CN114349876A - A Lycium barbarum polysaccharide for resisting retina injury caused by high sugar and hydrogen peroxide - Google Patents
A Lycium barbarum polysaccharide for resisting retina injury caused by high sugar and hydrogen peroxide Download PDFInfo
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Abstract
The invention provides the lycium barbarum homogeneous polysaccharide which has good effects of preventing and treating age-related macular degeneration and retinopathy caused by diabetes. Separating the water extract of the Chinese wolfberry by an ultrafiltration membrane with the molecular weight of 10kDa and 5kDa, freeze-drying the water extract component of the Chinese wolfberry which permeates through a 10kDa membrane component and is intercepted by a 5kDa membrane component, removing protein and pigment, and then dialyzing; performing column chromatography on the purified product, eluting with sodium chloride solution, collecting the components, dialyzing with 1000Da dialysis bag, freeze-drying the dialyzed product, dissolving in distilled water again, further separating with dextran gel, collecting the components, and freeze-drying to obtain the final product. The lycium barbarum polysaccharide has the effect of preventing and treating age-related macular degeneration and retinopathy caused by diabetes, and can be used for preparing health-care food, special medical application food or new drugs.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to lycium barbarum polysaccharide capable of resisting retina damage caused by high sugar and hydrogen peroxide.
Background
Age-related macular degeneration (AMD), a degenerative disease characterized by chronic inflammation, oxidative stress, and aging, is ubiquitous throughout the world among people over the age of 55. AMD mainly affects the foveal macular region, and the progression of disease can lead to permanent visual impairment or blindness, seriously affecting quality of life. Research has shown that oxidative stress is a major trigger for AMD, leading to cellular damage through the production of free radicals, reactive species, and oxidation-related reactions. It is widely believed that free radical disorders are associated with oxygen metabolism or oxidative stress, which plays an important role not only in normal aging, but also in the progression of retinal degeneration. Retinal pigment epithelial cells play a key role in maintaining retinal function between photoreceptors and choroidal capillaries; while reactive oxygen species gradually damage retinal pigment epithelial cells during aging, leading to protein misfolding, cellular dysfunction and retinal pigment epithelial cell damage, which are characteristic pathological indicators of AMD.
Wolfberry is becoming increasingly popular because of its good nutritional and medical value. Modern pharmacological and natural medicinal chemical studies have shown that polysaccharides are one of the main active ingredients of lycium barbarum, and their health benefits are closely related to their antioxidant properties. A large number of literature reports show that the lycium barbarum polysaccharide has a remarkable effect on protecting eyesight, is beneficial to reducing apoptosis and oxidative stress of retina, and can also improve the survival rate of retinal ganglion cells of newborn rats, so that retinal dysfunction caused by ischemia is effectively relieved, related neuron death and glial cell activation are reduced, and the occurrence of early ADM is prevented and treated. However, the existing research is only at the level of crude polysaccharide, and the exact structure of the functional single polysaccharide and the mechanism of how it prevents AMD has not been reported.
Disclosure of Invention
The invention provides lycium barbarum homogeneous polysaccharide with a function of resisting retinal damage caused by high sugar and hydrogen peroxide, and the lycium barbarum polysaccharide has an effect of protecting vision and can be used for preparing health-care food, special medical application food or a new medicine.
The lycium barbarum polysaccharide LICP009-3F-2a provided by the invention has the following structural formula:
the lycium barbarum polysaccharide LICP009-3F-2a provided by the invention has a molecular weight of 13720Da determined by gel permeation chromatography.
In the provided lycium barbarum polysaccharide LICP009-3F-2a, rhamnose: arabinose: galactose: glucose: the molar ratio of galacturonic acid to glucuronic acid is: 39.1:7.4:22.5:8.3:13.7:4.0.
The preparation method of the lycium barbarum polysaccharide LICP009-3F-2a provided by the invention comprises the following steps:
centrifuging the water extract of fructus Lycii, ultrafiltering the supernatant with ultrafiltration membrane with molecular weight of 10kDa and 5kDa, freeze drying the retentate, and re-dissolving with distilled water; after the solution is subjected to protein removal and pigment removal treatment, dialysis is carried out; and (3) performing column chromatography separation on the purified product, eluting by using distilled water and a sodium chloride solution in sequence, collecting the components, dialyzing the collected components again, dissolving the dialyzed product in distilled water again after freeze drying, separating by using a sephadex chromatographic column, collecting the eluent components, and performing freeze drying to finish the preparation.
