Application of pitaya branch polysaccharide in preventing and treating cognitive dysfunction

Abstract

The present invention relates to the application of pitaya branch polysaccharides in preventing and treating cognitive dysfunction diseases, and relates to the field of pharmaceutical technology. The pitaya branch polysaccharide is an extract obtained from pitaya branches by water extraction, and has the significant effects of inhibiting the expression of β-amyloid protein (β-amyloid, Aβ), improving the cognitive function of AD model rats, and alleviating hippocampal nerve damage.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to application of pitaya branch polysaccharide in preventing and treating cognitive dysfunction diseases. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder characterized by decreased cognitive function, reduced functional ability, and behavioral and mental abnormalities. It is the most common type of dementia in the elderly. With the increasing aging of society, the prevalence of AD has increased significantly: currently, approximately 5.8 million people aged 65 and over in the United States have AD, and this number is expected to rise to 13.8 million by 2050. AD not only reduces the quality of life of patients but also places a heavy burden on society and families. According to surveys, the total cost of Alzheimer's disease in the United States was approximately $244 billion in 2019 and is expected to rise to $305 billion in 2020.

[0003] The exact pathogenesis of AD remains largely undetermined, leading researchers to propose various hypotheses. The amyloid cascade hypothesis holds sway. Because the hallmark pathological feature of AD is the extracellular deposition of senile plaques (SP) within the brain, the primary component of which is β-amyloid (Aβ). Consequently, most researchers believe that overexpression of Aβ and its aggregation, forming senile plaques, is central to AD pathology, triggering a series of other pathological processes that, in turn, accelerate Aβ accumulation, creating a vicious cycle and exacerbating AD progression. Therefore, inhibiting Aβ aggregation is a crucial strategy for the prevention and treatment of AD, offering promising therapeutic approaches. However, with approximately 9.5 million dementia patients in my country, the country has the highest number of dementia cases worldwide. AD has become a serious medical and social problem in my country, yet there are currently no effective products or methods for its prevention and treatment. Summary of the Invention

[0004] In response to the above problems, the present invention provides a use of pitaya branch polysaccharide in the prevention and treatment of cognitive dysfunction diseases. The pitaya branch polysaccharide has significant effects of inhibiting the expression of β-amyloid protein (β-amyloid, Aβ), improving the cognitive function of AD model rats, and alleviating hippocampal nerve damage.

[0005] In order to achieve the above object, the present invention provides a use of pitaya branch polysaccharide in preventing and treating cognitive dysfunction diseases. The pitaya branch polysaccharide is an extract obtained from pitaya branches by water extraction.

[0006] During their research, the inventors discovered that polysaccharides extracted from pitaya branches, obtained through a water extraction method, exhibited significant Aβ-inhibitory effects and improved cognitive function in Alzheimer's disease (AD) model animals. Therefore, pitaya branch polysaccharides have great potential for development as a drug for the prevention and treatment of AD. Using β-amyloid (Aβ) transgenic Caenorhabditis elegans CL4176 as a model, the inventors tested the effects of pitaya branch polysaccharides on Aβ-induced toxicity. The results showed that pitaya branch polysaccharides at concentrations of 0.016 mg / ml and 0.08 mg / ml effectively delayed the paralysis phenotype induced by Aβ toxicity. Furthermore, the inventors evaluated the effects of pitaya branch polysaccharides on cognitive function in rats induced by D-galactose (D-gal) and aluminum chloride (AlCl3) in a model of Alzheimer's disease, characterized by memory and learning impairment, using the Morris water maze. Brain biomarkers and histopathological changes were also analyzed. Results showed that pitaya branch polysaccharides significantly reduced the escape latency of rats with dementia in a water maze during acquisition training and increased the number of platform crossings in a spatial exploration test. Pitaya branch polysaccharides not only effectively reduced neuronal Aβ expression in the hippocampus of rats with dementia but also protected against morphological changes in the hippocampus induced by D-gal and AlCl₃ toxicity. This suggests that pitaya branch polysaccharides may be used to prevent and treat cognitive impairment.

