A composition for preventing Parkinson's disease and its application

Abstract

The present invention relates to the field of nutritional supplements, and discloses a composition for preventing Parkinson's disease and its application, wherein the composition comprises 30 to 44 parts of probiotics, 5 to 7 parts of plant extracts, and 0 to 1 part of nutritional supplements. The probiotics include Lactobacillus helveticus R0052 and Bifidobacterium longum R0175, and the total viable bacterial concentration of the probiotics is not less than 8×10 8 CFU / g. The plant extracts include cranberry extract, ashwagandha extract, and saffron extract. The anthocyanin content of the cranberry extract is 30-50 mg / g, and the proanthocyanidin content is 23-35 mg / g. The composition for preventing Parkinson's disease of the present invention can improve the ability of the intestine to resist oxidative stress, improve anxiety, depression and sleep conditions in the prodromal stage of PD, alleviate constipation and anosmia symptoms in the prodromal stage of PD, and can prevent and delay the continuous development of symptoms in the prodromal stage before Parkinson's disease is diagnosed.

Description

Technical Field

[0001] The invention relates to a composition for preventing Parkinson's disease and application thereof. Background Art

[0002] Parkinson's disease (PD) has the characteristics of insidious onset and slow progression. Studies have reported that the prevalence of PD increases with age, but only 4% of PD patients are diagnosed before the age of 50. PD patients usually have motor symptoms such as resting tremor, muscle rigidity, posture changes and decreased spontaneous movement as the main clinical features. Currently, the clinical diagnosis of PD mainly relies on these motor symptoms. However, when the above motor symptoms appear, it also indicates that the disease has entered an irreversible clinical stage.

[0003] PD is a progressive disease, and current treatments can only improve the patient's clinical symptoms. PD can be divided into the following four stages: (1) Risk stage. Before neurodegenerative changes occur, PD genetic and environmental risk factors are present. (2) Preclinical stage. It is characterized by the occurrence of progressive neurodegenerative pathological changes, but the lack of any related symptoms and signs. (3) Prodromal stage. Observable symptoms or signs of neurodegeneration, i.e. non-motor symptoms, appear. Prodromal markers include rapid behavioral disorder (RBD), anosmia, autonomic dysfunction, depression, anxiety, mild motor signs, and imaging markers of the presynaptic dopaminergic system and cardiac sympathetic nervous system. Among them, depression, anxiety, constipation, hyposmia and sleep disorders have a relatively high proportion. (4) Clinical stage. Motor symptoms appear and meet the clinical diagnostic criteria for PD. Prior art reports that healthy intestinal microbiota has significant antioxidant and anti-inflammatory effects, while intestinal flora imbalance can manifest as low-grade inflammation, cell degeneration, and cellular energy imbalance, followed by increased oxidative stress. Oxidative stress may activate enteric neurons and glial cells, leading to the accumulation and misfolding of α-SYN in the ENS. α-SYN accumulation in the intestine can be transferred to the brain, leading to microglial activation, resulting in increased oxidative stress, thereby exacerbating neuroinflammation.

[0004] The accumulation of reactive oxygen species (ROS) is considered a major threat to physical health, and their activation disrupts the body's redox balance. The production of ROS exacerbates the misfolding of α-synuclein (α-SYN), leading to PD and other synaptic protein diseases. The endogenous antioxidant system includes superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), etc., which can maintain the body's oxidative balance, remove free radicals and other substances that cause oxidative stress, and protect cells. When this balance mechanism is disrupted, oxidative stress occurs, causing various neurodegenerative diseases. When SOD activity is disrupted, the pathogenesis associated with Parkinson's disease (PD) and oxidative stress (OS) is activated. α-SYN accumulation in the intestine can be transferred to the brain, leading to microglial activation, resulting in increased oxidative stress, thereby exacerbating neuroinflammation and exacerbating AD symptoms.