The deproteinization is carried out by using trichloroacetic acid as a specific example.
As a specific description of examples, the pigment removal was performed by using 30% hydrogen peroxide.
As a specific description of the examples, the dialysis was carried out using a dialysis bag having a molecular weight of 1000 Da.
As a specific description of examples, the column chromatography described above uses a DEAE-52 column and a Sephadex G-50 column.
The concentration of the sodium chloride solution in the eluent of the column chromatography is 0.2M.
The lycium barbarum polysaccharide provided by the invention can be used for preparing products for preventing and treating age-related macular degeneration or retinopathy caused by diabetes.
The product is health food, food with special medical application or medicine.
Drawings
FIG. 1: gel permeation chromatogram of LICP009-3F-2 a.
FIG. 2: (A) ion chromatograms of monosaccharide standards (1: Fuc; 2: GalN; 3: Rha; 4: Ara; 5: GlcN; 6: Gal; 7: Glc; 8: GlcNAc; 9: Xyl; 10: Man; 11: Fru; 12: Rib; 13: GalA; 14: GulA; 15: GlcA; 16: ManA); (B) ion chromatogram of LICP009-3F-2 a.
FIG. 3: (A) infrared spectrum of LICP009-3F-2 a; (B) XRD spectrum of LICP009-3F-2 a.
FIG. 4: NMR spectrum of LICP009-3F-2 a.
FIG. 5: two-dimensional nuclear magnetic spectrum of LICP009-3F-2 a.
FIG. 6: (A) graph of the Effect of LICP009-3F-2a (100, 200 and 400. mu.g/mL) on ARPE-19 cell proliferation, (B) H detected in ARPE-19 cells treated with LICP009-3F-2a (100, 200 and 400. mu.g/mL)2O2Fluorescence image of induced ROS. (C) With H in the presence of different concentrations of LICP009-3F-2a2O2Survival of treated ARPE-19 cells. LICP009-3F-2a (100, 200 and 400. mu.g/mL) vs. H2O2Induced effects of (D) CAT, (E) SOD1 and (F) MMP2 levels in ARPE-19 cells.
FIG. 7: (A) effect of LICP009-3F-2a (50, 100, 200 and 400. mu.g/mL) on viability of high-sugar treated ARPE-19 cells. (B) Effect of LICP009-3F-2a (100, 200 and 400. mu.g/mL) on the levels of the high-sugar induced apoptosis marker, Bax, in ARPE-19 cells.
Detailed Description
The invention adopts H2O2And a high-sugar induced human retinal epithelial cell test, and discovers a novel lycium barbarum polysaccharide with a uniform structure, which has good effects of preventing and treating age-related macular degeneration and retinopathy caused by diabetes.
The preparation method of the lycium barbarum polysaccharide comprises the steps of centrifuging a water extracting solution of lycium barbarum, and then carrying out ultrafiltration separation on supernate by using ultrafiltration membranes with molecular weights of 10kDa and 5 kDa.
Freeze drying the trapped fluid, and re-dissolving with distilled water; after the solution is subjected to protein removal and pigment removal treatment, dialysis is carried out; and (3) performing column chromatography separation on the purified product, eluting by using distilled water and a sodium chloride solution in sequence, collecting the components, dialyzing the collected components again, dissolving the dialyzed product in distilled water again after freeze drying, separating by using a sephadex chromatographic column, collecting the eluent components, and performing freeze drying to finish the preparation.
The obtained lycium barbarum polysaccharide is subjected to structural characterization and efficacy evaluation, and the lycium barbarum polysaccharide is determined to be used for preparing products for preventing and treating age-related macular degeneration and retinopathy caused by diabetes.
The present invention will be described in detail below with reference to examples and the accompanying drawings.
Example 1: preparation of Lycium barbarum homogeneous polysaccharide LICP009-3F-2a for preventing and treating senile macular degeneration and diabetic retinopathy
1. Preparation method of lycium barbarum homogeneous polysaccharide LICP009-3F-2a
1.1 instruments, reagents and materials
DEAE-52 cellulose, Sephadex G-50, standard monosaccharides (fucose, galactosamine, rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, N-acetyl-D-glucosamine, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid and mannuronic acid). All other chemicals were at least of analytical grade. The solvent of the high performance liquid chromatography is chromatographic purity.