[0007] The present invention also provides a use of pitaya branch polysaccharide in preparing a product for preventing and treating cognitive dysfunction diseases. The pitaya branch polysaccharide is an extract obtained from pitaya branches by water extraction; the product includes a medicine.

[0008] In one embodiment, the cognitive dysfunction disease includes Alzheimer's disease.

[0009] In one embodiment, the product includes pharmaceutically acceptable excipients and the pitaya branch polysaccharide; the dosage form of the product is an injection preparation or an oral preparation.

[0010] In one embodiment, the oral preparation includes tablets, capsules, powders, pills, and oral liquid preparations.

[0011] In one embodiment, the application includes oral administration or injection of the product, and the dosage of the pitaya branch polysaccharide is 1-1000 mg per kilogram of body weight.

[0012] The present invention also provides a product for preventing and treating cognitive dysfunction diseases. The product is a medicine, which includes pharmaceutically acceptable excipients and pitaya branch polysaccharides. The pitaya branch polysaccharides are extracts obtained from pitaya branches by water extraction.

[0013] In one embodiment, the excipient comprises at least one of the following raw materials: an excipient, a lubricant, an antioxidant, a preservative, a binder, a filler, or a thickener.

[0014] In one embodiment, the product can prevent and treat cognitive dysfunction by inhibiting the expression level of β-amyloid protein.

[0015] In one embodiment, the product can prevent and treat cognitive dysfunction by reversing the irregular arrangement of pyramidal cells in the hippocampal CA1 region.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The application of the dragon fruit branch polysaccharide of the present invention in preventing and treating cognitive dysfunction diseases, the polysaccharide is an extract obtained by water extraction of the dragon fruit branch, without any toxic and side effects, can be used safely for a long time, the pharmacological action of the dragon fruit branch polysaccharide is obvious, with the significant effect of inhibiting beta-amyloid protein expression, improving AD model rat cognitive function, and alleviating hippocampal nerve damage, and the raw material of the dragon fruit branch polysaccharide is easy to obtain, cheap, can be obtained by a simple preparation method, less process, production efficiency is high, safe to use, convenient, can form a variety of dosage forms, alleviate the related symptoms of cognitive dysfunction diseases, reduce patient pain, significant efficacy, low cost. The application of the dragon fruit branch polysaccharide in the field of preventing and treating cognitive dysfunction diseases has opened up a new application field for the plant of dragon fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the timeline of processing and behavioral testing in Example 3;

[0019] Figure 2 The graph shows the average escape latency results during the training phase in Example 3, where n=10, #p<0.05, ##p<0.01 (compared with the control group); *p<0.05, **p<0.01 (compared with the AD model group); the model group is the AD model group, the low-dose group is the low-dose group of pitaya branch polysaccharide, and the high-dose group is the high-dose group of pitaya branch polysaccharide;

[0020] Figure 3 The swimming trajectory results of representative individual rats in the space exploration test in Example 3, wherein n=10, A to D represent the control group, AD model group, pitaya branch polysaccharide low-dose group, and pitaya branch polysaccharide high-dose group respectively;

[0021] Figure 4This figure shows the results of the number of platform positions crossed in the space exploration test in Example 3, where n=10, #p<0.05, ##p<0.01 (compared with the control group); *p<0.05, **p<0.01 (compared with the AD model group); from left to right: control group, AD model group, low-dose pitaya branch polysaccharide group, and high-dose pitaya branch polysaccharide group;

[0022] Figure 5 The figure shows the results of the immunoblotting experiment in Example 3, from left to right: the control group, the AD model group, the low-dose pitaya branch polysaccharide group, and the high-dose pitaya branch polysaccharide group;

