present embodiment provides and spina date seed complex polysaccharide, it prepares by the following method: rhodiola root and spina date seed medicinal material are mixed according to weight ratio 1:1, wherein rhodiola root refers to the medicinal part rhizome of rhodiola root, spina date seed refers to the medicinal part seed of wild jujube, lower same, add water according to solid-liquid ratio 1:10 (g/mL) after pulverizing, at 90 DEG C, add thermal agitation extract 4h.Filtering the dregs of a decoction obtained is that 1:5 (g/mL) adds water according to solid-liquid ratio, continues to extract 4h at 90 DEG C.Merge extracted twice liquid, through reduced pressure concentration and centrifugal acquisition clarification concentrate, according to 1:4 volume ratio by 4 times of volume 95% (volumn concentrations, down together) ethanol adds in concentrate, after mixing, at 4 DEG C, overnight stand precipitates, centrifugal collecting precipitation, namely obtains complex polysaccharide through vacuum freeze drying.
Or add water according to solid-liquid ratio 1:10 (g/mL) after rhodiola root is pulverized, at 90 DEG C, add thermal agitation extract 4h.Filtering the dregs of a decoction obtained is that 1:5 (g/mL) adds water according to solid-liquid ratio, continues to extract 4h at 90 DEG C.Merge extracted twice liquid, through reduced pressure concentration and centrifugal acquisition clarification concentrate, according to 1:4 volume ratio, 4 times of volume 95% ethanol are added in concentrate, after mixing, at 4 DEG C, overnight stand precipitates, centrifugal collecting precipitation, namely obtains Radix Phodiolae Polyose through vacuum freeze drying, the same Radix Phodiolae Polyose of preparation method of spina date seed polysaccharide, the quality proportioning of spina date seed polysaccharide and Radix Phodiolae Polyose being pressed 1:1 mixes, and prepares rhodiola root and spina date seed complex polysaccharide.
The rhodiola root adopting first method and second method to prepare and spina date seed complex polysaccharide, the two performance etc. are substantially identical, no matter adopt any method to prepare rhodiola root and spina date seed complex polysaccharide.
Embodiment 2
A kind of rhodiola root that the present embodiment provides and spina date seed complex polysaccharide, it prepares by the following method: rhodiola root and spina date seed medicinal material are mixed according to weight ratio 2:3, add water according to solid-liquid ratio 1:15 (g/mL) after pulverizing, at 90 DEG C, add thermal agitation extract 6h.Centrifugal acquisition clarification concentrate after reduced pressure concentration, add in concentrate according to 1:3 volume ratio by 3 times of volume 95% ethanol, after mixing, at 4 DEG C, overnight stand precipitates, and centrifugal collecting precipitation, namely obtains complex polysaccharide through vacuum freeze drying.
The present embodiment also can adopt rhodiola root or spina date seed to be raw material with in embodiment 1 equally respectively, prepares rhodiola root and spina date seed complex polysaccharide.
Embodiment 3
A kind of rhodiola root that the present embodiment provides and spina date seed complex polysaccharide, it prepares by the following method: rhodiola root and spina date seed medicinal material are mixed according to weight ratio 3:2, add water according to solid-liquid ratio 1:10 (g/mL) after pulverizing, at 95 DEG C, add thermal agitation extract 3h.Filter the dregs of a decoction obtained to continue to extract once according to above-mentioned solid-liquid ratio, temperature and time.Merge extracted twice liquid, through reduced pressure concentration and centrifugal acquisition clarification concentrate, add in concentrate according to 1:2 volume ratio by 2 times of volume 95% ethanol, after mixing, at 4 DEG C, overnight stand precipitates, centrifugal collecting precipitation, namely obtains complex polysaccharide through vacuum freeze drying.
The present embodiment also can adopt rhodiola root or spina date seed to be raw material with in embodiment 1 equally respectively, prepares rhodiola root and spina date seed complex polysaccharide.
