CN109369815B - Wolfberry fruit arabinogalactan and preparation method and application thereof - Google Patents
Wolfberry fruit arabinogalactan and preparation method and application thereof Download PDFInfo
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- CN109369815B CN109369815B CN201710671638.1A CN201710671638A CN109369815B CN 109369815 B CN109369815 B CN 109369815B CN 201710671638 A CN201710671638 A CN 201710671638A CN 109369815 B CN109369815 B CN 109369815B
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- Prior art keywords
- lbp1a1
- polysaccharide
- lycium barbarum
- medlar
- beta
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- 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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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a polysaccharide LBP1A1-1 extracted from medlar, an application thereof in preparing a medicament or a health product for preventing and/or treating neurodegenerative diseases, and an application thereof in preparing a medicine or a health product for inhibiting Abeta42The use of the prepared medicine or health care product and the use in preparing the medicine or health care product for preventing and treating the Alzheimer disease. Specifically, the application relates to arabinogalactan extracted from wolfberry, and the preparation method comprises the following steps: firstly, extracting crude polysaccharide in the medlar by adopting cellulase, amylase and papain combined with water at 55 ℃, and then precipitating with alcohol. Combining with various column chromatography purification methods and spectral analysis to obtain the arabinogalactan. In vitro experiments prove that the polysaccharide can inhibit A beta in CHO/APPACE 1 cells stably transfected with APP and BACE1 in a dose-dependent manner42And (4) generating. Therefore, the polysaccharide has potential effect of treating Alzheimer's disease, and is expected to be developed into a carbohydrate medicament for treating Alzheimer's disease.
Description
Technical Field
The invention relates to polysaccharide substances, an extraction method thereof and application thereof in preparing medicaments, in particular to a method for extracting arabinogalactan polysaccharide LBP1A1-1 from red barbary wolfberry fruit (fruits of Lycium barbarum L.), the arabinogalactan polysaccharide extracted by the method and application of the arabinogalactan polysaccharide in preparing medicaments or health products for preventing and/or treating neurodegenerative diseases, and the application of the arabinogalactan polysaccharide in preparing medicaments or health products for inhibiting A beta42The use of the prepared medicine or health care product and the use in preparing the medicine or health care product for treating the Alzheimer disease.
Background
Alzheimer's Disease (AD), also known as Alzheimer's disease, is a progressive neurodegenerative disease, and the main clinical manifestations of AD are gradually hypomnesis, cognitive dysfunction, behavioral disorders, social disorders, etc. With the increasing aging of the population, the incidence of AD has also increased year by year, and has become one of the most important health concerns.
Deposition and abnormal expression of amyloid beta-protein (a β) in the brain are currently considered to be the central links in the initiation of AD. One of the characteristic pathological changes in AD is the formation of SPs, and A β is the core component of SPs, including A β40And Abeta42Wherein A β42Amyloidosis is more likely to occur. Thus, aggregation and abnormal deposition of a β are the primary and central links in the pathogenesis of AD. A beta is a normal metabolite of the body, and is hydrolyzed from beta-Amyloid Precursor Protein (APP), and its production and degradation are in dynamic equilibrium under normal conditions, and when some reasons cause APP metabolism to be abnormal, the A beta is increased and/or the degradation is reduced, which causes A beta to be deposited in a large amount. Therefore, the method takes A beta as an action target and reduces the generation of the A beta from the source by interfering the APP metabolism, and is a research hotspot of the current AD treatment drugs.
The understanding of the former on the medlar is summarized in Bencao gang mu by the Ming dynasty pharmacologist Li Shizhen, and the leaf of medlar, named Tianjing grass, is collected in spring; summer flower, named Changsheng grass; qicaizi, named wolfberry fruit; collected root in winter, named cortex lycii radicis. The Chinese wolfberry is called as an first-class medicinal material with the effects of nourishing liver and kidney, supplementing energy, improving eyesight and the like because the medicinal parts of the Chinese wolfberry are different and the nature and taste functions of the Chinese wolfberry are obviously different. And is listed as the top grade as early as in Shen nong Ben Cao Jing, which is called as 'long-time taking for light weight, not old, cold-resistant and summer-heat-resistant'; has antiaging and antiaging effects. The Chinese wolfberry is a general name of the species under the lycium genus such as commercial Chinese wolfberry, plant Ningxia wolfberry, Chinese wolfberry and the like. The wolfberry fruits which are edible and medicinal in daily life are mostly the fruits of Ningxia wolfberry fruit, and Ningxia wolfberry fruit is the only one loaded in Chinese pharmacopoeia 2010 edition and is called as the traditional Chinese medicine four-treasure together with Qiongzhen glossy ganoderma, Changbai mountain ginseng and Dong' donkey-hide gelatin. Mainly distributed in the west and north areas (such as Xinjiang, Tibet, Qinghai, Gansu, inner Mongolia, Ningxia, Shaanxi, Shanxi, Hebei, etc.) of China. Modern medical and pharmacological studies have found that polysaccharides in wolfberry are an important active substance. Has the functions of reducing blood fat, reducing blood sugar, protecting liver, resisting tumor, regulating immunity and the like, but the function of the lycium barbarum polysaccharide in treating the Alzheimer disease is not reported. Therefore, the lycium barbarum polysaccharide LBP1A1-1 has a huge application prospect in the aspect of candidate drugs for treating the Alzheimer disease.