[0005] Based on the above research on the mechanism of the efficacy of probiotics on Parkinson's disease, the prior art also proposes a composition for preventing and treating Parkinson's disease and a preparation method thereof, wherein blueberry extract is added to the culture medium, and then used for the first fermentation culture of Clostridium butyricum, Lactobacillus acidophilus, Lactobacillus fermentum and Bifidobacterium lactis JYBR-190, and then the enzymatic hydrolysate of cicada shell, cornus officinalis and antelope horn is added for the second fermentation culture, and the obtained probiotic fermentation Chinese medicine composition is evenly mixed with the antioxidant composition, prebiotics and short-chain fatty acids to obtain a composition for preventing and treating Parkinson's disease. However, it can be seen from Test Example 2 of the patent that it verifies the samples in the clinical stage with motor symptoms, and it cannot reflect the preventive effect before the clinical stage.

[0006] The prior art discloses a probiotic composition for treating rapid eye movement sleep behavior disorder in patients with Parkinson's disease, which is prepared from Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus, Enterococcus faecalis and pharmaceutically acceptable excipients. The number of viable Bacillus licheniformis is not less than 10.0×10 8 CFU / g; the number of viable Bifidobacterium longum is not less than 4.0×10 8 CFU / g; the number of viable Lactobacillus acidophilus should not be less than 1.0×10 8 CFU / g; the number of viable Enterococci is not less than 0.5×10 8 CFU / g.

[0007] However, the above-mentioned prior art does not disclose the technical solutions and technical inspirations for preventing prodromal patients. PD is a disease involving multiple systems. When neuronal loss reaches a certain level and reaches the clinical stage, typical motor symptoms may appear. The non-motor symptoms that appear in the early pathological stage provide a clinical theoretical basis for prodromal PD. Due to the lack of relevant markers for identifying individuals in the preclinical stage, the prodromal markers can be used to diagnose PD 10 to 20 years earlier. Therefore, intervention in prodromal patients with non-motor symptoms of PD may be the key to delaying or even blocking the onset of the disease. If the intervention is too late, the prevention effect cannot be achieved. Summary of the invention

[0008] In view of the fact that the prior art does not disclose intervention for non-motor symptoms in the prodromal stage of PD, the present invention provides a composition for preventing Parkinson's disease;

[0009] Another object of the present invention is to provide an application of the composition for preventing Parkinson's disease.

[0010] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0011] A composition for preventing Parkinson's disease is prepared from the following materials in parts by weight:

[0012] Probiotics 30~44 servings

[0013] 5~7 parts of plant extract

[0014] Nutritional supplements 0~1 portion

[0015] The probiotics include Lactobacillus helveticus R0052 and Bifidobacterium longum R0175; and the plant extract includes cranberry extract.

[0016] Furthermore, the total viable bacterial concentration of the probiotics is not less than 8×10 8 CFU / g.

[0017] Preferably, the total viable bacteria concentration of the probiotics is not less than 10 9 CFU / g.

[0018] The present invention innovatively uses Lactobacillus helveticus R0052 and Bifidobacterium longum R0175, providing a new technical solution for the application of probiotics in neurodegenerative diseases. The two probiotics act simultaneously to significantly inhibit the level of reactive oxygen species, thereby reducing the production of α-SYN aggregates, and increase the antioxidant capacity of the intestine to directly and indirectly inhibit oxidative stress, which can significantly alleviate the non-motor symptoms of patients in the prodromal stage of PD.

[0019] Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 are present in healthy intestines and benefit the human body through different mechanisms, including enhancing immunity, promoting nutrient absorption, and helping to maintain the microecological balance of the intestine. The inventor unexpectedly discovered that when these two probiotics act at the same time, they can significantly inhibit the level of reactive oxygen species (ROS). Reactive oxygen is a molecule produced during cell metabolism. It is at low levels under normal circumstances, but if it is produced too much, it will damage cells and tissues and cause oxidative stress. Oxidative stress is considered an important factor in neurodegenerative diseases such as Parkinson's disease.

[0020] By suppressing the level of reactive oxygen species, these two probiotics can reduce the production of α-SYN aggregates. α-SYN is a protein that often forms aggregates in the midbrain dopaminergic neurons of Parkinson's patients, which is a key step in the pathological process of Parkinson's disease. Reducing the production of α-SYN aggregates may help slow the progression of Parkinson's disease.