Embodiment 4
A kind of rhodiola root that the present embodiment provides and spina date seed complex polysaccharide, it prepares by the following method: rhodiola root and spina date seed medicinal material are mixed according to weight ratio 4:1, add water according to solid-liquid ratio 1:5 (g/mL) after pulverizing, at 75 DEG C, add thermal agitation extract 3h.Filter the dregs of a decoction obtained and continue extraction twice according to above-mentioned solid-liquid ratio, temperature and time.Merge No. three extracts, through reduced pressure concentration and centrifugal acquisition clarification concentrate, add in concentrate according to 1:9 volume ratio by 9 times of volume 95% ethanol, after mixing, at 4 DEG C, overnight stand precipitates, centrifugal collecting precipitation, namely obtains complex polysaccharide through vacuum freeze drying.
The present embodiment also can adopt rhodiola root or spina date seed to be raw material with in embodiment 1 equally respectively, prepares rhodiola root and spina date seed complex polysaccharide.
Embodiment 5
A kind of rhodiola root that the present embodiment provides and spina date seed complex polysaccharide, it prepares by the following method: rhodiola root and spina date seed medicinal material are mixed according to weight ratio 1:2, add water according to solid-liquid ratio 1:15 (g/mL) after pulverizing, at 50 DEG C, add thermal agitation extract 1h, extract 3 times, merging filtrate, centrifugal acquisition clarification concentrate after reduced pressure concentration, according to 2:1 volume ratio, 0.5 times of volume 95% ethanol is added in concentrate, after mixing, at 4 DEG C, overnight stand precipitates, centrifugal collecting precipitation, namely obtains complex polysaccharide through vacuum freeze drying.
The present embodiment also can adopt rhodiola root or spina date seed to be raw material with in embodiment 1 equally respectively, prepares rhodiola root and spina date seed complex polysaccharide.
Embodiment 6
Transgenic C nematode CL2355 expresses A β 42 albumen in neuron in vivo, its gathering can produce significant neurotoxicity, thus cause the chemotactic capacity of C. Elegans Automatic Screening to decline, therefore can utilize the mitigation that this scale-model investigation complex polysaccharide is coerced toxic protein.By complex polysaccharide obtained for embodiment 1 and single polysaccharide, add respectively in L1 phase CL2355 larva with 2mg/mL concentration, control group adds isopyknic S Medium.Cultivate 36h at 16 DEG C after, be transferred in 23 DEG C and again cultivate 36h.Get 9cm culture dish, draw bar straight line with 1mol/L glycerine in centre, add ethanolic solution and the 1.5 μ L1mol/L sodium azide solutions of 1.5 μ L0.5% benzaldehydes in left semicircle shape region, be labeled as A district.Add 1.5 μ L ethanol and 1.5 μ L 1mol/L sodium azide solutions in the same position in right semi-circle region, be labeled as B district.At 23 DEG C, hatch 1h after forwarding CL2355 C. Elegans Automatic Screening to culture dish central authorities, then add up the nematode population in A district and B district respectively, chemotactic rate=(number-B district of A district number)/line insect number.Data represent with mean ± SD, adopt T inspection to compare the otherness of polysaccharide group and control group.
Result as shown in Figure 1A, compared with the control, adopts the CL2355 nematode of complex polysaccharide process more at the number in benzaldehyde region.Figure 1B statistical result showed, the chemotactic rate of untreated CL2355 nematode is only 34.8%, and chemotactic rate can significantly improve to 55%, 47.3% and 44.1% when 2mg/mL concentration by complex polysaccharide, Radix Phodiolae Polyose and spina date seed polysaccharide respectively
(p≤0.05)。Compared to single polysaccharide, the activity that complex polysaccharide improves the beta induced behavioral deficiency of toxic protein A is better, shows that it has the effect alleviating Neurodegenerative conditions.
