Application of bupleurum chinense polysaccharide in medicine for preventing brain aging

Abstract

The invention discloses an application of bupleurum chinense polysaccharide in a drug for preventing brain aging, particularly a protection effect of BCP on D-gal induced aging HT22 cells, and discovers that BCP has in-vitro anti-oxidation and anti-D-gal induced HT22 cell aging effects, and can reduce the occurrence of nervous system degenerative diseases and delay aging.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and in particular relates to the application of Bupleurum chinense polysaccharide in medicines for preventing brain aging. Background Art

[0002] Brain aging is the earliest to occur in all organs of the body. The incidence of neurodegenerative diseases related to brain aging is also gradually increasing, becoming an important issue affecting the quality of life of the elderly and the national socio-economic development. Alzheimer's disease (AD) , Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, which can cause changes in cognitive function and even lead to dementia in most people.

[0003] In normal metabolic activities, mitochondria will form ROS. Aging will disrupt the dynamic balance of free radicals in the human body, leading to the accumulation of ROS in the body. ROS is a strong oxidant. Excessive accumulation in the body will damage DNA coding sequences, change DNA methylation modification, change gene expression, and cause apoptosis, aging, and even cancer in the body's cells. Many neurodegenerative diseases related to brain aging are related to oxidative stress.

[0004] The pathological characteristics of AD are the formation of extracellular senile plaques and NFTs. ROS plays a key role in β-mediated neuronal apoptosis. Mitochondrial dysfunction will further release ROS, stimulate the production of pro-apoptotic proteins, and lead to neuronal apoptosis. Oxidative stress plays a key role in the pathogenesis of neurodegenerative diseases, especially in the phosphorylation of tau protein and the formation of neurofibrillary tangles. Under pathological conditions, the production of ROS will interfere with the normal function of tau protein, leading to its hyperphosphorylation, weakening the ability of tau protein to bind to microtubules, thereby accelerating the depolymerization process of microtubules, and ultimately leading to the interruption of neural signaling. Therefore, it is of great significance to prevent and treat neurodegenerative diseases by reducing oxidative stress and inhibiting the hyperphosphorylation of tau protein.

[0005] According to the Chinese Pharmacopoeia, Bupleurum is the dried root of Bupleurum (Apiaceae), which is divided into "Southern Bupleurum" and "Northern Bupleurum" according to different properties. It is a very important herbal medicine in China with a long history. Modern pharmacological studies have shown that Bupleurum has antipyretic, sedative, analgesic, antitussive, anti-inflammatory, antibacterial, antiviral, anti-tumor, antioxidant, antidepressant, anti-epileptic, liver-protective, choleretic effects, and has regulatory effects on the central nervous system, cardiovascular, digestive and immune systems. Chinese herbal polysaccharides are active polysaccharides extracted from Chinese herbal medicines. They have antioxidant, anti-inflammatory, anti-aging, anti-tumor, and immunomodulatory functions and have become a hot topic of research. Current research focuses on the fields of hepatitis, myocardial injury, nephritis, pneumonia, etc., and there is no literature report on its application in the field of anti-brain aging. Summary of the invention

[0006] The purpose of the present invention is to provide an application of Bupleurum chinense polysaccharide in a drug for preventing brain aging, especially the protective effect of BCP on D-gal-induced senescent HT22 cells. The present invention finds that BCP has in vitro antioxidant and anti-D-gal-induced HT22 cell senescence effects, can reduce the occurrence of neurodegenerative diseases and delay aging.

[0007] The present invention is achieved through the following technical solutions: Application of Bupleurum chinense polysaccharide in drugs for preventing brain aging.

[0008] The Bupleurum chinense polysaccharide comprises rhamnose, arabinose, galactose, glucose and galacturonic acid.

[0009] The application of the Bupleurum chinense polysaccharide in the drug for preventing brain aging is achieved by the following method: The crude polysaccharide of Bupleurum chinense was extracted by water extraction and alcohol precipitation, and then purified by repeated freezing and thawing, sevage method and running water dialysis. The in vitro antioxidant capacity of BCP was observed by testing its DPPH free radical scavenging ability, hydroxyl free radical inhibition ability and total antioxidant capacity. The HT22 cell senescence model was induced by D-gal, and the CCK8 method was used to observe whether BCP had a protective effect on the HT22 senescence cell model; the reactive oxygen species detection kit was used to observe whether BCP had an anti-senescence effect on HT22 cells.

