CN103800348B - Application of mannose glucuronic acid oligosaccharide in preparation of medicine and/or healthcare product for treating or preventing Parkinson's disease and/or senile dementia - Google Patents

Abstract

The invention relates to the application of mannoglucuronide oligosaccharides in the preparation of medicines and health care products for the prevention and patent of senile dementia and Parkinson's disease. Mannoglucuronide oligosaccharides are mannoglucuronide oligosaccharides formed by alternate linkages of 2-linked mannose and 4-linked glucuronic acid or their salts, which have significant neuroprotective effects on Alzheimer's and Parkinson's diseases It can be used to prepare medicines and health products for preventing and treating neurodegenerative diseases.

Description

Application of mannoglucuronic acid oligosaccharides in preparation of medicines and/or health products for treating or preventing Parkinson's disease and/or senile dementia

technical field

The invention belongs to the field of biomedicine and relates to the application of mannose glucuronide oligosaccharides in treating Parkinson's disease and senile dementia.

Background technique

With the average life expectancy of human beings increasing year by year, the world is entering the era of global aging. Age-related neurodegenerative diseases have also increased. In the central nervous system, the main manifestations are weakened excitation and inhibition processes, decreased brain function, memory loss and loss, followed by progressive decline in recognition function and aggravation of emotional disorders wait. Parkinson's disease, also known as Parkinson's paralysis, is a common degenerative disease of the central nervous system in middle-aged and elderly people. The main pathological feature of Parkinson's disease is that after pathological changes occur in cells located in the "substantia nigra" of the midbrain, the release of dopamine Synthesis decreases, the function of inhibiting acetylcholine decreases, and the excitatory effect of acetylcholine is relatively enhanced. The result of the imbalance between the two is "paralysis agitans". The latest data show that there are 1.7 million Parkinson's disease patients in China, among which there is one Parkinson's disease in every 100 people over the age of 55, and it is estimated that there are about 100,000 new Parkinson's patients every year. Parkinson's disease has brought enormous pressure to patients, families and society, which has attracted a lot of attention from all over the world. The research on therapeutic drugs for Parkinson's disease has become an important topic in the field of geriatric medicine.

Alzheimer's Disease (Alzheimer's Disease) is the so-called senile dementia, also known as Alzheimer's disease. It is a fatal neurodegenerative disease with progressive development. The clinical manifestations are deteriorating cognitive and memory functions, progressive decline in daily life ability, and various neuropsychiatric symptoms and behavioral disturbances. According to the survey results released by the China Alzheimer's Association in 2011, there are about 36.5 million people suffering from dementia in the world, and one person suffers from dementia every 7 seconds. The average survival period is only 5.9 years, which is a threat to the health of the elderly. One of the "Four Big Killers".

In recent years, considering its efficacy and safety, the development trend of carbohydrate drugs has gradually shifted from the research on polysaccharides to the research on oligosaccharides and oligosaccharides. Oligosaccharides have a huge demand space in the healthcare field. Oligosaccharide drugs have uniform structure, controllable quality, and clear mechanism of action, and have become one of the main directions of sugar drug development. Ocean University of China has reported that oligomannuronic acid HS971 with a carboxyl group at the reducing end can significantly improve memory and resist senile dementia, and is intended to be used clinically for the prevention and treatment of senile dementia.

This patent reports that mannoglucuronide oligosaccharides composed of mannose and glucuronic acid have significant therapeutic effects on Parkinson's disease and Alzheimer's disease in cell and animal experiments, and can be applied to the treatment of Parkinson's disease and Alzheimer's disease prevention and treatment.

Contents of the invention

Application of the mannose glucuronic acid oligosaccharide in the preparation of drugs for preventing and treating Parkinson's disease or senile dementia and health care products. Described mannoglucuronic acid oligosaccharide has the following characteristics:

(1) Constituent sugars: mannose and glucuronic acid or their salts;

(2) 2-linked mannose and 4-linked glucuronic acid or its salts are alternately linked;

(3) Its structural formula is the following general formula (I) or (II)

In the formula, R is H or Na, K and other inorganic salts, n is an integer between 0-8

In the formula, R is H or Na, K and other inorganic salts, n is an integer between 0-8

(II)

The medicine is a pharmaceutical composition containing mannoglucuronide oligosaccharides and pharmaceutically acceptable carriers and/or excipients.