Disclosure of Invention
The invention adopts a simple and effective polysaccharide extraction process and method, and obtains a polysaccharide which is an arabinogalactan by taking medlar as a raw material. Pharmacological experiments show that the polysaccharide can inhibit A beta in CHO/APPACE 1 cells stably transfected with APP and BACE1 (beta-site APP-cleavage enzyme 1, beta-site APP cleavage enzyme 1) in a dose-dependent manner42And (4) generating. Therefore, the polysaccharide has potential effect of treating Alzheimer's disease, and is expected to be developed into a carbohydrate medicament for treating Alzheimer's disease.
One aspect of the present invention provides a lycium barbarum polysaccharide LBP1a1-1, having the structure:
wherein the weight average molecular weight of the lycium barbarum polysaccharide LBP1A1-1 is in the range of 10-450kDa, preferably 30-100kDa, more preferably 38-50 kDa.
Wherein a is an integer of 0 to 15; n is an integer from 2 to 75.
The monosaccharide composition of the lycium barbarum polysaccharide LBP1A1-1 comprises galactose, arabinose, glucose and rhamnose, and the mass ratio of the galactose, the arabinose, the glucose and the rhamnose is 51.72:45.39:2.04: 0.81.
The main stretching vibration absorption peak of the infrared characteristic spectrum of the lycium barbarum polysaccharide LBP1A1-1 is basically consistent with that in the infrared characteristic spectrum shown in figure 1, preferably 34 in the infrared characteristic spectrum of the lycium barbarum polysaccharide LBP1A1-125.94cm-1Is the O-H stretching vibration absorption peak, 2924.50cm-1Is C-H stretching vibration absorption peak, 1451.30-1029.39cm-1Nearby is C-O and sugar ring vibration signal, 1720cm-1There was no absorption peak nearby, indicating that the polysaccharide contained no uronic acid.
The lycium barbarum polysaccharide LBP1A1-1, which13The main signal values of the C NMR spectrum are shown in FIG. 213The C NMR spectrum is substantially identical, preferably, in said Lycium barbarum polysaccharide LBP1A1-113In the C NMR spectrum, the terminal carbon signals at delta 110-delta 108 are the C1 signals for 1, 5-arabinose and alpha-terminal arabinose, respectively; the terminal carbon signals at δ 106- δ 104 are the C1 signals for 1,3, 6-galactose, 1, 3-galactose, 1, 6-galactose and terminal galactose, respectively; the terminal carbon signals at delta 103-delta 102, the C1 signals for beta-terminal arabinose and terminal rhamnose, respectively; the signal peak for the rhamnose methyl carbon is at δ 17.56.