[0021] In addition, these two probiotics can also increase the antioxidant capacity of the intestine. The intestine is the largest immune organ in the human body and the main habitat of probiotics. Increasing the antioxidant capacity of the intestine can not only directly inhibit oxidative stress, but also indirectly affect the health of the whole body by improving the intestinal microecology.

[0022] These two probiotics also showed significant alleviating effects on non-motor symptoms in patients in the prodromal stage of Parkinson's disease. The prodromal stage of Parkinson's disease refers to a stage in which obvious motor symptoms have not yet appeared, but there are already some non-motor symptoms (such as hyposmia, sleep disorders, emotional problems, etc.). Patients at this stage are usually not easy to diagnose. Therefore, intervention through non-drug means such as probiotics may help delay the progression of the disease and improve the quality of life of patients. Furthermore, the extraction process of the cranberry extract includes the following steps:

[0023] (1) Select cranberries in the red fruit stage, freeze-dry and then crush;

[0024] (2) adding an extracting solution prepared by mixing ethanol and acetic acid, wherein the volume concentration of ethanol in the extracting solution is 50-60%, the volume concentration of acetic acid is 0.3-0.8%, and the ratio of cranberry to the extracting solution is 1:8-15 kg / L;

[0025] (3) Adjust the pH to 1-3;

[0026] (4) extracting by ultrahigh pressure assisted method to obtain an extract;

[0027] (5) The extract is dried at a temperature not exceeding 40° C. to obtain cranberry extract.

[0028] Preferably, in step (2), the volume concentration of ethanol is 52-58%, the volume concentration of acetic acid is 0.4-0.6%, and the ratio of cranberry to extract is 1:10-13 kg / L.

[0029] Preferably, steps (2) to (5) are performed under light-proof conditions.

[0030] Furthermore, the conditions of the ultrahigh pressure assisted method are: temperature 20~35°C, pressure 250~400 MPa, and extraction time 2~8min.

[0031] Preferably, the conditions of the ultrahigh pressure assisted method are: temperature 25-30° C., pressure 300-380 MPa, and extraction time 3-5 min.

[0032] Furthermore, the cranberry extract includes the following components and contents: anthocyanin content is 30-50 mg / g, proanthocyanidin content is 23-35 mg / g;

[0033] Preferably, the content of quercetin in the cranberry extract is 8-10 mg / g, and the content of resveratrol is 0.7-1 mg / g.

[0034] The present invention creatively adds cranberry extract to relieve non-motor symptoms in the prodromal stage of Parkinson's disease. Cranberry extract is rich in anthocyanins, proanthocyanidins, quercetin and other substances. In the research of the present invention, cranberry extract can increase the activity of intestinal GSH and SOD and significantly improve the ability to resist oxidative stress.

[0035] Cranberries are rich in antioxidants and anti-inflammatory ingredients, which fight free radical activity and reduce oxidative damage, thereby protecting brain cells from damage. In addition, the anti-inflammatory ingredients in cranberries can also reduce pain and muscle inflammation, thereby improving muscle stiffness and tremors in Parkinson's patients.

[0036] Cranberry extract can increase the activity of intestinal GSH (glutathione) and SOD (superoxide dismutase). GSH and SOD are important antioxidant enzymes in cells, which can remove reactive oxygen species (ROS) and free radicals and maintain the redox balance of cells. Increasing the activity of these two enzymes in the present invention enhances the ability of cells to resist oxidative stress.

[0037] Parkinson's disease is a neurodegenerative disease closely related to oxidative stress. In the prodromal stage of Parkinson's disease, patients may experience some non-motor symptoms, which may be related to oxidative stress and imbalance of intestinal microbiota. Therefore, the present invention increases the ability to resist oxidative stress by increasing the activity of intestinal GSH and SOD through cranberry extract, and effectively alleviates the non-motor symptoms of Parkinson's disease in the prodromal stage.