Embodiment 7
C. Elegans Automatic Screening transgenic models HA759 utilizes osmotic pressure related gene osm-10 to impel polyQ40 fragment to express in its head ASH neuron, focusing energy due to polyQ40 produces remarkable toxicity, to be there is Progressive symmetric erythrokeratodermia decline (showing as fluorescence to disappear) in ASH neuron, therefore by observing the mitigation that ASH neuron survival rate research complex polysaccharide is coerced toxic protein.By complex polysaccharide obtained for embodiment 2 and single polysaccharide, add in the HA759 larva of L1 phase respectively with 2mg/mL concentration, control group adds isopyknic S Medium.Cultivate at 15 DEG C after 3 days and collect nematode, add sodium azide solution and nematode is benumbed, drip on agarose pad after mixing, at the neuronic GFP phosphor dot of fluorescence microscopy Microscopic observation every bar C. Elegans Automatic Screening ASH.Each processed group random statistical ~ 50 nematode, nematode count/(nematode count of the nematode count+ASH neuronal death of ASH neuronal survival) × 100% of ASH neuronal survival percentage=ASH neuronal survival.Survival rate represents with mean ± SEM, uses one-way ANOVA analysis and Tukey post-hoc test to compare the otherness of polysaccharide group and control group.
Result as shown in Figure 2 A, without the HA759 C. Elegans Automatic Screening of polysaccharide process ASH neuron because polyQ assemble and become feeble and die (fluorescence disappearance), after adding complex polysaccharide, ASH neuron still survives (fluorescence existence).Fig. 2 B shows, the ASH neuron survival rate after 3 days of untreated HA759 C. Elegans Automatic Screening is 36.4%, after adopting 2mg/mL complex polysaccharide and Radix Phodiolae Polyose process, ASH neuron survival rate significantly improves to 49.2% and 45.1% (p≤0.05) respectively, and spina date seed polysaccharide (41.3%) can not significantly improve ASH neuron survival rate under 2mg/mL concentration.This result shows that complex polysaccharide can suppress the toxicity stress of easy polyprotein polyQ to react, and has the effect alleviating Neurodegenerative conditions.
Avoidance function is oozed because ASH neuron has height, therefore be subject to luring when stopping with hypertonic solution at the same time, the C. Elegans Automatic Screening that ASH neuron does not become feeble and die can retain the behavioural characteristic of avoiding hypertonic solution, and the avoidance ability that the C. Elegans Automatic Screening of ASH neuronal death will be lost hypertonic solution.Therefore HA759 C. Elegans Automatic Screening model is utilized can to detect the mitigation of complex polysaccharide to toxic protein induction behavioral deficiency.By complex polysaccharide obtained for embodiment 2 and single polysaccharide, add in the HA759 larva of L1 phase respectively with 2mg/mL concentration, control group adds isopyknic S Medium.Cultivate at 15 DEG C after 3 days and collect nematode.Prepare chemotactic avoid dull and stereotyped with 8M glycerine (height oozes reagent), biacetyl (luring reagent), sodium azide (anesthetic), C. Elegans Automatic Screening after process is placed on flat board, cultivate 1.5h in 20 DEG C after, takes out the distribution number of statistics C. Elegans Automatic Screening.Rate of avoidance computing formula is as follows: rate of avoidance=(in region of avoidance nematode count/line insect number) × 100%, rate of avoidance represents with mean ± SD, uses one-way ANOVA analysis and Tukey post-hoc test to compare the otherness of polysaccharide group and control group.
Result as shown in Figure 2 C, control group has the C. Elegans Automatic Screening of 42.3% to show avlidance behavior near hypertonic solution glycerine, after adopting 2mg/mL complex polysaccharide and Radix Phodiolae Polyose process, the rate of avoidance of HA759 C. Elegans Automatic Screening significantly improves to 61% and 52.3% (p≤0.05) respectively, and spina date seed polysaccharide (47.2%) can not significantly improve the rate of avoidance of HA759 C. Elegans Automatic Screening under 2mg/mL concentration, show compared to single polysaccharide, complex polysaccharide can improve the behavioral deficiency that toxic protein polyQ induces, consistent with Fig. 2 A and Fig. 2 B result.
The display of above result, what complex polysaccharide can suppress A β and polyQ toxic protein on neuronal cell and Animal Behavior Science level coerces damage, shows that it has the effect of good control nerve degenerative diseases.