The invention also aims to provide a preparation method of the lycium barbarum polysaccharide LBP1A1-1, which comprises the following steps:
a. polysaccharide extraction: crushing dried medlar fruits, carrying out enzyme-water combined extraction, inactivating enzymes, centrifuging, concentrating the obtained filtrate, dialyzing, concentrating, centrifuging, precipitating with ethanol, centrifuging, washing, and drying in vacuum to obtain enzyme-water combined extraction medlar crude polysaccharide;
preferably, the step a includes: crushing dried medlar fruits by a crusher, adding 15-30 times of deionized water, extracting at 50-60 ℃ for 1h by respectively adding 3% of cellulase, 1% of amylase and 0.5% of papain, then heating to 100 ℃ to inactivate the enzymes, centrifuging, concentrating the filtrate, dialyzing, concentrating again, adding 95% (mass fraction) ethanol which is five times of the volume of the concentrated solution, centrifuging to obtain precipitates, alternately washing the precipitates with absolute ethanol and acetone for three times respectively, and drying in vacuum to obtain enzyme-water co-extracted medlar crude polysaccharide;
b. polysaccharide purification: extracting fructus Lycii crude polysaccharide with enzyme-water, dissolving in water, centrifuging, and subjecting the supernatant to DEAE SepharoseTMPerforming primary fractional purification on Fast Flow anion exchange column, eluting with water, 0.05M, 0.1M and 0.2M sodium chloride, and collectingEluting the fraction with 0.05M sodium chloride, and purifying the fraction with Sephacryl HR S-200 gel chromatography column to obtain Lycium barbarum polysaccharides LBP1A 1-1;
preferably, the step b includes: extracting fructus Lycii crude polysaccharide with enzyme-water, dissolving in 10-15 times of water, centrifuging, and passing the supernatant through DEAE SepharoseTMSeparating with Fast Flow anion exchange column, eluting with deionized water, 0.05M, 0.1M and 0.2M sodium chloride, detecting with sulfuric acid-phenol, collecting and mixing 0.05M sodium chloride eluate, concentrating, dialyzing, and freeze drying to obtain primarily purified polysaccharide LBP1A 1; then LBP1A1 is dissolved in 0.01 times weight of 0.2M sodium chloride (equilibrium liquid), centrifugation is carried out, the supernatant is separated by a Sephacryl HR S-200 gel chromatographic column, elution is carried out by 0.2M sodium chloride, sulfuric acid-phenol detection is carried out, an elution curve is drawn under 490nm wavelength, 3-4 absorption peaks are obtained from the elution curve, the first absorption peak (namely, eluent with elution speed of 3-6ml/15min is collected from 20 to 45 tubes) is collected and combined, HPGPC detection is carried out, the absorption peak with weight average molecular weight of 10-450kDa and single symmetry is the component LBP1A1-1, concentration, dialysis and freeze drying are carried out, and the lycium barbarum polysaccharide LBP1A1-1 is obtained.
And (3) polysaccharide structure identification: the lycium barbarum polysaccharide LBP1A1-1 is subjected to comprehensive analysis on monosaccharide composition, methylation, infrared, partial acid hydrolysis, nuclear magnetism and the like to determine the structure of the lycium barbarum polysaccharide.
Another object of the present invention is to provide the use of the lycium barbarum polysaccharide LBP1a1-1 in the preparation of a medicament or health product for preventing and/or treating neurodegenerative diseases, which may be diseases caused by abnormal expression and deposition of amyloid beta in brain.
Preferably, the neurodegenerative disease is Alzheimer's disease, namely the invention provides application of the lycium barbarum polysaccharide LBP1A1-1 in preparing a medicament or health-care product for preventing and/or treating Alzheimer's disease.
In another aspect of the invention, the invention also provides the application of the lycium barbarum polysaccharide LBP1A1-1 in preparation of medicines for inhibiting A beta42The produced medicine or health product.
It is yet another aspect of the present invention to provide a method for inhibiting A β42And a method for preventing and/or treating alzheimer's disease, which comprises administering a therapeutically effective amount of the above-mentioned polysaccharide, drug or health product to a patient in need thereof.
Drawings
FIG. 1 is a characteristic infrared spectrum of Lycium barbarum polysaccharide LBP1A 1-1;
FIG. 2 shows the characteristics of lycium barbarum polysaccharide LBP1A1-113A C NMR spectrum;
FIG. 3 is a characteristic HMBC profile of Lycium barbarum polysaccharide LBP1A 05I;
FIG. 4 is a characteristic HMBC profile of Lycium barbarum polysaccharide LBP1A 1-1;
FIG. 5 shows that LBP1A1-1 of Lycium barbarum polysaccharides inhibits A.beta.in CHO/APPACE 1 cells42Histograms of secretion volume;
FIG. 6 is a line graph of the effect of Lycium barbarum polysaccharides LBP1A1-1 on CHO/APPACE 1 cell viability;
FIG. 7 is a bar graph of the reduction of ThT (Thioflavin T) fluorescence intensity by LBP1A1-1 at various concentrations.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are given by way of illustration only. It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention within the scope and spirit of the invention.
High performance gel permeation chromatography using an ultrahydrogel (TM) 2000(25 cm. times.0.75 cm, Waters Corp.); and Ultrahydrogel 500(25 cm. times.0.75 cm, Waters, USA) in series column to prepare standard curve with T-series standard Dextran (Dextran) of different molecular weight;
high performance liquid gel permeation chromatography (HPGPC) was performed using an Agilent 1260Seri high performance liquid chromatography system (Agilent Corp., USA); infrared analysis was performed using a Perkin-Elmer model 599B infrared spectrophotometer (Perkin-Elmer, USA);
NMR analysis was carried out by means of a Brucker AM-500 NMR spectrometer (Brucker, Germany).