[0023] Figure 6 The results of Example 3 show the effect of pitaya branch polysaccharide on Aß expression in the hippocampus of AD rats, where #p<0.05, ##p<0.01 (compared with the control group); *p<0.05, **p<0.01 (compared with the AD model group); from left to right are the control group, AD model group, pitaya branch polysaccharide low-dose group, and pitaya branch polysaccharide high-dose group

[0024] Figure 7 This is the hematoxylin and eosin staining result of the rat hippocampal CA1 region histopathological analysis (H&E, x400), where A~D represent the control group, AD model group, pitaya branch polysaccharide low-dose group, and pitaya branch polysaccharide high-dose group, respectively. DETAILED DESCRIPTION

[0025] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; and the experimental methods, unless otherwise specified, are all conventional experimental methods in the art.

[0028] Example 1

[0029] A pitaya branch polysaccharide and an extraction method thereof.

[0030] The method for extracting dragon fruit branch polysaccharide comprises the following steps:

[0031] 1. Slice the dragon fruit branches and dry them under reduced pressure until the quality is constant.

[0032] 2. Soak and extract with petroleum ether and 95% by volume ethanol for 4 hours to remove fat-soluble components such as pigments and small molecular components, and evaporate the solvent to obtain a pretreated sample.

[0033] 3. Accurately weigh the pretreated sample, add water at a solid-liquid ratio of 1:20 (g / mL), place it in a pressure cooker extraction device, and extract at 121°C for 30 minutes.

[0034] 4. Concentrate the supernatant to 1 / 5 of the original volume, add 5 times the volume of anhydrous ethanol, precipitate for 24 hours, and centrifuge to obtain the precipitate.

[0035] 5. Dissolve the precipitate with an appropriate amount of deionized water to remove insoluble protein, dialyze with deionized water to remove small molecular impurities, and freeze-dry to obtain dragon fruit branch polysaccharide.

[0036] Example 2

[0037] The feasibility of pitaya branch polysaccharides in preventing and treating AD was evaluated using the nematode AD model.

[0038] The specific experimental work of this example was carried out by Shanghai Model Organisms Center, Inc., and the specific experimental methods and results are shown below.

[0039] 1. Nematodes: The nematode strain CL4176 (Bristol strain) used in this example was purchased from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, USA. It is an Aβ model nematode. The following information is provided:

[0040]

[0041] 2. Sample information: The freeze-dried pitaya branch polysaccharide powder used in this example is the pitaya branch polysaccharide prepared in Example 1, which is water-soluble.

[0042] 3. Nematode culture conditions: Nematodes were cultured on NGM (nematode growth medium) solid culture medium with Escherichia coli OP50 at a temperature of 16°C and a humidity of 40-60%.

[0043] 4. Plate preparation:

[0044] Pitaya branch polysaccharides were diluted with ddH2O to the corresponding experimental concentrations (0.0032 mg / ml, 0.016 mg / ml, and 0.08 mg / ml, respectively). Mixed with 1 ml of E. coli OP50 pellet, 200 μL was spread on the surface of NGM solid medium (3 cm Petri dish) and allowed to dry before use in experiments. Caffeine was used as a positive control at a concentration of 6.27 mM.

[0045] 5. After synchronizing the CL4176 strain on the control plate and the drug plate, culture at 16°C for 48 hours, then induce at 23°C, and begin counting after 38 hours.

[0046] 6. Statistical Methods: The Log-rank (Mantel-Cox) test was used to compare the differences in paralysis degree. GraphPad.Prism.v5.0 was used for data analysis. p A value <0.05 was considered statistically significant.

[0047] 7. Experimental results: Pitaya branch polysaccharide at concentrations of 0.016 mg / ml and 0.08 mg / ml can inhibit nematode paralysis. After culturing nematodes with 0.0032 mg / ml of pitaya branch polysaccharide, there is no significant effect on the level of nematode paralysis, as shown in the following table.