[0038] Furthermore, the plant extract also includes ashwagandha extract and saffron extract;

[0039] In the ashwagandha extract, the content of withanolide is 50~80 mg / g; in the saffron extract, the content of safranal is 2~5 mg / g, and the content of crocetin is 0.4~0.8 mg / g.

[0040] Preferably, the content of withanolide in the ashwagandha extract is 60-70 mg / g.

[0041] The present invention innovatively uses saffron aldehyde to prevent and alleviate Parkinson's disease. The abnormal aggregation of α-synuclein protein is a core link in the pathogenesis of Parkinson's disease. In the saffron extract of the present invention, saffron aldehyde binds to the hydrophobic residues of α-synuclein protein, causing the formation of β-folded structure to be delayed, effectively inhibiting the aggregation of α-synuclein.

[0042] Crocetin also has the effect of resisting abnormal aggregation of α-synuclein protein, further enhancing the effect of saffron extract in preventing and alleviating Parkinson's disease.

[0043] The antioxidant effect of the ashwagandha extract innovatively used in the present invention helps maintain the healthy state of brain cells, thereby alleviating the symptoms of Parkinson's disease. The ashwagandha extract is rich in withanolides. When the pathological changes caused by chronic stress in PD are caused by oxidative stress, the oxygen free radical scavenging enzymes and lipid peroxidation in the anterior cortex and striatum of the brain are disordered, and withanolides play an antioxidant role. Withanolides can scavenge free radicals and reduce the damage of oxidative stress to cells. Furthermore, the mass ratio of cranberry extract, ashwagandha extract, and saffron extract in the plant extract is (100~160): (80~120): (5~7).

[0044] Preferably, the mass ratio of cranberry extract, ashwagandha extract and saffron extract in the plant extract is (120-140): (90-110): (5-7).

[0045] Furthermore, the nutritional supplements include one or more of dopamine, γ-aminobutyric acid, melatonin, vitamins, zinc, and magnesium.

[0046] Furthermore, the vitamins include one or more of vitamin B1, B2, B6, B12, vitamin C, and folic acid.

[0047] Preferably, the prevention of Parkinson's disease includes alleviating non-motor symptoms in the prodromal stage of Parkinson's disease.

[0048] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0049] (1) Improve the ability of the intestine to resist oxidative stress. The experiment showed that after the MPTP-induced PD model mice were gavaged with the composition of the present invention, the ROS level in the colon tissue decreased by 27.00%, the antioxidant GSH increased by 98.90%, and the SOD increased by 86.27%, and the oxidative stress level in the intestine decreased.

[0050] (2) Improve negative emotions and sleep conditions in the prodromal stage of PD. According to the survey results, after taking the Parkinson's disease prevention composition of the present invention for 8 months, the anxiety and depression of the test subjects were significantly relieved, the sleep quality was improved, and the sleepiness was improved. The NMSS score was reduced by 9.18%, the HAMA score was reduced by 20.97%, the HAMD score was reduced by 19.82%, the PSQI score was reduced by 11.13%, and the ESS score was reduced by 18.59%.

[0051] (3) Alleviate constipation and hyposmia symptoms in the prodromal stage of PD. According to the survey results, after taking the Parkinson's disease prevention composition of the present invention for 8 months, the proportion of constipation in the test subjects decreased by 36.25%, and 48 test subjects had constipation, and 60.42% of them had significant improvement in constipation. The proportion of hyposmia in the test subjects decreased by 30.00%, and 45 test subjects had hyposmia, and 53.33% of them had significant improvement in hyposmia.