Carriers include sodium alginate microspheres, liposomes, and the like.

Excipients: such as mannitol, magnesium stearate, starch, cyclodextrin, etc.

The dosage form of the medicine is injection, oral preparation or local administration preparation.

The present invention has the following advantages:

The mannose glucuronide oligosaccharide has preventive and therapeutic effects on Parkinson's disease and senile dementia, has little toxic and side effects, is safe and effective, and can be used to prepare medicines and health care products for treating Parkinson's disease and senile dementia.

Description of drawings

The gel chromatogram of alcohol eluent in the preparation process of Fig. 1 oligosaccharide;

Figure 2 ESI-MS and HPLC images of mannoglucuronide oligosaccharides G1-G4; a is G4; b is G3; c is G2; d is G1;

The hydrogen spectrum of Figure 3G2;

The carbon spectrum of Figure 4G2;

The two-dimensional HMBC spectrum of Figure 5G2;

The two-dimensional HSQC spectrum of Figure 6G2;

The two-dimensional TOCSY spectrum of Figure 7G2;

The hydrogen spectrum of Figure 8G3;

Carbon spectrum of Figure 9G3;

The hydrogen spectrum of Figure 10G4;

Carbon spectrum of Figure 11G4;

Figure 12 Mannoglucuronide oligosaccharide G2 is on the influence of the number of mistakes and the latency of the dark avoidance test in dementia mice;

Figure 13 Mannoglucuronide oligosaccharide G2 is on the influence of the number of mistakes and the latency of the dementia mouse water maze avoidance test;

Figure 14 The effect of mannoglucuronide oligosaccharide G2 on the activity of dementia mouse hippocampal acetylcholine transferase;

Fig. 15 Effect of mannoglucuronide oligosaccharide G2 on the activity of acetylcholinesterase in the hippocampus of dementia mice.

Detailed ways

The present invention is specifically described below with examples, but the present invention is not limited to the scope of the following examples.

The preparation of embodiment 1 mannoglucuronic acid oligosaccharides

Extract the dried kelp with 30 times the mass of distilled water at 100°C for 3 hours, filter the extract, concentrate, add ethanol to a final concentration of 75% to precipitate, let it stand for 12 hours, collect the precipitate, sink to the point of vacuum drying to obtain laminarin . Dissolve the laminarin sample in a sulfuric acid solution with a mass concentration of 4% (the ratio of solid to liquid is 60mg/mL), heat and reflux for 5 hours, neutralize it with barium hydroxide to PH=6-7, centrifuge, and concentrate the supernatant to the original One-fifth of the volume, the concentrated solution is subjected to activated carbon column chromatography, firstly equilibrated with distilled water, and then eluted with a gradient of 50%-90% ethanol, and the 50%-90% ethanol eluate is concentrated to one-fifth of the original volume 1. Ethanol was evaporated, directly applied to Bio-gel P4 column chromatography, and six components were separated (Figure 1), and the samples collected above were analyzed by ESI-MS and HPLC (Figure 2). NMR (Figure 3-11) results further confirmed the structure of oligosaccharides. The results showed that G1-G4 were mannoglucuronic acid octasaccharide, hexasaccharide, tetrasaccharide and disaccharide, respectively. Its structure conforms to the structural formula shown in the figure below:

Where G1 is mannoglucuronic acid octaose, where n=3

G2 is mannoglucuronic acid hexasaccharide, where n=2

G3 is mannoglucuronide tetrasaccharide, where n=1

G4 is mannoglucuronide disaccharide, where n=0

Example 2: Protective effect of mannoglucuronide oligosaccharides on MPP ⁺ damaged nerve cells

The neurotoxin MPTP is widely used in the research of Parkinson's disease (PD). The MPTP entering the human body is converted into MPP ⁺ by monoamine oxidase B, which inhibits the NADH CoQ reductase (complex I) of the mitochondrial electron transport chain, thereby making the intracellular ATP decreases, oxygen free radicals are formed, apoptosis is initiated, and eventually cell death occurs. PC12 cells derived from rat adrenal medullary pheochromocytoma are widely used in in vitro studies of nervous system diseases, and are widely used in the study of Parkinson's disease. Since there is no monoamine oxidase in the cultured cell line, MPP ⁺ is added directly to simulate the cell model of Parkinson's disease caused by MPTP. The present invention studies the protective effect of mannuronic acid oligosaccharides on nerve cell PC12 cells damaged by MPP ⁺ by taking the cell survival rate as an index, so as to explore the possibility of treating Parkinson's disease with mannuronic acid oligosaccharides.