[0048] Table 1 Effects of pitaya branch polysaccharides on the onset of paralysis in nematode CL4176

[0049]

[0050] 8. Result analysis:

[0051] The pitaya branch polysaccharide of Example 1 significantly prolonged the onset time of nematode paralysis caused by Aβ toxicity by inhibiting the expression of β-amyloid (Aβ) protein in AD nematodes.

[0052] Example 3

[0053] 1. Animals.

[0054] Female SD rats weighing 170–200 g were purchased from the Guangdong Medical Experimental Animal Center, and feed was purchased from the Experimental Animal Center of Guangdong Medical University.

[0055] 2. Grouping and processing.

[0056] According to the method reported in the prior art, an AD rat model was established by injecting D-gal and AlCl3. The rats were randomly divided into a control group (n=10) and an AD group (rats treated with D-gal and AlCl3) (n=30). The control group was intraperitoneally injected with normal saline (0.9% NaCl) (0.2 ml / rat) every day, and the AD group was intraperitoneally injected with D-gal (60 mg / kg / day) and AlCl3 (10 mg / kg / day) every day for 7 weeks. The AD group was further divided into 3 groups (n=10), namely the AD model group, the low-dose pitaya branch polysaccharide group, and the high-dose pitaya branch polysaccharide group, with 10 rats in each group. The control group and the AD model group were gavaged with tap water every day, while the low-dose pitaya branch polysaccharide group and the high-dose pitaya branch polysaccharide group were gavaged with pitaya branch polysaccharide (20 mg / kg and 60 mg / kg) every day. The drug administration and modeling were synchronized and lasted for 49 days. The specific process and subsequent experiments are as follows. Figure 1 shown.

[0057] Control group: intraperitoneal injection of normal saline, gavage with tap water; AD model group: intraperitoneal injection of D-gal and AlCl3, gavage with tap water; low-dose pitaya branch polysaccharide group: intraperitoneal injection of D-gal and AlCl3, gavage with pitaya branch polysaccharide (20 mg / kg); high-dose pitaya branch polysaccharide group: intraperitoneal injection of D-gal and AlCl3, gavage with pitaya branch polysaccharide (60 mg / kg).

[0058] 3. Morris water maze (MWM) experiment.

[0059] The MWM experimental apparatus consisted of a circular tank (120 cm diameter, 80 cm height) filled with warm water (23 ± 1°C) to a depth of 40 cm. The water was rendered opaque using a non-toxic white dye. The tank was divided into four quadrants (NE, SE, SW, and NW) by four points equally spaced along the tank's circumference. An invisible platform (12 cm diameter) was fixed at the center of the SW-III quadrant (the target quadrant) and submerged 2 cm below the water surface. Animal behavior was recorded with a camera suspended above the tank and analyzed using a video computational tracking system (Institute of Medical Sciences, Chinese Academy of Medical Sciences).

[0060] On the first day of the seventh week, prior to testing, rats were trained to locate and climb onto a submerged platform in response to various environmental cues. During the acquisition phase (days 2–5 of the seventh week), rats were tested four times daily, using each of the four starting positions (NE-I, NW-II, SW-III, and SE-IV) once, with the platform always located in the same quadrant (SW-III). Rats were placed headfirst into the pool and given 60 seconds to search for the hidden platform. The time it took to reach the platform was recorded as the escape latency. If the platform could not be found after 60 seconds, the rat was guided to the platform and remained there for 10 seconds. On the sixth day of the seventh week, a spatial exploration test was conducted. The platform was removed and the rat was placed in a quadrant other than the platform. The number of times the rat crossed the original platform position within 60 seconds was recorded.

[0061] 4. Specimen collection.

[0062] Twenty-four hours after the MWM experiment, all rats were anesthetized with pentobarbital (30 mg / kg), and the entire brain, excluding the cerebellum, was removed and washed in 0.9% NaCl. Three brains from each group were fixed in 10% formalin and embedded in paraffin for pathological examination. The hippocampal regions of seven additional brains from each group were obtained and stored at −80°C for immunoblotting analysis of biomarkers.