Example 1: preparation of lycium barbarum polysaccharide LBP1A1-1
a. Polysaccharide extraction:
pulverizing 1000g of dried fructus Lycii, adding 20L of deionized water, extracting at 55 deg.C with 30g of cellulase, 10g of amylase and 5g of papain respectively for 1h, heating to 100 deg.C to inactivate enzyme, centrifuging, concentrating the filtrate, and dialyzing against flowing water for 3 days. Heating and concentrating the dialyzed internal solution to 3L, centrifuging to remove precipitate, adding 95% ethanol with the volume of five times 15L into the supernatant under stirring, standing overnight, centrifuging, washing the obtained precipitate with anhydrous ethanol and acetone alternately for 3 times, centrifuging, and vacuum drying the precipitate at 50 ℃ to obtain 25.8g of enzyme-water co-extracted lycium barbarum crude polysaccharide.
b. Polysaccharide purification:
dissolving the prepared crude polysaccharide 6g in 60mL deionized water, centrifuging at 4000r/min for 10min to remove insoluble substances, and passing the supernatant through DEAE SepharoseTMSeparating with Fast Flow anion exchange column, eluting with deionized water, 0.05M, 0.1M and 0.2M sodium chloride, detecting with sulfuric acid-phenol, drawing elution curve, collecting and combining 0.05M sodium chloride eluate according to the elution curve, concentrating, dialyzing, and freeze-drying to obtain primarily purified polysaccharide LBP1A 1. 200mg of LBP1A1 was dissolved in 2mL of 0.2M sodium chloride, centrifuged at 4000r/min for 10min, and the supernatant was chromatographed on a Sephacryl HR S-200 gel column eluting with 0.2M NaCl at a controlled flow rate of 3-6mL/15 min. Detecting with sulfuric acid-phenol method, drawing elution curve at 490nm wavelength, obtaining 3-4 absorption peaks from the elution curve, collecting and combining the first absorption peak (collecting eluate from 20-45 tubes with elution speed of 3-6ml/15 min), detecting by HPGPC, obtaining component LBP1A1-1 as single symmetrical absorption peak with weight average molecular weight of 10-450kDa, concentrating, dialyzing, and freeze drying to obtain Lycium barbarum polysaccharide LBP1A1-1 about 40 mg.
c. Polysaccharide structure identification and analysis:
firstly, an efficient gel permeation chromatography system, Ultrahydrogel (TM) 2000(25 cm. times.0.75 cm, Waters Corp., USA) is adopted; and Ultrahydrogel 500(25 cm. times.0.75 cm, Waters, USA) in series column to prepare standard curve with T-series standard Dextran (Dextran) of different molecular weight; the column temperature was set at 40 ℃ and 0.1M NaNO was added3As mobile phase, the flow rate is 5ml/min, and the sample concentration of lycium barbarum polysaccharide LBP1A1-1 is2mg/ml, and the molecular weight of the lycium barbarum polysaccharide LBP1A1-1 is determined.
The relative molecular mass of the lycium barbarum polysaccharide LBP1A1-1 is determined to be 45 kDa. The monosaccharide composition analysis is carried out, namely polysaccharide is completely hydrolyzed, reduced, acetylated, extracted and concentrated, and then the polysaccharide is sent to GC (gas chromatography) for analysis. The analysis result of monosaccharide composition shows that the lycium barbarum polysaccharide LBP1A1-1 mainly contains arabinose, galactose and trace amounts of rhamnose and glucose. LBP1A1-1 was determined to be an arabinogalactan by a combination of infrared, partial acid hydrolysis, methylation and NMR analysis (see FIG. 1, FIG. 2, FIG. 3, FIG. 4).
② analysis of monosaccharide composition shows that the monosaccharide composition of LBP1A1-1 is: the mass ratio of the galactose, the arabinose, the glucose and the rhamnose is 51.72:45.39:2.04: 0.81.
③ infrared spectrum (figure 1), 3425.94cm-1Is the O-H stretching vibration absorption peak, 2924.50cm-1Is C-H stretching vibration absorption peak, 1451.30-1029.39cm-1Nearby is C-O and sugar ring vibration signal, 1720cm-1There was no absorption peak nearby, indicating that the polysaccharide contained no uronic acid.
Partial acid hydrolysis of polysaccharides
LBP1A1-1150mg was dissolved in 15mL of 0.1M trifluoroacetic acid, sealed and hydrolyzed at 100 ℃ for 1 h. After the reaction is finished, repeatedly decompressing and evaporating methanol to dryness, dialyzing with deionized water for 4 times, wherein each time is 1L (the molecular weight cut-off of a dialysis bag is 3500Da), and concentrating and freeze-drying the dialyzed internal and external liquid to obtain partial acid hydrolysis products LBP1A1O1I (dialyzed internal liquid) and LBP1A101O (dialyzed external liquid).