1.2 preparation of crude polysaccharide of Lycium barbarum
Adding 1L distilled water into 100g fructus Lycii powder, and extracting with high speed shearing dispersing emulsifying machine at 15000 r/min and 60 deg.C for 30 min. After the extraction is finished, centrifuging at 8000 rpm for 15min, removing residues, and collecting supernatant containing crude polysaccharide. And then intercepting and separating by using ultrafiltration membrane separation equipment with molecular weights of 10kDa and 5kDa, wherein the membrane separation process comprises pumping the supernatant of the crude lycium barbarum polysaccharide obtained after centrifugation into a membrane module with the molecular weight cutoff of 10kDa through a peristaltic pump, the flow rate is 150mL/min, and the membrane area is 100 x 100 mm. Separating into two parts of interception liquid and permeation liquid, and obtaining the permeation liquid LICP009a after the separation is finished when the final interception volume is 35 mL. The permeate LICP009a was pumped into a membrane module with molecular weight cut-off of 5kDa by a peristaltic pump at a flow rate of 150mL/min and a membrane area of 100X 100 mm. Separating into two parts of interception liquid and permeation liquid, and finally obtaining trapped liquid LICP009 after finishing separation when the trapped volume is 35 mL. Placing LICP009 into vacuum freeze drying machine at-50 deg.C, sample temperature of-35 deg.C, and vacuum degree of 10 Pa.
5 g of LICP009 was dissolved in 100mL of distilled water and the protein was precipitated with 20mL of trichloroacetic acid. Then 10mL of ammonia water is added to adjust the pH value, 10mL of 30% hydrogen peroxide is added, and the mixture is stirred and decolored at 60 ℃ with the stirring speed of 120 r/min. Dialyzing with dialysis bag with molecular weight of 1000Da for 3 days. Freeze drying to obtain purified polysaccharide LICP 009-3F.
Dissolving 0.8g of purified lycium barbarum polysaccharides in 20mL of distilled water, pumping into a pretreated DEAE-52 chromatographic column by using a peristaltic pump, and respectively eluting with distilled water and 0.2 and 0.5 mol of sodium chloride solution step by step at the flow rate of 2mL/min, and collecting 8mL of the purified lycium barbarum polysaccharides in each tube. The eluted fractions were monitored by phenol-sulfuric acid method. Then, fractions eluted with 0.2M NaCl were collected, dialyzed against 1000Da dialysis bag for 2 days, and freeze-dried to obtain LICP009-3F-2 fraction. The above operation was repeated to accumulate LICP009-3F-2 fraction.
0.2G of the above-mentioned LICP009-3F-2 fraction was dissolved in 6mL of distilled water, and added to a Sephadex G-50 column using a dropper, followed by elution with water. The flow rate was 2mL/min, and 8mL was collected per tube. The eluted fractions were monitored by phenol-sulfuric acid method. Collecting 29-41 tubes, concentrating to 10mL with rotary evaporator under reduced pressure, and freeze drying to obtain LICP009-3F-2 a.
Example 2: lycium barbarum Hook homogeneous polysaccharide LICP009-3F-2a structure analysis
1.1 determination of molecular weight
Subjecting to gel permeation chromatography with dextran of molecular weight 1152,5000,11600,23800,48600,80900,14800,27300,409800, 670000Da as standardAnd (3) drawing a standard curve of lgMw-RT according to a calibration curve equation: -0.2014x +12.661, R20.9912; the molecular weight of LICP009-3F-2a was determined to be 13720Da according to the retention time shown in FIG. 1, and as can be seen from FIG. 1, the chromatographic peak symmetry was good, indicating that the polysaccharide was a homogeneous polysaccharide.
1.2 monosaccharide composition analysis
The composition and ratio of LICP009-3F-2a were analyzed by ion chromatography. As shown in FIGS. 2A and 2B, according to the retention time of standard monosaccharides, the monosaccharide composition in Lycium barbarum polysaccharide LICP009-3F-2A is rhamnose: arabinose: galactose: glucose: the molar ratio of galacturonic acid to glucuronic acid is: 39.1:7.4:22.5:8.3:13.7:4.0. The composition and ratio of LICP009-3F-2a was significantly different from the previously reported polysaccharides. Therefore, LICP009-3F-2a is a novel acidic heteropolysaccharide.