Materials: Human neuroblastoma cell line PC12 was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM high-glucose medium (Invitrogen) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin was used to culture in an incubator at 37°C with 5% CO ₂ . The mannuronic acid oligosaccharides used the samples prepared in Example 1.

Method: The experiment was divided into control group, MPP ⁺ injury group, each oligosaccharide sample on normal cell treatment group and each oligosaccharide sample on MPP ⁺ injury PC12 cell group, and the concentration of oligosaccharide sample treatment was 10, 100 and 1000 μg/ml respectively. , 8 wells per group, incubated at 37°C in an incubator containing 5% CO ₂ for 24h and 48h, then replaced 180μL of fresh culture medium, added 20mL of MTT (5mg/mL, Sigma), and added 200μL of dimethyl sulfoxide (DMSO) after 4h , Sigma), shaken for 10 minutes, and measured the optical density (OD) at 550 nm with a microplate reader (Diagnostic Pasteur LP400).

Table 1 shows the results of the protective effect of each oligosaccharide on MPP+ injured nerve cells. The above MTT assay results indicated that the damage rate of MPP+ to cells was (49.50±5.48)%. The four mannoglucuronide oligosaccharides tested have obvious repairing effects on nerve cell damage induced by MPP+, cell viability has been significantly improved, and the system has a good neuroprotective effect. PC12 cells are DA-ergic neuroblastoma cell lines, which can well represent various physiological characteristics of DA-ergic neurons in the substantia nigra region of the midbrain, so they are widely used in the research of Parkinson's disease. Each mannuronic acid oligosaccharide can protect PC12 cells from MPP ⁺ damage, indicating that it has a protective effect on nerve cells and can be used for the prevention and treatment of Parkinson's disease.

Table 1 Neuroprotective effect of mannoglucuronide oligosaccharides on MPP ⁺ injured PC12 cells

Note: The damage rate of MPP ⁺ to PC cells was (49.50±5.48)%

Example 3 Protective effect of mannoglucuronide oligosaccharides on neuroblastoma line SH-SY5Y damaged by β-amyloid peptide

Materials: SH-SY5Y cell line; Aβ _25-35 fragments were synthesized by Capital Medical University and purified by high performance liquid chromatography with a purity of over 98%. Serum-free MEM solution was prepared, filtered through a 0.22 μm filter membrane, and stored at -20°C. The mannuronic acid oligosaccharides used the samples prepared in Example 1.

Test method: Cell culture conditions are MEM (Gibco BRL, 41500-067) medium with 10% fetal bovine serum (Hyclone, SH30070.03), 15mmol.L ^-1 HEPES (DNN company), penicillin 1x10 ⁵ IU.L ^-1 , Streptomycin 1x10 ⁵ IU.L ^-1 , 5% CO ₂ , 37°C, change the medium twice a week, pass passage once.

Measurement of MTT metabolic rate: SY5Y cells were inoculated in 96-well plates (Costar products) at a density of 1× ¹⁰³ cells, and divided into control group, Aβ _25-35 group and Aβ _25-35 +P2 group (10, 40, 80 μg/ml), Aβ _25-35 + each oligosaccharide (10, 100, 1000 μg/ml) drug group, 8 wells in each group. 24 hours after inoculation, add 20 μl of MTT (5 mg/ml) to each well, incubate at 37°C for 4 hours, aspirate the culture medium, add 200 μl of DMSO to each well, shake for 10 minutes, and measure the optical density at 550 nm with a microplate reader (Diagnostic Pasteur LP400) (OD).