Embodiment 8
Due to the key pathogenetic albumen that A β 42 and polyQ40 is in Alzheimer's disease and Huntington's chorea respectively, its gathering can produce significant neurotoxicity, suppresses its gathering can effectively alleviate its neurotoxicity undoubtedly.By complex polysaccharide obtained for embodiment 3, add in 50 μMs of A β 42 and polyQ40 monomer solution respectively with 1mg/mL, 2mg/mL and 4mg/mL concentration, hatch at mixing is placed on 37 DEG C.The same process of control group, but replace polysaccharide solution with isopyknic water.Take out mixed liquor at regular intervals, add thioflavin T solution and Glycine-NaOH solution, utilize fluorescence microplate reader to detect the fluorescence intensity of sample after shaken well, excitation wavelength and emission wavelength are respectively 450nm and 485nm.Relative intensity of fluorescence represents with mean ± SD, adopts T inspection to compare the otherness of polysaccharide group and control group.
As shown in Figure 3 A and Figure 3 B, A β 42 and polyQ40 assembles gradually along with time lengthening, and adding after rhodiola root and spina date seed complex polysaccharide can effective CKIs aggregation velocity, shows that this complex polysaccharide has the effect suppressing toxic protein to be assembled.
Embodiment 9
Heat shock protein (Heat Shock Proteins, HSPs) be that a large class extensively exists the molecular chaperones family with high conservative, folding, assemble, transport, locate and playing a significant role in degradation process mainly at protein, also take part in the adjustment of many A signal pathways in cell simultaneously.Research shows, in protein aggregation related neurodegenerative disease, intracellular protein balance presents imbalance state, the activity of some heat shock proteins also declines to some extent, and can reach minimizing protein abnormal aggregation thus the effect alleviating Neurodegenerative conditions by improving heat shock protein expression.By complex polysaccharide obtained for embodiment 4, coat on NGM culture dish after mixing according to 2mg/mL concentration and OP50, then add L1 phase CL2070 nematode (expression in vivo hsp-16.2::GFP fusion).At 20 DEG C, be cultured to L4 after date, collect nematode and fix by sodium azide paralysis, being placed on 2% agarose pad.At the pharyngeal photo of fluorescence microscope shooting nematode, use the pharyngeal fluorescence intensity of Image J software statistics.Each processed group random statistical ~ 10 nematode, data represent with mean ± SD, adopt T inspection to compare the otherness of polysaccharide group and control group.
As shown in Figure 4 A, compared to control group, after adopting complex polysaccharide process, the pharyngeal fluorescence level of CL2070 nematode significantly improves result.Fig. 4 B statistical result showed, adopts the relative fluorescent intensity after the process of 1mg/mL, 2mg/mL and 4mg/mL complex polysaccharide significantly to rise to 1.2,1.28 and 1.27 (p≤0.05) respectively by 0.98.This result shows, complex polysaccharide can promote the expression of heat-shock protein family important member HSP16.2, coerces contribute to some extent alleviation toxic protein.
Embodiment 10
Toxic protein due to abnormal aggregation can produce a large amount of active oxygen radical (reactive oxygen species, ROS), thus produce oxidative stress damage, therefore the ROS level reduced in disease model body will contribute to reducing oxidative stress injury, thus alleviate Neurodegenerative conditions.By complex polysaccharide obtained for embodiment 5, add respectively in CL2355 and the HA759 larva of L1 phase with 1mg/mL, 2mg/mL and 4mg/mL concentration.Control group is for adding isopyknic S Medium.Cultivate at 15 DEG C after 3 days and collect C. Elegans Automatic Screening, add the PBS containing 1% polysorbas20, DCFH-DA probe solution (the horizontal indicator of ROS) is added after homogenate on ice, at 37 DEG C, lucifuge is hatched, then fluorescent value is detected with fluorescence microplate reader, excitation wavelength is 485nm, and emission wavelength is 530nm.Relative intensity of fluorescence represents with mean ± SEM, adopts T inspection to compare the otherness of complex polysaccharide group and control group.
As shown in Figure 5, the DCF relative fluorescence of CL2355 C. Elegans Automatic Screening is 63.9 to result, and after adopting the process of 1mg/mL, 2mg/mL and 4mg/mL complex polysaccharide, DCF relative fluorescence significantly drops to 50.8,45.2 and 42.4 (p≤0.05) respectively; The DCF relative fluorescence of HA759 C. Elegans Automatic Screening is 49.2,1mg/mL, the process of 2mg/mL and 4mg/mL complex polysaccharide can distinguish significantly reduction DCF relative fluorescence to 41.6,39.1 and 36.4 (p≤0.05).These results show that complex polysaccharide significantly can reduce the ROS level in C. Elegans Automatic Screening body, have potential neuroprotective activity.