LBP1A101I 60mg was dissolved in 6mL of 0.5M trifluoroacetic acid and sealed before hydrolysis at 100 ℃ for 1 h. After the reaction is finished, repeatedly decompressing and evaporating methanol to dryness, dialyzing with deionized water for 4 times, wherein each time is 1L (the molecular weight cut-off of a dialysis bag is 3500Da), and concentrating and freeze-drying the dialyzed internal and external liquid to obtain partial acid hydrolysis products LBP1A1O5I (dialyzed internal liquid) and LBP1A105O (dialyzed external liquid).
NMR analysis
Adding polysaccharide LBP1A05I (30mg) and LBP1A1(50mg) respectively with D2O0.4 mL, 2.5. mu.L acetone as internal standard (. delta.)H＝2.29ppm，δC31.5ppm), one-and two-dimensional nmr spectra were measured on a BrukerAVANCE III 500M nmr at 25 ℃ and structure confirmation of LBP1a1-1 was performed with reference to the nmr spectra, the results are shown in fig. 3 and 4. The structure of LBP1a1-1 was confirmed by the HMBC spectrum (heteronuclear polycarbon correlation spectrum) of fig. 3 and the HMBC spectrum (heteronuclear polycarbon correlation spectrum) of fig. 4, and the results are shown in fig. 3 and 4.
13In the C NMR spectrum (FIG. 2), the terminal carbon signals at δ 110- δ 108 are the C1 signals for 1, 5-arabinose and α -terminal arabinose, respectively; the terminal carbon signals at δ 106- δ 104 are the C1 signals for 1,3, 6-galactose, 1, 3-galactose, 1, 6-galactose and terminal galactose, respectively; the terminal carbon signals at delta 103-delta 102 are the C1 signals for beta-terminal arabinose and terminal rhamnose, respectively. From the above results, it was found that LBP1A1-1 is an arabinogalactan.
In the HMBC map of LBP1a105I (fig. 3), the order of linkage between glycosidic linkages can be determined. Correlation peak A (Δ 100.55/Δ 4.02) represents the C-1 and H-6 correlation of 1,3,6- β -galactose for 1,4- α -glucose, and correlation peak B (Δ 70.75/Δ 5.08) represents the C-6 and H-1 correlation of 1,3,6- β -galactose for 1,4- α -glucose; the correlation peak C (δ 76.76/δ 4.51) represents the C-4 and H-1 correlation of 1,4- α -galactose of 1,4- α -glucose, and the correlation peak D (δ 104.65/δ 3.84) represents the C-1 and H-4 correlation of 1,4- α -glucose of 1,6- β -galactose; the correlation peak E (delta 83.20/delta 4.69) represents the C-3 correlation of 1, 3-beta-galactose and the H-1 correlation of 1,3, 6-beta-galactose, and the correlation peak F (delta 105.31/delta 3.93) represents the C-1 correlation of 1,3, 6-beta-galactose and the H-3 correlation of 1, 3-beta-galactose; the correlation peak G (delta 70.75/delta 4.69) represents the C-6 and H-1 correlation of 1,3, 6-beta-galactose, and the correlation peak H (delta 105.31/delta 4.10) represents the C-1 and H-6 correlation of 1,3, 6-beta-galactose; the correlation peak I (delta 83.20/delta 4.75) represents the C-3 correlation of 1,3, 6-beta-galactose and the H-1 correlation of 1, 3-beta-galactose, and the correlation peak J (delta 104.96/delta 3.93) represents the C-1 correlation of 1, 3-beta-galactose and the H-3 correlation of 1,3, 6-beta-galactose; the correlation peak K (. delta.70.75/. delta.4.59) represents the C-6 and H-1 correlation of the beta-terminal galactose for 1,3, 6-beta-galactose, and the correlation peak L (. delta. 103.89/. delta.4.02) represents the C-1 and H-6 correlation of the 1,3, 6-beta-galactose for beta-terminal galactose.