[0052] (4) The composition for preventing Parkinson's disease of the present invention improves the ability of the intestine to resist oxidative stress, relieves non-motor symptoms in the prodromal stage of PD, and can play a significant role in preventing and delaying the continuous development of symptoms in the prodromal stage before Parkinson's disease is diagnosed. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0054] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0055] Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 in the embodiments of the present invention are products of Canadian Lallemand Healthcare Technology Co., Ltd. Example 1

[0056] A composition for preventing Parkinson's disease is prepared from the following materials in parts by weight:

[0057] Probiotics 30 servings

[0058] 5 parts of plant extract

[0059] Nutritional supplements 0.8 parts

[0060] Probiotics include Lactobacillus helveticus R0052 and Bifidobacterium longum R0175, and the total live bacterial concentration of probiotics is 10 9 CFU / g;

[0061] The plant extracts include cranberry extract, ashwagandha extract and saffron extract, with a mass ratio of 130:100:6.

[0062] The content of anthocyanins in cranberry extract is 50 mg / g, the content of proanthocyanidins is 30 mg / g, the content of quercetin is 10 mg / g, and the content of resveratrol is 1 mg / g.

[0063] In the ashwagandha extract, the content of withanolide is 75 mg / g;

[0064] In saffron extract, the content of safranal is 5 mg / g and the content of crocetin is 0.7 mg / g.

[0065] Nutritional supplements include dopamine, γ-aminobutyric acid, melatonin, vitamin C, zinc and magnesium.

[0066] After uniformly mixing the above components at the above weight fractions, the mixture is packaged into 3.5 g portions to obtain a composition for preventing Parkinson's disease.

[0067] The extraction process of cranberry extract includes the following steps:

[0068] (1) Select cranberries in the red fruit stage, freeze-dry and then crush;

[0069] (2) adding an extracting solution composed of ethanol and acetic acid, wherein the volume concentrations of ethanol and acetic acid in the extracting solution are 56% and 0.7%, respectively, and the ratio of cranberry to the extracting solution is 1:12 kg / L;

[0070] (3) Adjust the pH to 2;

[0071] (4) Extraction was performed by ultrahigh pressure assisted extraction at 28 °C, 365 MPa, and extraction time of 3 min.

[0072] (5) The extract liquid is converted into powder by low-temperature spray drying at 40°C to obtain cranberry extract.

[0073] Steps (2) to (5) are carried out in a light-proof condition. Example 2

[0074] A composition for preventing Parkinson's disease is prepared from the following materials in parts by weight:

[0075] Probiotics 44 servings

[0076] 7 parts of plant extract

[0077] Nutritional supplements 0

[0078] Probiotics include Lactobacillus helveticus R0052 and Bifidobacterium longum R0175, and the total live bacterial concentration of probiotics is 8×10 8 CFU / g;

[0079] The plant extracts include cranberry extract, ashwagandha extract and saffron extract, and the mass ratio thereof is 160:120:7.

[0080] The content of anthocyanins in cranberry extract is 30 mg / g, the content of proanthocyanidins is 23 mg / g, the content of quercetin is 8 mg / g, and the content of resveratrol is 0.7 mg / g.

[0081] In the ashwagandha extract, the content of withanolide is 50 mg / g;

[0082] In saffron extract, the content of safranal is 3 mg / g, and the content of crocetin is 0.4 mg / g.

[0083] After uniformly mixing the above components at the above weight fractions, the mixture is packaged into 3.5 g portions to obtain a composition for preventing Parkinson's disease.

[0084] The extraction process of cranberry extract includes the following steps:

[0085] (1) Select cranberries in the red fruit stage, freeze-dry and then crush;

[0086] (2) adding an extracting solution composed of ethanol and acetic acid, wherein the volume concentrations of ethanol and acetic acid in the extracting solution are 50% and 0.8%, respectively, and the ratio of cranberry to the extracting solution is 1:15 kg / L;

[0087] (3) Adjust the pH to 1;

[0088] (4) Extraction was performed by ultrahigh pressure assisted extraction at 20 °C, 250 MPa, and 8 min.

[0089] (5) The extract liquid is converted into powder by low-temperature spray drying at 40°C to obtain cranberry extract.

[0090] Steps (2) to (5) are carried out in a light-proof condition. Example 3

[0091] A composition for preventing Parkinson's disease is prepared from the following materials in parts by weight:

[0092] Probiotics 32 servings

[0093] 6 parts of plant extract

[0094] Nutritional supplement 1 serving

[0095] Probiotics include Lactobacillus helveticus R0052 and Bifidobacterium longum R0175, and the total live bacterial concentration of probiotics is 2×10 9 CFU / g;

[0096] The plant extracts include cranberry extract, ashwagandha extract and saffron extract, and the mass ratio thereof is 100:80:5.