Confirm through above-mentioned C. Elegans Automatic Screening model experiment results, prepared complex polysaccharide can the neurotoxicity (embodiment 6 and embodiment 7) of effective more than enough polyglutamine and beta-amyloid aggregation induction, its active function mode relate to suppress toxic protein to assemble (embodiment 8), the active oxygen radical level (embodiment 10) improved in heat shock protein expression (embodiment 9) and reduction disease model body.The complex polysaccharide that these result sufficient proof are prepared by rhodiola root and spina date seed has the effect of good control nerve degenerative diseases.
Embodiment 11
By complex polysaccharide obtained for embodiment 1, add appropriate lactalbumin as required, maltodextrin preparation becomes the pulverulence healthy food or health food with neuroprotective efficacy, other customary adjuvant can also be added and make regular dosage form.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (9)
1. rhodiola root and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation.
2. rhodiola root according to claim 1 and the spina date seed complex polysaccharide application in the anti-nerve degenerative diseases health food of preparation, is characterized in that: in described rhodiola root and spina date seed complex polysaccharide, the quality proportioning of Radix Phodiolae Polyose and spina date seed polysaccharide is 1:4 ~ 4:1.
3. rhodiola root according to claim 2 and the spina date seed complex polysaccharide application in the anti-nerve degenerative diseases health food of preparation, is characterized in that: in described rhodiola root and spina date seed complex polysaccharide, the quality proportioning of Radix Phodiolae Polyose and spina date seed polysaccharide is 2:3 ~ 3:2.
4. the rhodiola root according to any one of claim 1-3 and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation, it is characterized in that: described rhodiola root and spina date seed complex polysaccharide prepare by the following method: with rhodiola root or spina date seed for raw material, through containing water extraction, concentrated and alcohol precipitation operation prepares Radix Phodiolae Polyose or spina date seed polysaccharide, again the two is mixed by metering ratio, prepare rhodiola root and spina date seed complex polysaccharide, or described rhodiola root and spina date seed complex polysaccharide prepare by the following method: with rhodiola root and spina date seed for raw material, through containing water extraction, concentrated and alcohol precipitation operation prepares rhodiola root and spina date seed complex polysaccharide, described complex polysaccharide has neuroprotection, can be used for preparing anti-nerve degenerative diseases health food.
5. rhodiola root according to claim 4 and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation, it is characterized in that: during with rhodiola root or spina date seed for raw material, the quality proportioning of described Radix Phodiolae Polyose and spina date seed polysaccharide is 1:4 ~ 4:1, during with rhodiola root and spina date seed for raw material, the quality proportioning of described rhodiola root and spina date seed is 1:4 ~ 4:1.
6. rhodiola root according to claim 5 and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation, it is characterized in that: during with rhodiola root or spina date seed for raw material, the quality proportioning of described Radix Phodiolae Polyose and spina date seed polysaccharide is 2:3 ~ 3:2, during with rhodiola root and spina date seed for raw material, the quality proportioning of described rhodiola root and spina date seed is 2:3 ~ 3:2.
7. rhodiola root according to claim 4 and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation, it is characterized in that: during water extraction, the mass volume ratio of rhodiola root and/or spina date seed medicinal material and water is 1:2 ~ 1:20, Extracting temperature is 50 ~ 100 DEG C, and extraction time is 1 ~ 10h; Time concentrated, thickening temperature is 40 ~ 100 DEG C; During alcohol precipitation, the volume ratio of ethanol and concentrate is 2:1 ~ 9:1.
8. rhodiola root according to claim 1 and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation, is characterized in that: described nerve degenerative diseases refers to and comprises the nervous system disease that Alzheimer's disease and Huntington's chorea etc. are major pathologic features with toxic protein abnormal aggregation.
9. rhodiola root according to claim 1 and the application of spina date seed complex polysaccharide in the anti-nerve degenerative diseases health food of preparation, is characterized in that: the formulation of described health food is capsule, pulvis, granule, tablet or oral liquid.
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