[0010] The research results of the present invention show that: Bupleurum polysaccharide BCP has in vitro antioxidant activity, can promote HT22 cell proliferation, and reduce D-gal stimulation of HT22 by inhibiting cell aging, thereby reducing the generation of ROS. Therefore, Bupleurum polysaccharide BCP can inhibit brain aging neurodegenerative diseases caused by oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a BCP Congo red experimental analysis diagram in the present invention; Figure 2 It is the ultraviolet spectrum of BCP in the present invention; Figure 3 It is the infrared spectrum of BCP in the present invention; Figure 4 This is the electron microscope scanning analysis of BCP in the present invention, A: BCP magnification 500×; B: BCP magnification 2000×; C: BCP magnification 5000×; Figure 5 This is a comparison chart of the DPPH free radical scavenging ability determination in the present invention; Figure 6This is a comparative diagram of the hydroxyl radical scavenging ability determination in the present invention; Figure 7 This is a comparison chart of the total antioxidant capacity determination in the present invention; Figure 8 This is a graph showing the effect of BCP on the proliferation rate of HT22 cells in the present invention; Fig. 9 The figure is the effect of D-gal on the proliferation rate and half lethality (IC50) of HT22 cells in the present invention, wherein: A is a comparison of the effect of different concentrations of D-gal on the proliferation rate of HT22 cells, B is the half lethality of HT22 cells treated with different concentrations of D-gal for 12 hours, C is the half lethality of HT22 cells treated with different concentrations of D-gal for 24 hours, D is the half lethality of HT22 cells treated with different concentrations of D-gal for 48 hours, E is the half lethality of HT22 cells treated with different concentrations of D-gal for 72 hours, and F is the half lethality of HT22 cells treated with different concentrations of D-gal for 96 hours; Fig.10 Comparison of the protective effects of different concentrations of BCP on the HT22 senescent cell model in the present invention; Fig.11 Graph showing the effect of BCP on the level of reactive oxygen species (ROS) in D-gal-induced HT22 cells in the present invention. DETAILED DESCRIPTION

[0012] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0013] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0014] The specific process of extracting Bupleurum chinense polysaccharide is as follows: 1. Take the slices of Radix Bupleuri and grind them, soak them in a ratio of 30:1 of distilled water to Radix Bupleuri for 24 hours, extract them 3 times at 80°C for 3 hours each time, filter, centrifuge and evaporate and concentrate the supernatant, add anhydrous ethanol to 80% ethanol content, centrifuge and precipitate and dry to obtain crude polysaccharide of Radix Bupleuri; 2. Dissolve the crude polysaccharide in distilled water at a ratio of 5% (5g sugar + 100g water), remove the protein through repeated freeze-thaw and centrifugation, prepare Sevage solution (chloroform: n-butanol analytical grade = 4:1), and mix it at a ratio of 2 times the volume of the sugar solution, repeatedly shake and centrifuge, retain the upper sugar solution and repeatedly centrifuge it, discard the protein precipitate, dialyze it in running water for 24 hours, and freeze-dry it to obtain the Bupleurum chinense polysaccharide BCP.

[0015] The invention adopts a water extraction and alcohol precipitation method to process 500g of dried northern bupleurum slices to obtain 30.58g of dark brown northern bupleurum crude polysaccharide, with a specific gravity of 6.12% of the dry material; after repeated freezing and thawing, sevage protein removal and dialysis, 9.4557g of BCP is obtained, with a yield of 30.92%.

[0016] The determination of Bupleurum chinense polysaccharide specifically includes the following contents: 1. The total sugar content of the Bupleurum chinense polysaccharide extracted in the above steps was detected by the phenol-sulfuric acid method and was 68.02%±1.62%; 2. The protein content in BCP was 27.2%±2.8% as determined by the Coomassie Brilliant Blue method; 3. The Congo red method was used to determine whether BCP has a spatial helical structure. Under the action of different concentrations of NaOH, Congo red reacted with polysaccharides with a spatial helical structure and formed specific chemical bonds, causing the maximum absorption wavelength (λmax) of ultraviolet scanning to red shift. When the NaOH concentration continued to increase, the structure of the complex formed was destroyed, and λmax gradually decreased. Figure 1 It can be seen that in the experimental determination of the present invention, as the NaOH concentration increases, BCP does not undergo a red shift, so BCP does not have a triple helical structure; 4. Prepare 0.01 mg / mL BCP solution, use a microplate reader, set the scanning band to 230-600 nm to scan the BCP solution to determine the purity of BCP. Figure 2 From the content, we can see that BCP has absorption peaks at 260nm and 280nm, indicating that BCP contains nucleic acids and proteins.