[0097] The content of anthocyanins in cranberry extract is 50 mg / g, the content of proanthocyanidins is 35 mg / g, the content of quercetin is 10 mg / g, and the content of resveratrol is 1 mg / g.

[0098] The content of withanolide in the ashwagandha extract is 80 mg / g;

[0099] In saffron extract, the content of safranal is 2 mg / g, and the content of crocetin is 0.8 mg / g.

[0100] Nutritional supplements include dopamine, gamma-aminobutyric acid, melatonin, vitamin B12, zinc and magnesium.

[0101] After uniformly mixing the above components at the above weight fractions, the mixture is packaged into 3.5 g portions to obtain a composition for preventing Parkinson's disease.

[0102] The extraction process of cranberry extract includes the following steps:

[0103] (1) Select cranberries in the red fruit stage, freeze-dry and then crush;

[0104] (2) adding an extracting solution composed of ethanol and acetic acid, wherein the volume concentrations of ethanol and acetic acid in the extracting solution are 60% and 0.3%, respectively, and the ratio of cranberry to the extracting solution is 1:8 kg / L;

[0105] (3) Adjust the pH to 3;

[0106] (4) Extraction was performed by ultrahigh pressure assisted extraction at a temperature of 35 °C and a pressure of 400 MPa for 2 min.

[0107] (5) The extract liquid is converted into powder by low-temperature spray drying at 40°C to obtain cranberry extract.

[0108] Steps (2) to (5) are carried out in a light-proof condition. Example 4

[0109] The technical solution of Example 4 is similar to that of Example 1, except that the plant extract added in Example 4 only includes cranberry extract.

[0110] Comparative Examples 1 to 10

[0111] The technical solutions of Comparative Examples 1 to 10 are similar to those of Example 1, with the specific differences being as follows:

[0112] Table 1 Technical solutions of comparative examples 1 to 10

[0113] Comparative Example 11

[0114] The technical scheme of Comparative Example 11 is similar to that of Example 1, except that the total viable bacterial concentration of probiotics is 5×10 8 CFU / g. Comparative Example 12

[0115] The technical solution of Comparative Example 12 is similar to that of Example 1, except that the content of anthocyanins in the cranberry extract is 25 mg / g, and the content of proanthocyanidins is 20 mg / g. Comparative Example 13

[0116] The technical scheme of Comparative Example 13 is similar to that of Example 1, except that, in the Ashwagandha extract, the content of withanolide is 40 mg / g; in the saffron extract, the content of safranal is 1 mg / g, and the content of crocetin is 0.3 mg / g.

[0117] Comparative Examples 14-18

[0118] The technical solutions of Comparative Examples 14 to 18 are similar to those of Example 1, except that the contents of the components of the cranberry extracts are different. The cranberry extracts are prepared by the following different preparation schemes:

[0119] Table 2 Technical solutions of comparative examples 14 to 18

[0120]

[0121] Detection Methods

[0122] 1. Determination of anthocyanin content

[0123] The total content of anthocyanins was determined by the pH differential method. Take 2 portions of 0.4 mL of cranberry extract sample, dilute to 10 mL with pH 1.0 potassium chloride and pH 4.5 sodium acetate buffer solutions, respectively, and place for 30 min. Measure the absorbance at 520 nm and 700 nm, respectively, and calculate the anthocyanin content (expressed as cyanidin-3-glucoside) according to the following formula.

[0124] Total anthocyanin content (mg / 100 g) = A×449.2×10×100×V×100 / (26900×100×m×L)

[0125] A: Absorbance A = (A520-A700) pH1.0-(A520-A700) pH4.5;

[0126] V: total volume of the extract, mL;

[0127] m: sampling volume, g;

[0128] L: cuvette optical path, 1 cm:

[0129] 26900: molar extinction coefficient of cyanidin-3-glucoside, L / (mol·cm);

[0130] 449.2: Molecular weight of cyanidin-3-glucoside, g / mol.