Results: As shown in Table 1, mannuronic acid oligosaccharides G1-G4 can significantly resist the toxicity of 20 μM Aβ _25-35 at three concentrations, increase the MTT metabolic rate of cultured cells, and its activity gradually increases with the increase of sample concentration , the results showed that mannuronic acid oligosaccharides can protect nerve cells SY5Y against the toxicity of Aβ _25-35 , and have a significant neuroprotective effect. Nerve cell apoptosis plays a very important role in nerve cell injury, and drugs can prevent its further development by intervening in the process of apoptosis and protect nerve cells. SH-SY5Y cells are derived from human neuroblastoma. Their shape and physiological function are similar to normal neurons. They are cone-shaped and have obvious axons. It is a kind of cell that is well researched on the function of nerve cells in the world. Mannuronate oligosaccharides have neuroprotective effect on SY5Y cells, indicating that mannuronate oligosaccharides can be used in the treatment of nervous system diseases such as Parkinson's disease and senile dementia.

Table 2 Neuroprotective effect of mannoglucuronide oligosaccharides on Aβ-injured SH-SY5Y cells

Note: The damage rate of Aβ to cells is (62.45±6.58)%

Example 4: The effect of mannuronic acid oligosaccharide G2 on senile dementia

Experimental animals: Kunming mice. Male KM mice with an average body weight of about 22 g were given free access to food and water for one week before the experiment to adapt to the laboratory environment.

Material: mannoglucuronide oligosaccharide G2, prepared in Example 1.

Aβ _1-40 was purchased from Sigma Company, dissolved in sterile normal saline to a concentration of 1mg/ml, sealed and placed in a 37°C incubator for 7 days to agglomerate.

Test method: After one week of adaptation, the mice were weighed and randomly divided into 5 groups, namely, the control group, the model group, and the low, medium and high levels of mannoglucuronide oligosaccharide G2 (25, 50, 100 mg/kg). Dosage group, positive control drug Donapezil hydrochloride (2.5mg/kg) group. Except for the control group, mice were injected with Aβ _1-40 unilaterally into the ventricle. According to the Maurice method, after anesthetized with chloral hydrate, the right ventricle was positioned (A: -2mm; L: 2mm; H: -4mm), the needle was inserted 3mm vertically into the skull, and 3μl/piece of Aβ _1-40 was slowly injected, and the control group equal volume of sterile saline.

The animals were given corresponding drugs on the second day after operation, the dosage was 0.5ml/20g, and the control group and model group were fed with the same amount of normal saline, once a day, continuously until the end of the experiment.

(1) Dark avoidance test for mice

The dark avoidance test was carried out on the 9th day after Aβ _1-40 modeling. The dark avoidance device is divided into two compartments, light (25×25×16cm) and dark (24×20×15cm), which are connected by an arch (8.9×11.4cm). The door can be closed or closed by the experimenter as needed. Open. The floor of the compartment is a stainless steel fence (3.175mm). The fence floor on one side of the dark room is connected to the stimulator and can give electric shocks, while the side of the bright room is not connected to the stimulator, so no current passes through it. Put the mice into the light chamber, and open the door between the light and dark chambers after 10 s. After the mice (limbs) completely entered the dark room, the middle door was closed immediately, and an electric shock was given (the current was 0.6 mA). Let the mouse stay in the dark room for 10s, and put the mouse back into the cage. After 24 hours of electrical stimulation training, the mice were put back into the bright room, the middle door was opened, and the number of times the mice entered the dark room and the latency period within 5 min were recorded.

(2) Mouse maze experiment

The water maze test was carried out on the 12th day after Aβ _1-40 modeling, and lasted for five days. The maze has 5 blind ends. On the first day of the experiment, a black plastic plate was used to divide the maze into two parts, and the mice were allowed to swim in the part containing only one blind end; The section containing three blind ends was swam; the plastic plate was removed from the third to fifth day of the experiment and the mice were allowed to swim the entire course. The latency to find the platform and the number of errors reaching the blind end were recorded respectively.

(3) Determination of cortical acetylcholinesterase (AChE) and acetylcholine transferase (ChAT) activities

After the behavioral test, the mice were decapitated to take the brains, and the hippocampal tissue was quickly peeled off in an ice bath, and 10% tissue homogenate was made with pre-cooled physiological saline. According to the instructions of the kit, the activities of acetylcholinesterase and acetylcholinetransferase in the tissue were determined.

result

Mouse dark avoidance test

1) As can be seen from Figure 12, compared with the control group, the incubation period of the mice in the Aβ model group was significantly shortened, and the number of errors was significantly increased (p<0.05). The latency and error times of the three dose groups of mannoglucuronide oligosaccharide G2 were significantly improved compared with the Aβ model group, and its effect was comparable to that of the positive control drug donepezil hydrochloride.