[0017] 5. The infrared spectrum of BCP is mainly used to analyze the functional groups of BCP. 1 mg of dry BCP and 100 mg of potassium bromide (KBr) are mixed in a mortar, ground and pressed into tablets. After tableting, the tablets are scanned using an infrared spectrum scanner with a scanning range of 400-4000 cm -1 ,according to Figure 3 The content shows that at 3408 cm -1 There is a broad and strong -OH stretching vibration band at 2935 cm -1 The absorption peak at 1615 cm is caused by CH stretching vibration.-1 and 1411 cm -1 There are absorption peaks at 1369 cm-1, which are the stretching vibrations of carboxyl carbonyl and CO, indicating that BCP contains uronic acid. -1 The absorption peak at 1151 cm is attributed to the CH bending vibration. -1 ,1081 cm -1 and 1025 cm -1 The absorption peak at 846 cm-1 is caused by COC, indicating that BCP contains a pyranose ring. -1 It is the absorption peak of α-glycosidic bond.

[0018] 6. Take a small amount of dry BCP powder and evenly apply it on the conductive rubber plate fixed with double-sided tape. Blow off the excess BCP powder floating on the surface and then spray gold. Use a scanning electron microscope to observe and photograph the morphology of BCP at different magnifications. Figure 4 From the content, we can see that BCP is layered and flaky, with an uneven surface and full of cracks.

[0019] 7. DPPH free radical scavenging ability determination: Use anhydrous ethanol to prepare a concentration of 2×10 -4 mol / L DPPH solution, VC and BCP solutions with concentrations of 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL, and 800μg / mL were prepared. The experimental group (different concentrations of BCP or VC solution + DPPH solution), the experimental control group (different concentrations of VC or BCP solution + distilled water), and the blank control group (DPPH solution + distilled water) were kept in the dark for 30 min, and the absorbance was measured at a wavelength of 517nm using an enzyme marker to calculate the clearance rates of BCP and VC. Figure 5 It can be seen that the DPPH free radical scavenging ability of BCP solution gradually increases with the increase of concentration, while the DPPH free radical scavenging ability of VC solution first increases and then decreases with the increase of concentration. Therefore, BCP has a certain DPPH free radical scavenging ability.

[0020] 8. Determination of hydroxyl radical scavenging ability: prepare 6mmol / L salicylic acid solution, FeSO4 solution and hydrogen peroxide (H2O2) solution, as well as VC and BCP solutions with concentrations of 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 400μg / mL and 800μg / mL. The experimental group (different concentrations of BCP or VC solution + FeSO4 + salicylic acid solution), the experimental control group (different concentrations of BCP or VC solution + FeSO4 + distilled water), and the blank control group (distilled water + FeSO4 + salicylic acid solution) were reacted at 37℃ in the dark for 10min, then 6mmol / L H2O2 solution was added and mixed quickly. The mixture was reacted at 37℃ in the dark for 30min. The absorbance was measured at 510nm using an enzyme reader. Figure 6 It can be seen that the ability of BCP solution to inhibit hydroxyl free radicals first increases and then decreases with the increase of concentration, while the ability of VC solution to inhibit hydroxyl free radicals increases with the increase of concentration. Therefore, BCP has a certain ability to scavenge hydroxyl free radicals.

[0021] 9. Total antioxidant capacity determination: Prepare VC and BCP solutions with concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL, and use the total antioxidant capacity determination kit to determine the total antioxidant capacity of BCP. Figure 7 It can be seen from the content that the total antioxidant capacity of BCP solution increases with the increase of concentration, and the total antioxidant capacity of VC solution increases with the increase of concentration. Therefore, BCP has a certain total antioxidant capacity.