1.3 Infrared Spectroscopy
As shown in FIG. 3A, LICP009-3F-2a is at 3413.9cm-1A broad and strong hydroxyl stretching peak occurs, indicating strong intermolecular and intramolecular interactions between polysaccharide chains. 2929.2cm-1The weak absorption band is due to the asymmetric stretching vibration of C-H, 1643.4 and 1425.3cm-1The strong absorption peak at (a) is due to asymmetric stretching vibration of C ═ O or COOH, indicating the presence of uronic acid in the polysaccharide, consistent with the monosaccharide composition analysis results. At 1000--1Absorption peaks in the range are assigned to bending or stretching vibrations of the C — O group. 1077.7and 1027.8cm-1Indicating the presence of pyranose, 897.5cm-1The absorption peak of (A) indicates the presence of a beta-glycosidic bond.
1.4X-ray diffraction analysis
X-ray diffraction analysis is widely used to detect the degree of crystallinity of a substance, and is able to sufficiently reflect the crystalline or amorphous nature of polysaccharides. As shown in FIG. 3B, the X-ray diffraction pattern showed the characteristic diffraction curve of LICP009-3F-2 a. There are two main reflected waves at 17.2 and 42.82 θ. Both diffraction peaks are arched, indicating that LICP009-3F-2a contains two major crystalline components. From these results, LICP009-3F-2a was identified as a semi-crystalline polymer.
1.5 methylation analysis
The ligation pattern of LICP009-3F-2a was analyzed by methylation followed by hydrolysis and reduction, the results of which are shown in Table 1. The non-reducing end of LICP009-3F-2a consisted of T-Araf (2.5%), T-Arap (1.8%), T-Glcp (6.1%) and T-Galp (8.3%); 1,2-Rhap (6.4%), 1,4-Galp (15.4%), 1,4-Glcp (9.2%), 1,3-Galp (7.4%), 1,6-Glcp (15.1%), 1,6-Galp (9.7%) and a small amount of 1,5-Araf (2.4%) as intrachain residues, indicating that LICP009-3F-2a comprises a main chain and branched chains. In addition, the major branch points occurred in 1,3,6-Galp (12.8%), 1,2,4-Rhap (1.7%) and 1,3,4-Glcp (1.4%). Methylation results indicate that LICP009-3F-2a is a highly branched structure with a variety of monosaccharide compositions and linkage types.
Table 1: methylation analysis Table of LICP009-3F-2a
1.6 nuclear magnetic analysis
The LICP009-3F-2a hydrogen spectrum signal is mainly concentrated between 3.0-5.5 ppm (FIG. 4). Delta 3.2-4.0ppm is sugar ring proton signal, and the signal peaks of main terminal group proton peaks 5.2,5.17,5.01,4.97,4.61,4.57 and 4.44 are intensively distributed in the region of 4.3-5.5 ppm.
The nuclear magnetic carbon spectrum signals are mainly concentrated between 60-120ppm (FIG. 5). By observing the carbon spectrum, the main anomeric carbon signal peak 108.69,105.22,104.83,103.84 can be seen, and the 99.8 anomeric carbon region is mainly between delta 93 and 110. And main signal peaks of 83.07,81.7,80.16,77.97,77.1,76.75,76.6,76.34,74.81,74.14,74.06,73.76,73.36,73.11,71.45,71.84,70.51,70.76,70.2,67.61,64.34,62.1,62.3,62.64,61.99 and 61.67 are distributed in a 60-85 ppm area.
From the HSQC spectrum (FIG. 5), it can be observed that the anomeric carbon signal is δ 104.9, the corresponding anomeric hydrogen signal in the HSQC spectrum is δ 4.43, and the signal through HH-COSY, H1-2 is 4.43/3.24; the signal of H2-3 is 3.24/3.4; the signal of H3-4 is 3.4/3.56; the signal of H4-5 is 3.56/3.36; we can conclude that H1, H2, H3, H4 and H5 are delta 4.43,3.24,3.4,3.56 and 3.36ppm, respectively. According to the correlation peaks of HSQC, C1/H1, C2/H2, C3/H3, C4/H4 and C5/H5, the corresponding C1-C5 are deduced to be 104.9, 74.53, 76.75, 76.42 and 70.9. Therefore, the signal should be assigned to the glycosidic bond → 6) - β -D-Glcp- (1 →.