2) Mouse maze test

It can be seen from Figure 13 that compared with the control group, the latency period for the mice to find the platform and the number of errors through the blind end were significantly prolonged in the model group (p<0.05), indicating that the dementia model was established in mice induced by unilateral intracerebroventricular injection of Aβ _1-40 success. With the extension of training time, the latency and the number of mistakes of the mice were gradually shortened. From the third day, compared with the control group and the three dose groups of mannoglucuronide oligosaccharide G2 in the Aβ _1-40 unilateral intracerebroventricular injection model group, the number of mistakes in the mice increased significantly, among which mannoglucuronide oligosaccharide G2 Compared with the model group, the incubation period of mice in the high concentration group was significantly shortened. On the fourth day of training, the incubation period of the control group and the medium and high dose groups of mannoglucuronide oligosaccharide G2 was significantly shortened compared with the model group, while the positive control drug, donepezil hydrochloride, had no effect, indicating that G2 had a rapid effect. On the fifth day of training, compared with the model group in the three dose groups of mannoglucuronide oligosaccharide G2 and the donepezil hydrochloride group, the latency of the mice was significantly shortened and the number of mistakes was significantly reduced.

3) Hippocampal acetylcholinesterase and acetylcholine transferase activity

Acetylcholinesterase and acetylcholinetransferase are decomposing enzymes and synthetizing enzymes of acetylcholine, respectively, and both jointly regulate the content of acetylcholine in the brain. As shown in Figure 14, in the hippocampal tissue, the activity of acetylcholine transferase in the group of donepezil hydrochloride and the three dosage groups of mannoglucuronide oligosaccharide G2 was higher than that of the Aβ _1-40 unilateral intracerebroventricular injection model group, But not significant. As shown in Figure 15, the acetylcholinesterase activity of the control group, the donepezil hydrochloride group and the three dose groups of mannoglucuronide oligosaccharide G2 was significantly lower than that of the model group, and it is worth noting that the acetylcholinesterase activity of the first two groups The activity was about 50% lower than that of the control group, indicating that the middle dose of mannoglucuronide oligosaccharide G2 and donepezil hydrochloride could significantly inhibit the decomposition of acetylcholine in the hippocampus, thereby increasing the content of acetylcholine in the hippocampus. This may be one of the ways that mannoglucuronide oligosaccharide G2 improves AD symptoms caused by unilateral intracerebroventricular injection of Aβ _1-40 .

Conclusion: Mannoglucuronide oligosaccharide G2 can improve the behavioral indicators of mice with Aβ _1-40 damage, improve the memory of mice, and reduce the number of mistakes. Increase the activity of acetylcholine transferase and reduce the activity of acetylcholinesterase. It has significant anti-senile dementia effect.

The specific examples of the present invention have been described above, but they are not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein that do not depart from the scope and spirit of the present invention.

Claims (4)

1. the application in the medicine and/or health product treating and/or preventing parkinson disease and/or senile dementia prepared by manna glucuronic acid oligosaccharide; Described its structural formula of manna glucuronic acid oligosaccharide is one in following general expression (I) or (II) or two kinds;

In formula, R is one or two or more kinds in H, Na or K, and n is the integer between 0-8

In formula, R is one or two or more kinds in H, Na or K, and n is the integer between 0-8

（II）。

2. application according to claim 1, is characterized in that:

Described manna glucuronic acid oligosaccharide has following feature:

(1) composition sugar: mannose and glucuronic acid or its salt;

(2) glucuronic acid that the mannose that 2-connects is connected with 4-or its salt alternately connect.

3. application according to claim 1, is characterized in that: described medicine is the pharmaceutical composition comprising mannoglucan aldehydic acid oligosaccharide and other pharmaceutically acceptable carrier and/or excipient.

4. application according to claim 1, is characterized in that: the dosage form of described medicine is injection, oral formulations or local administration preparation.