[0131] 2. Determination of proanthocyanidin content

[0132] Using catechin as the standard, a linear regression equation was established, and the vanillin-concentrated hydrochloric acid method was used to determine the absorbance of the sample at 500 nm, and the content of proanthocyanidins was converted according to the standard curve. Take 1 mL of cranberry extract sample, add 6 mL of 8% vanillin-methanol solution in a 10 mL colorimetric tube, and then add 3 mL of concentrated hydrochloric acid. After mixing evenly, react in a 30°C water bath in the dark for 40 min, and measure the absorbance of the reaction solution at 500 nm.

[0133] Test Results

[0134] According to Table 3, from Comparative Example 14, when the concentration of the extract is too high, the content of proanthocyanidins is significantly reduced, and the content of anthocyanins is also reduced to a certain extent. As the concentration of the extract increases, the water content decreases, the solvent cannot enter the cell membrane, and the target substance cannot be effectively leached out; if the concentration of the extract is too high, the polarity of the solvent will continue to increase, aggravating the dissolution of alcohol-soluble impurities and reducing the solubility of anthocyanins and proanthocyanidins. When the solid-liquid ratio is within an appropriate range, the concentration difference between the extracted substance and the extractant generates mass transfer power to promote the dissolution of anthocyanins. When the solid-liquid ratio is too large, the mass transfer power basically reaches a balance, the increase in the amount of anthocyanin extraction is not significant, and raw materials will be wasted.

[0135] From Comparative Example 15, when the extraction time is 9 min, the anthocyanin content and proanthocyanidin content are significantly reduced. This is because when the pressure rises to a certain level, the pressure is released instantly to rupture the cells, and the contents are more easily dissolved in the solvent, and the ideal extraction effect can be achieved in 2 to 8 min. As the pressure holding time increases, anthocyanins may degrade under high pressure or bind to proteins to reduce the extraction rate. In addition, as the extraction time increases, anthocyanins are easily unstable due to exposure to light and aerobic environment, causing degradation or binding to denatured proteins. The structure of proanthocyanidins is unstable, and increasing the extraction time will cause some proanthocyanidin structures to be destroyed and decomposed.

[0136] According to Comparative Example 16, the anthocyanin content is significantly reduced because the structure of anthocyanin is unstable in neutral and alkaline environments. As the pH increases from acidic, the anthocyanin structure is converted from 2-phenylbenzopyran to chalcone, and the color gradually disappears from red to colorless. The higher the pH value, the faster the anthocyanin degradation rate. When the pH is not greater than 3, the antioxidant activity of anthocyanin is also more stable, while the antioxidant activity and stability of the extract are significantly reduced with the increase of pH.

[0137] From Comparative Example 17, the presence of pressure accelerates the dynamic equilibrium of the dissolution process, and the pressure difference between the inside and outside of the cell accelerates the leaching of functional substances in the cell, so that the effective ingredients are quickly dissolved and the dissolution rate is increased. However, excessive pressure degrades anthocyanins and proanthocyanidins into other small molecules or denatures proteins in cranberries, producing more sites that can bind to anthocyanins or proanthocyanidins, thereby reducing the extraction amount of anthocyanins and proanthocyanidins.

[0138] According to Comparative Example 18, the extraction temperature is too high, resulting in the degradation of anthocyanins, the anthocyanin content and the antioxidant capacity gradually decrease, and the total reducing capacity also decreases to varying degrees. The higher the temperature and the longer the extraction time, the greater the decrease. In addition, proanthocyanidins are easily oxidized substances with unstable structures. When the optimal temperature is exceeded, the structure of proanthocyanidins will be increasingly damaged. The higher the degree of oxidation, the more damage to the effective ingredients.

[0139] In addition, cranberry extract should be prepared or stored away from light. Under light conditions, the content of anthocyanins and proanthocyanidins decreases faster and more significantly. Under light-proof conditions, cranberry extract has higher antioxidant stability.