From the HSQC spectrum (FIG. 5), it was observed that the anomeric carbon signal was δ 99.88, the corresponding anomeric hydrogen signal in the HSQC spectrum was δ 5.20, and the signal by HH-COSY, H1-2 was 5.20/4.05; the signal of H2-3 is 4.05/3.82; the signal of H3-4 is 3.82/3.34; the signal of H4-5 is 3.34/3.69; we can conclude that H1, H2, H3, H4, H5 are delta 5.20,4.05,3.82,3.34,3.69ppm, respectively. According to the correlation peaks of HSQC, C1/H1, C2/H2, C3/H3, C4/H4 and C5/H5, the corresponding C1-C5 are deduced to be 99.88, 77.69, 70.12, 73.56 and 71.45. Therefore, the signal should be assigned to the glycosidic bond → 2) - α -L-Rha- (1 →. All glycosidic bond signals were assigned according to a similar rule and in combination with HMBC and NOESY, as shown in the following table:
table 2: nuclear magnetic characteristic peak attribution table of LICP009-3F-2a
In the two-dimensional map (fig. 5), the glycosidic bond signal of the polysaccharide is assigned according to the nuclear magnetic one-dimensional two-dimensional map;
backbone analysis:
(A) → 2) - α -L-Rha- (1 → anomeric hydrogen has a correlation peak with its own C2, indicating the presence → 2) - α -L-Rha- (1 →.
(A) → 2) - α -L-Rha- (1 → anomeric hydrogen has a peak associated with its (B) → 2,4) - α -L-Rha- (1 → C2, indicating the presence → 2) - α -L-Rha- (1 → 2,4) - α -L-Rha- (1 →.
(B) → 2,4) - α -L-Rha- (1 →. anomeric hydrogen has a peak associated with its (E) → 4) - α -D-GalAp- (1 → C4 indicating the presence → 2,4) - α -L-Rha- (1 → 4) - α -D-GalAp- (1 →.
(E) → 4) - α -D-GalAp- (1 → anomeric hydrogen with its (K) → 3,6) - β -D-Galp- (1 → C6 has a peak associated therewith, indicating the presence → 4) - α -D-GalAp- (1 → 3,6) - β -D-Galp- (1 →.
(K) → the anomeric hydrogen of 3,6) - β -D-Galp- (1 → has a correlation peak with its own C6, indicating the presence → 3,6) - β -D-Galp- (1 →.
(K) The anomeric hydrogen of → 3,6) - β -D-Galp- (1 → has a correlation peak with (J) → 6) - β -D-Galp- (1 → C6, indicating the presence → 3,6) - β -D-Galp- (1 →.
The main chain is as follows:
→2)-α-L-Rha-(1→2,4)-α-L-Rha-(1→4)-α-D-GalAp-(1→3,6)-β-D-Galp-(1→3,6)-β-D-Galp-(1→6)-β-D-Galp-(1→(A→B→E→K→K→J)
branched chain analysis:
branched chain 1:
NOESY profile: the anomeric hydrogen of the glycosidic bond alpha-L-Araf- (1 → H5 of → 5) -alpha-L-Araf- (1 → has a signal peak, indicating the existence of the linkage mode of alpha-L-Araf- (1 → 5) -alpha-L-Araf- (1 → H).
Glycosidic bond → 5) - α -L-Araf- (1 → anomeric hydrogen and → 6) - β -D-Glcp- (1 → H6 have associated signal peaks; indicating the presence of the → 5) - α -L-Araf- (1 → 6) - β -D-Glcp- (1 → linkage.
In HMBC mapping: → 6) - β -D-Glcp- (1 → anomeric hydrogen and → 2,4) - α -L-Rha- (1 → C4 have related signal peaks; indicating the presence of the → 6) - β -D-Glcp- (1 → 2,4) - α -L-Rha- (1 → linkage.
In summary, the following results can be obtained: there is α -L-Araf- (1 → 5) - α -L-Araf- (1 → 6) - β -D-Glcp- (1 → 2,4) - α -L-Rha- (1 → (C → D → L → B)
Branched chain 2:
the related signal peak exists between the anomeric carbon of beta-D-Glcp- (1 → and H4 of → 4) -beta-D-Glcp- (1 →), and the existence of the linkage mode of beta-D-Glcp- (1 → 4) -beta-D-Glcp- (1 → is indicated.