[0063] 5. Immunoblotting analysis.

[0064] Hippocampal tissue was lysed on ice using RIPA buffer containing 1 mM PMSF. Protein was extracted using a protein extraction kit. Concentrations were determined using a BCA protein assay kit. SDS-PAGE was performed, the membrane was transferred, and the membrane was blocked with skim milk for 2 hours. Anti-Aβ antibody (1:1000; Abcam) was added and incubated overnight at 4°C. The membrane was washed and a secondary antibody was added, incubated for 2 hours at room temperature. After washing, the membrane was exposed and developed, and target protein expression levels were analyzed using Quantity One software.

[0065] 6. Hematoxylin and eosin staining.

[0066] Paraffin-embedded specimens were serially sectioned at 4 μm thickness, dewaxed, and stained with hematoxylin and eosin (H&E).

[0067] 7. Statistical processing.

[0068] SPSS 12.0 software system was used for statistical analysis, and the experimental data were Multiple group comparisons were performed using one-way analysis of variance. Further pairwise comparisons between groups were performed using the LSD test. p A difference of <0.05 was considered statistically significant.

[0069] 8. Experimental results and analysis.

[0070] During the acquisition training phase, the escape latency of the AD model group was significantly prolonged during the 4-day training compared with the control group ( p <0.01), indicating that the learning ability of AD model rats was severely impaired, suggesting that AD modeling was successful. Compared with the AD model group, from the first day of acquisition training, both the low-dose and high-dose groups of pitaya branch polysaccharides significantly shortened the escape latency of AD rats ( p <0.01), suggesting that oral administration of pitaya branch polysaccharide can alleviate the learning disability of rats caused by D-gal and AlCl3 treatment. Figure 2 shown.

[0071] The results of space exploration experiments are as follows Figure 3 、 Figure 4 As shown in the figure, it is consistent with the results of the above-mentioned acquisition training. The number of times the rats in the AD model group crossed the original platform position was significantly lower than that in the control group ( p <0.01), while intervention with pitaya branch polysaccharide significantly increased the number of times AD rats crossed the original platform location ( p <0.01), indicating that oral administration of pitaya branch polysaccharide improved the ability of AD rats to maintain the spatial location memory of the platform.

[0072] The results of immunoblotting experiments were as follows Figure 5 、 Figure 6 As shown, the results showed that both low-dose and high-dose pitaya branch polysaccharides could significantly downregulate the expression of Aβ protein in the rat hippocampus ( p <0.01)

[0073] The results of hematoxylin and eosin staining experiments were as follows Figure 7 Results showed that compared with the control group, pyramidal cells in the CA1 region of the hippocampus in the AD model group were loosely and disorganized. Treatment with pitaya branch polysaccharides improved the irregular arrangement of pyramidal cells in the CA1 region of AD rats. The combined use of D-gal and AlCl₃ altered the arrangement of pyramidal cells in the CA1 region of the rat hippocampus, while oral administration of pitaya branch polysaccharides effectively reversed these changes.

[0074] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An application of pitaya branch polysaccharide in the preparation of a medicament for preventing and treating Alzheimer's disease, characterized in that: The pitaya branch polysaccharide is an extract obtained from the pitaya branch through a water extraction method.

2. The use according to claim 1, characterized in that The medicine further comprises pharmaceutically acceptable excipients; the dosage form of the medicine is an injection preparation or an oral preparation.

3. The use according to claim 2, characterized in that The auxiliary material includes at least one of the following raw materials: a lubricant, an antioxidant, a preservative, an adhesive, a filler, or a thickener.

4. The use according to claim 2, characterized in that The oral preparations include tablets, capsules, powders, pills, and oral liquid preparations.

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