In the HMBC map of LBP1A1-1 (FIG. 4), the order of linkage between glycosidic linkages can be determined. The correlation peak M (delta 83.23/delta 5.32) represents the C-3 of 1,3, 6-beta-galactose and the H-1 correlation of 1, 5-alpha-arabinose, and the correlation peak N (delta 110.50/delta 3.84) represents the C-1 of 1, 5-alpha-arabinose and the H-3 correlation of 1,3, 6-beta-galactose; the correlation peak O (delta 108.67/delta 3.86) represents the C-1 of the alpha-terminal arabinose and the H-5 correlation of the 1, 5-alpha-arabinose, and the correlation peak P (delta 67.96/delta 5.15) represents the C-5 of the 1, 5-alpha-arabinose and the H-1 correlation of the alpha/beta-terminal arabinose; the correlation peak R (delta 102.03/delta 4.02) represents the C-1 and H-1 correlation of 1,3, 6-alpha-galactose for the beta-terminal rhamnose, and the correlation peak Q (delta 70.55/delta 4.82) represents the C-6 and H-1 correlation of beta-terminal rhamnose for the 1,3, 6-alpha-galactose. As described above, the polysaccharide has galactose residues and glucose residues as main chains, and other sugar residues as branched chains are directly or indirectly connected to the C-3 or C-6 position of the main chain sugar residues.
The above results indicate that the structure of LBP1A1-I is:
wherein a is an integer of 0 to 15; n is an integer from 2 to 75.
Example 2 arabinogalactan LBP1A1-1 inhibits A.beta.42Generation of
CHO/APPACE 1 and HEK293-APPswA beta in cells42ELISA detection of
CHO/APPACE 1 cells (from Shanghai pharmaceutical research institute of Chinese academy of sciences) were cultured in Ham's F12 medium (from Hyclone, USA) containing 10% fetal bovine serum (from Gibco, USA), 100U/ml penicillin and 100. mu.g/ml streptomycin. HEK293-APPsw(given to the subject group of Octopus octopus) cells were cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 100U/ml penicillin, 100. mu.g/ml streptomycin and 200. mu.g/ml, and when the cells grew to 80% -90% confluency, the cells were cultured at 5X 105Density of one/well in 24-well plates at 5% CO2After 24 hours of incubation in an incubator at 37 ℃, wolfberry in different concentrations (0. mu.g/ml, 62.5. mu.g/ml, 125. mu.g/ml, 250. mu.g/ml, 500. mu.g/ml and 1000. mu.g/ml) was addedLycium polysaccharide LBP1A1-1 (prepared in example 1), and after 24h, cell supernatant was collected.
Using HumanA beta42ELISA kit (purchased from Invitrogen, USA) for detecting A beta in supernatant42The specific method comprises the following steps:
1) the standard (A beta) diluted by standard diluent (provided in the kit)42Preparing a standard mother solution by using 55mM sodium bicarbonate (pH 9.0), subpackaging and storing at-80 ℃ or adding a sample to be detected into an ELISA (enzyme-linked immunosorbent assay) pore plate (the ELISA pore plate is coated with a capture antibody, and the antibody is carried by a kit), wherein each pore is 50 mu l;
2) adding 50 mul of detection antibody (carried by the kit) into each hole, and incubating for 3h on a shaking table at room temperature;
3) washing for 5 times (washing solution, diluted by 1:25 (diluted with ultrapure water)) from washing solution concentrate carried in the kit), adding HRP-streptavidin (diluted 100 times with antibody diluent, which is carried in the kit), incubating for 30min at room temperature with 100 μ l of each well;
4) washing for 5 times, adding 3,3 ', 5, 5' -tetramethylbenzidine dihydrochloride chromogenic substrate (TMB), incubating for 30min at room temperature in dark place with 100 μ l of each well, and adding stop solution (provided by the kit);
5) the A.beta.in the cells was calculated by taking OD readings per well at a wavelength of 450nm using a microplate reader (purchased from BMG Labtech, Germany)42Concentration of (g/ml). The results are shown in FIG. 5, and Lycium barbarum polysaccharides LBP1A1-1 can inhibit CHO/APPACE 1 cell (A) and HEK293-APP concentration-dependentlyswA.beta.in cells (B)42Wherein p < 0.05, and p < 0.01 indicates the degree of significant difference compared to the control group (0 μ g/ml).
MTT experiment for detecting influence of lycium barbarum polysaccharide LBP1A1-1 on CHO/APPACE 1 cell growth
CHO/APPACE 1 cells (5X 10) in logarithmic growth phase3One/hole) are planted into a 96-hole plate, three-hole multiple holes are arranged, and the culture is carried out in an incubator for 24 hours; the cell supernatant was aspirated off, LBP1A1-1 solutions were added to final concentrations of 7.8125. mu.g/ml, 15.625. mu.g/ml, 31.25. mu.g/ml, 62.5. mu.g/ml, 125. mu.g/ml, 250. mu.g/ml, 500. mu.g/ml and 1000. mu.g/ml, and after further incubation for 24h, 48h and 72h, 5 mg/well was addedmu.l of MTT solution (purchased from Sigma, USA, prepared by PBS, and filtered by 0.22 mu m microporous membrane), continuously culturing for 4h, sucking out cell culture solution in each well, adding 100 mu.l DMSO (dimethyl sulfoxide) into each well to dissolve formed purple crystal, namely methyl , and collecting absorbance at 490nm by using a microplate reader. Cell viability was calculated according to the following formula: the cell survival rate was (experimental OD value-blank OD value)/(control OD value-blank OD value) × 100%. The results are shown in FIG. 6, and the survival rate of the cells after the treatment of the cells for 24h, 48h and 72h with different concentrations of LBP1A1-1 is over 85%, which indicates that LBP1A1-1 is basically non-toxic.