→ 4) - β -D-Glcp- (1 → anomeric carbon and → 3,6) - β -D-Galp- (1 → H3 have related signal peaks; indicating the presence of the → 4) - β -D-Glcp- (1 → 3,6) - β -D-Galp- (1 → linkage.
In summary, the following results can be obtained: branched chain beta-D-Glcp- (1 → 4) -beta-D-Glcp- (1 → 3,6) -beta-D-Galp- (1 → (M → I → K)
Branched chain 3:
the anomeric hydrogen of the glycosidic linkage β -D-Galp- (1 → and C3 of → 3) - β -D-Galp- (1 → have a signal peak indicating the presence of the linkage of β -D-Galp- (1 → 3) - β -D-Galp- (1 →;
glycosidic bond → 3) - β -D-Galp- (1 → anomeric hydrogen and → 3,6) - β -D-Galp- (1 → C3 have a signal peak; indicating the presence of the → 3) - β -D-Galp- (1 → 3,6) - β -D-Galp- (1 → linkage. (H → F → K)
From the above, we can conclude that the main glycosidic bond structure of the polysaccharide is as follows: the backbone linkage is the glycosidic bond → 2) - α -L-Rha- (1 → 2,4) - α -L-Rha- (1 → 4) - β -D-GalAp- (1 → 6) - β -D-Galp- (1 → and the terminal branch is attached to the backbone by the O-4 bond → 3,6) - β -D-Galp- (1 → O-3 bond → 2,4) - α -L-Rha- (1 → 4 bond. The structural formula is as follows:
example 3: evaluation of activity for preventing and treating age-related macular degeneration and diabetic retinopathy
1. LICP009-3F-2a promotes proliferation of ARPE-19 cells in human retinal epithelial cells
Retinal epithelial cells are critical to the integrity and function of the retina. ARPE-19 is a human retinal epithelial cell line that maintains normal nuclear and functional properties of retinal epithelial cells in vivo. As shown in FIG. 6A, the fluorescence intensity of the ARPE-19 cells treated with LICP009-3F-2a was increased dose-dependently and higher than the positive control, indicating that ARPE-19 cells in the LICP009-3F-2a group proliferated more, and particularly at a dose of 400. mu.g/mL, a significant increase in ARPE-19 cell proliferation was observed. The above results demonstrate that LICP009-3F-2a can induce ARPE-19 cell proliferation.
2. LICP009-3F-2a protects ARPE-19 cells from H2O2Induced oxidative damage
ARPE-19 cells were pretreated with various concentrations of LICP009-3F-2a for 24 hours, followed by 1mM hydrogen peroxide for 0.5 hours. As shown in FIG. 6C, andARPE-19 cells pretreated with LICP009-3F-2a showed significantly increased viability in a dose-dependent manner compared to the lesion group. The viability of cells pretreated with 400. mu.g/mL LICP009-3F-2a was 156% (p) of the control group<0.05), suggesting that pretreatment with LICP009-3F-2a promotes ARPE-19 cell proliferation and is resistant to H2O2Resulting in oxidative damage. Meanwhile, the detection that the LICP009-3F-2a is in H under different concentrations2O2Concentration of Reactive Oxygen Species (ROS) in ARPE-19 cells under induction. As shown in FIG. 6B, the fluorescence signal of the group containing LICP009-3F-2a was weaker than that of the control group, and the fluorescence signal of LICP009-3F-2a was the weakest at 400. mu.g/mL. It was shown that LICP009-3F-2a can be used to reduce ROS levels and protect ARPE-19 cells.
The accumulation of excess ROS results in a decrease in enzyme (e.g., SOD and CAT) activity. Given the importance of oxidative stress on normal ARPE-19 cell status, the levels of SOD1 and CAT were examined in ARPE-19 cells treated with various concentrations of LICP009-3F-2 a. As shown in FIGS. 6D and 6E, ARPE-19 cells pretreated with different concentrations of LICP009-3F-2a had higher SOD and CAT activities (p <0.05) compared to the control group.
Matrix metalloprotein kinases (MMPs) are produced by retinal pigment epithelial cells; they are the main factors for maintaining the balance between extracellular matrix degradation and synthesis, and play an important role in maintaining the balance of ocular tissue matrix components. As shown in fig. 6F, expression of MMP2 was detected and it was found that LICP009-3F-2a protected ARPE-19 cells by down-regulating MMP2 expression, a clear dose-effect relationship was observed when polysaccharide concentration was increased from 100 to 400 μ g/mL.