[0022] D-gal is an aldohexose, a reducing sugar naturally present in the human body, and is also present in many foods. When its content is high, it will be metabolized in the body to produce ROS. ROS are associated with oxidative stress, inflammation, mitochondrial dysfunction and apoptosis. During the D-gal-induced brain aging process, advanced glycation end products are formed and oxidative stress is caused, leading to increased malondialdehyde levels and decreased superoxide dismutase and glutathione peroxidase activities. The present invention has demonstrated the protection of BCP on aging HT22 cells by constructing an aging HT22 cell model with D-gal, and the specific process is as follows: Cell culture: Mouse hippocampal neuronal cells HT22 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% double antibodies (penicillin and streptomycin) at 37°C in a 5% CO2 incubator.

[0023] CCK8 method to measure cell proliferation: HT22 cells were seeded in 96-well plates, and the CCK8 method was used to detect the effect of BCP on the proliferation rate of HT22 cells. The specific experiment set up a control group and a BCP experimental group. The concentrations of BCP in the BCP experimental group were 25µg / mL, 50µg / mL, 100µg / mL, 200µg / mL, 400µg / mL, and 800µg / mL, respectively. The control group and the BCP experimental group were treated with HT22 cells for 24 hours, and the cell proliferation rate was detected by CCK8 method. Figure 8 It can be seen that as the BCP concentration increases, the cell proliferation rate increases, and the difference is significant.

[0024] Construction of D-gal-induced HT22 senescence model: CCK8 method was used to screen the optimal concentration and action time of D-gal to induce HT22 cell senescence. The control group and D-gal group were established in the experiment. The concentrations of D-gal added to the D-gal group were 25mM, 50mM, 100mM, 200mM, and 400mM, respectively. The control group and D-gal group were treated with HT22 cells for 12h, 24h, 48h, 72h, and 96h, respectively. Fig. 9 It can be seen that compared with the control group, the cell proliferation rate of HT22 decreased with the increase of D-gal concentration, and the difference was significant. Further calculation of the cell half lethality (IC50) showed that 50mM, 48h ( Fig. 9 D) or 100 mM, 24 h ( Fig. 9 Therefore, the concentration of 50 mM and treatment for 48 h were selected as the conditions for the D-gal-induced HT22 senescent cell model.

[0025] BCP protects HT22 senescent cell model: A control group, a 100mM D-gal group, and BCP groups with concentrations of 25µg / mL, 50µg / mL, 100µg / mL, 200µg / mL, 400µg / mL, and 800µg / mL were set up. After pre-treating cells with different concentrations of BCP for 24h, the culture medium was discarded, and 50mM D-gal was added to the BCP group and D-gal group, respectively. After continuous culture for 48h, the cell viability was determined by CCK8 method. Fig.10 It can be seen that compared with the control group, the proliferation rate of HT22 cells in the D-gal group was decreased (P<0.05); compared with the D-gal group, the proliferation rates of cells in the BCP group at 25μg / ml, 50μg / ml, 100μg / ml, and 800μg / ml were not statistically significant (P>0.05), while at concentrations of 200μg / ml and 400μg / ml, the proliferation rate of HT22 cells induced by D-gal was increased (P<0.01), indicating that BCP pre-protection has an effect on anti-aging of HT22 cells.

[0026] Reactive oxygen detection: control group, D-gal group, BCP group and VC group were set up. All groups were cultured in the same environment, and the cells were covered with 10umol / L DCFH-DA fluorescent probe, incubated in a 37℃, 5% CO2 incubator for 20min, washed three times with serum-free cell culture medium, and observed and counted under a fluorescence microscope. Fig.11 It can be seen that compared with the control group, the number of ROS-stained cells in the D-gal group increased significantly; compared with the D-gal group, the number of positively stained cells in the BCP group and VC group decreased significantly, scale: 200 μm, so it can be seen that D-gal 50mM induced senescence of HT22 cells, and BCP can reduce D-gal stimulation of HT22 cells to reduce the production of ROS.

[0027] After the preferred embodiments are described in detail, those familiar with the technology can clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above patent application, and any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention. The present invention is not limited to the example implementation methods given in the specification.

Claims

1. Application of Bupleurum chinense polysaccharide in drugs for preventing brain aging.

2. The use according to claim 1, characterized in that: The Bupleurum chinense polysaccharide comprises rhamnose, arabinose, galactose, glucose and galacturonic acid.

3. The use according to claim 1, characterized in that: The Bupleurum chinense polysaccharide is extracted by water extraction and alcohol precipitation, and then the extracted Bupleurum chinense polysaccharide is purified by repeated freezing and thawing, sevage method and running water dialysis.

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