3. Detection of Abeta by Thioflavin T binding experiment42Is gathered
1) Fresh A beta taken out from a refrigerator at-80 DEG C42The powder (amyloid 42) (available from rpeptide, USA) was dissolved in 110. mu.l of anhydrous DMSO (available from Fluka, USA) to prepare 2mM A.beta.42Storing the liquid;
2) take 1. mu. lA. beta42The stock solution was dissolved in 19. mu.l of fibril-formation buffer (50mM sodium phosphate, pH 7.5; 100mM NaCl, ultrapure water, used after filtration through a 0.22 μm filter) or 1. mu. lA. beta42Dissolving the stock solution in 9 μ l of fibril-formatting buffer, adding 10 μ l of LBP1A1-1 solution (0.25mg/ml,0.5mg/ml,1mg/ml, and polysaccharide solution prepared by fibril-formatting buffer) with different concentrations, mixing, placing in 37 deg.C incubator, and incubating for 30 min;
3) to each group was added 80 μ l of 6.25 μ M ThT solution (with 50mM glycine solution, pH 8.5), mixed well and added to a black fluorescence detection 96-well plate (purchased from Greiner, usa) and incubated at 37 ℃ in an incubator;
4) taking out the pore plate at certain intervals, and detecting the reading of each pore by using a microplate reader, wherein the detection wavelength is 430nm for Ex, and 490nm for Em.
As shown in FIG. 7, different concentrations of LBP1A1-1 reduced the ThT fluorescence intensity to different degrees with the passage of the detection time, and the inhibition was more obvious with higher concentration, that is, LBP1A1-1 inhibited A β concentration-dependently42To (3) is performed.
To sum up, lead toAccording to the examples, arabinogalactan LBP1A1-1 polysaccharide extracted from Lycium barbarum can inhibit A beta in CHO/APPACE 1 cells in a concentration-dependent manner42And LBP1A1-1 can inhibit A beta concentration-dependently42To (3) is performed. LBP1A1-1 is a potential polysaccharide drug for treating and/or preventing Alzheimer's disease.
Finally, it should be noted that the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be construed as limiting the scope of the present invention, and any non-essential modifications and improvements made in the above-mentioned contents of the present invention belong to the scope of the present invention.
Claims (7)
1. A lycium barbarum polysaccharide LBP1A1-1, characterized by having the following structure:
wherein a is an integer of 0 to 15; n is an integer of 2 to 75,
the lycium barbarum polysaccharide LBP1A1-1 comprises monosaccharides such as galactose, arabinose, glucose and rhamnose, and the mass ratio of the galactose, the arabinose, the glucose and the rhamnose is 51.72:45.39:2.04: 0.81.
2. The lycium barbarum polysaccharide LBP1a1-1 of claim 1, wherein: the main stretching vibration absorption peak of the infrared characteristic spectrum is basically consistent with that in the infrared characteristic spectrum shown in the attached figure 1.
3. The lycium barbarum polysaccharide LBP1a1-1 of claim 1, wherein: it is composed of13The main signal values of the C NMR spectrum are shown in FIG. 213The C NMR spectrum was substantially uniform.
4. A process for the preparation of the lycium barbarum polysaccharide LBP1a1-1 according to any one of claims 1-3, characterized in that it comprises the following steps:
a. crushing dried medlar fruits by a crusher, adding deionized water 15-30 times the weight of the crushed medlar fruits, extracting for 1 hour at 50-60 ℃, then respectively adding cellulase 3% of the weight of medlar, amylase 1% of the weight of medlar and papain 0.5% of the weight of medlar, raising the temperature to 100 ℃ to inactivate the enzymes, centrifuging, concentrating the filtrate, dialyzing, concentrating again, adding 95% ethanol five times the volume of the concentrated solution, centrifuging to obtain precipitates, alternately washing the precipitates with absolute ethanol and acetone for three times respectively, and drying in vacuum to obtain enzyme-water co-extracted medlar crude polysaccharide;
b. extracting fructus Lycii crude polysaccharide with enzyme-water, dissolving in 10-15 times of water, centrifuging, and passing the supernatant through DEAE SepharoseTMSeparating with Fast Flow anion exchange column, eluting with deionized water, 0.05M, 0.1M and 0.2M sodium chloride, detecting with sulfuric acid-phenol, collecting and mixing 0.05M sodium chloride eluate, concentrating, dialyzing, and freeze drying to obtain primarily purified polysaccharide LBP1A 1; then LBP1A1 is dissolved in 0.01 times weight of 0.2M sodium chloride, centrifugation is carried out, the supernatant is separated by a Sephacryl HR S-200 gel chromatographic column, elution is carried out by 0.2M sodium chloride, sulfuric acid-phenol detection is carried out, an elution curve is drawn under 490nm wavelength, 3-4 absorption peaks are obtained from the elution curve, the first absorption peak is collected and merged, HPGPC detection is carried out, the absorption peak with the weight average molecular weight of 10-450kDa and single symmetry is the component LBP1A1-1, concentration, dialysis and freeze drying are carried out, and the lycium barbarum polysaccharide LBP1A1-1 is obtained.