3. LICP009-3F-2a protects ARPE-19 cells from high sugar induced damage
LICP009-3F-2a was effective in protecting ARPE-19 cells from high sugar induced damage. As shown in FIG. 7A, after treatment with LICP009-3F-2a, the survival of high-sugar induced ARPE-19 cells was significantly higher and dose-dependent (p <0.0), and further examination of the molecular apoptosis marker Bax showed that LICP009-3F-2a significantly reduced the expression of Bax (FIG. 7B). Thus, it was shown that LICP009-3F-2a protected ARPE-19 cells from hyperglycemia damage by inhibiting apoptosis.
In conclusion, the lycium barbarum homogeneous polysaccharide provided by the invention has the effect of resisting retina damage caused by high sugar and hydrogen peroxide, has the effect of protecting vision, and can be used for preparing health-care food, special medical application food or new drugs.
Claims (10)
1. The lycium barbarum polysaccharide is characterized by being represented by the following structural formula:
2. the lycium barbarum polysaccharide of claim 1, having a molecular weight of 13720Da as determined by gel permeation chromatography.
3. The lycium barbarum polysaccharide of claim 1, wherein the lycium barbarum polysaccharide has a rhamnose: arabinose: galactose: glucose: the molar ratio of galacturonic acid to glucuronic acid was 39.1:7.4:22.5:8.3:13.7: 4.0.
4. The lycium barbarum polysaccharide of claim 1, wherein the preparation method of the lycium barbarum polysaccharide comprises the following steps:
centrifuging the water extract of fructus Lycii, ultrafiltering the supernatant with ultrafiltration membrane with molecular weight of 10kDa and 5kDa, freeze drying the retentate, and re-dissolving with distilled water; after the solution is subjected to protein removal and pigment removal treatment, dialysis is carried out; and (3) performing column chromatography separation on the purified product, eluting by using distilled water and a sodium chloride solution in sequence, collecting the components, dialyzing the collected components again, dissolving the dialyzed product in distilled water again after freeze drying, separating by using a sephadex chromatographic column, collecting the eluent components, and performing freeze drying to finish the preparation.
5. The lycium barbarum polysaccharide of claim 4, wherein said deproteinizing is deproteinizing using trichloroacetic acid.
6. The lycium barbarum polysaccharide of claim 4, wherein said depigmentation is performed using 30% hydrogen peroxide.
7. The lycium barbarum polysaccharide of claim 4, wherein the column chromatography is performed using a DEAE-52 column and a Sephadex G-50 column; the concentration of the sodium chloride solution in the eluent is 0.2M.
8. The use of lycium barbarum polysaccharide of claim 1 in the preparation of a product for the prevention and treatment of age-related macular degeneration or diabetic retinopathy.
9. The use according to claim 8, wherein the product is a health food, a food for special medical use or a pharmaceutical product.
10. A product for the prevention and treatment of age-related macular degeneration or retinopathy caused by diabetes mellitus, comprising the lycium barbarum polysaccharide of claim 1 in a pharmacologically effective concentration.
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| CN115947875A (en) * | 2022-09-30 | 2023-04-11 | 中国科学院兰州化学物理研究所 | Lycium barbarum polysaccharide extracted by ionic liquid and application of lycium barbarum polysaccharide in reducing blood sugar |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112851829A (en) * | 2021-01-15 | 2021-05-28 | 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112851829A (en) * | 2021-01-15 | 2021-05-28 | 中国科学院兰州化学物理研究所 | A fructus Lycii polysaccharide with blood lipid reducing effect |
Non-Patent Citations (1)
| Title |
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| JIANFEI LIU ET AL: ""A polysaccharide from Lycium barbarum L.: Structure and protective effects against oxidative stress and high-glucose-induced apoptosis in ARPE-19 cells"", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115947875A (en) * | 2022-09-30 | 2023-04-11 | 中国科学院兰州化学物理研究所 | Lycium barbarum polysaccharide extracted by ionic liquid and application of lycium barbarum polysaccharide in reducing blood sugar |
| CN115947875B (en) * | 2022-09-30 | 2024-02-02 | 中国科学院兰州化学物理研究所 | Lycium barbarum polysaccharide extracted by ionic liquid and application thereof in reducing blood sugar |
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