5. Use of the lycium barbarum polysaccharide LBP1a1-1 according to any one of claims 1-3 for the preparation of a medicament for the prevention and/or treatment of a neurodegenerative disease.
6. Use according to claim 5, characterized in that: the neurodegenerative disease is a disease caused by abnormal expression and deposition of beta amyloid in the brain.
7. Use of the lycium barbarum polysaccharide LBP1a1-1 according to any one of claims 1-3 for the preparation of a medicament for inhibiting Α β42The use of the resulting medicament.
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| CN111671089A (en) * | 2020-06-24 | 2020-09-18 | 天津市泉又今生物科技有限公司 | Application of biological enhanced cell nutrient in preparation of anti-beta-amyloid protein cytotoxicity functional food |
| CN112851829B (en) * | 2021-01-15 | 2021-09-24 | 中国科学院兰州化学物理研究所 | A fructus Lycii polysaccharide with blood lipid reducing effect |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012612A (en) * | 2012-12-14 | 2013-04-03 | 天津大学 | Extraction method of wolfberry polysaccharide |
| CN104072627A (en) * | 2014-06-04 | 2014-10-01 | 沈阳化工大学 | Preparation method for typhonium giganteum polysaccharide extract |
| CN104987433A (en) * | 2015-07-23 | 2015-10-21 | 中国科学院西北高原生物研究所 | Preparation method of RG-I type lycium barbarum pectin with anti-aging activity |
| CN106554427A (en) * | 2015-09-29 | 2017-04-05 | 李博琪 | A kind of extracting method of lycium barbarum polysaccharide |
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| CN100464756C (en) * | 2007-04-27 | 2009-03-04 | 山东大学 | Application of fucoidan in the preparation of medicine and health-care products for preventing and treating neurodegenerative diseases |
| US8835141B2 (en) * | 2011-06-09 | 2014-09-16 | The United States Of America As Represented By The Secretary Of Agriculture | Methods for integrated conversion of lignocellulosic material to sugars or biofuels and nano-cellulose |
| CN106317243A (en) * | 2016-08-30 | 2017-01-11 | 宁夏沃福百瑞枸杞产业股份有限公司 | Preparation method of lycium barbarum polysaccharides |
-
2017
- 2017-08-08 CN CN201710671638.1A patent/CN109369815B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012612A (en) * | 2012-12-14 | 2013-04-03 | 天津大学 | Extraction method of wolfberry polysaccharide |
| CN104072627A (en) * | 2014-06-04 | 2014-10-01 | 沈阳化工大学 | Preparation method for typhonium giganteum polysaccharide extract |
| CN104987433A (en) * | 2015-07-23 | 2015-10-21 | 中国科学院西北高原生物研究所 | Preparation method of RG-I type lycium barbarum pectin with anti-aging activity |
| CN106554427A (en) * | 2015-09-29 | 2017-04-05 | 李博琪 | A kind of extracting method of lycium barbarum polysaccharide |
Non-Patent Citations (4)
| Title |
|---|
| Neuroprotective effects of anti-aging oriental medicine Lycium barbarum against β-amyloid peptide neurotoxicity;Man-ShanYu等;《Experimental Gerontology》;20050930;第40卷(第8-9期);第716-725页 * |
| 张民等.枸杞多糖的纯化及相对分子质量研究.《中国食品添加剂》.2012,第50页. * |
| 枸杞多糖对β淀粉样蛋白1-40致PC12细胞线粒体损伤的影响;成江等;《宁夏医科大学学报》;20161230;第38卷(第12期);第1371-1374页 * |
| 枸杞多糖的纯化及相对分子质量研究;张民等;《中国食品添加剂》;20120215;第50页 * |
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