[0140] Table 3 Anthocyanin and proanthocyanidin content

[0141] Anthocyanin content (mg / g) Proanthocyanidin content (mg / g) Comparative Example 14 28.36 20.57 Comparative Example 15 26.35 21.23 Comparative Example 16 25.21 22.48 Comparative Example 17 28.94 21.39 Comparative Example 18 25.21 23.26

[0142] 3. Determination of antioxidant levels in colon tissue of C57BL / 6J mice

[0143] Two-month-old C57BL / 6J mice were used as experimental animals. The mice were kept in a feeding box at a temperature of 21-25°C and a humidity of 50-60%. The light-dark alternation time was 12 h:12 h. They were free to eat and drink. The antioxidant content of mouse colon tissue was determined using a commercially available kit.

[0144] The study set up the following groups:

[0145] Blank group: 0.2 mL of normal saline was gavaged daily and normal saline solution was injected intraperitoneally for 14 days;

[0146] Model group: mice were gavaged with 0.2 mL of saline daily, intraperitoneally injected with saline for 3 days, and then intraperitoneally injected with saline containing 30 mg / kg MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) starting on the 4th day for 5 consecutive days. Finally, mice were intraperitoneally injected with saline for 6 days.

[0147] Experimental group: 60 mg / kg of the composition of Examples 1 to 3 or Comparative Examples 1 to 12 dissolved in 0.2 mL of normal saline was gavaged daily, and normal saline was intraperitoneally injected for 3 days. On the 4th day, 30 mg / kg of MPTP was intraperitoneally injected with normal saline, and the injection was continued for 5 days. Finally, the mice were given intraperitoneal injection of normal saline for 6 days (experimental groups A1 to A4 were gavaged with the composition of Examples 1 to 4, respectively, and experimental groups B1 to B13 were gavaged with the composition of Comparative Examples 1 to 13, respectively).

[0148] (1) Reactive oxygen species (ROS) detection

[0149] After homogenization of mouse colon tissue, centrifuge at 12000r / min at 4℃ for 10min in precooled phosphate buffered saline, and the supernatant is used for ROS determination. According to the instructions of the kit, the oxidation-sensitive probe 2,7-dichlorofluorescein diacetate is used to evaluate the generation of ROS, and the absorbance value at 530nm is measured using a microplate reader.

[0150] (2) Glutathione (GSH) detection

[0151] According to the instructions of the kit, the required application solution was prepared, the mouse colon tissue was treated, 10% tissue homogenate was prepared, the precipitated cells were collected by low-speed centrifugation, 0.3~0.5 mL of isotonic PBS buffer was added to suspend the cells, and the cells were ultrasonically disrupted for testing. 0.1 mL of 10% tissue homogenate or the cell suspension after disruption was taken, 0.1 mL of reagent was added to mix, 3500r / min, centrifuged for 10min, and the supernatant was taken. The corresponding blank wells, standard wells and test wells were configured, mixed, and allowed to stand at room temperature for 5 min. The absorbance at 405 nm was measured using an ELISA reader for data analysis.

[0152] (3) Superoxide dismutase (SOD) activity detection

[0153] According to the instructions of the kit, the required application solution was prepared, 10 mg of colon tissue was weighed, 100 μL of sample preparation solution was added, and the mixture was homogenized on ice. The mixture was centrifuged at 12000 g for 5 min at 4°C, and the supernatant was taken as the sample to be tested. WST-8 working solution and reaction start working solution were prepared, sample wells and blank control wells were set, and the sample to be tested and other solutions were added respectively. After adding the reaction start working solution, the mixture was mixed, and the mixture was incubated in a 37°C incubator for 30 min. The absorbance at 450 nm was measured using an ELISA reader for data analysis.

[0154] Test Results

[0155] As shown in Table 4, compared with the blank group, the ROS content in the colon of the model group mice increased (p<0.05), and the activities of SOD and GSH were significantly reduced (p<0.05). After oral administration of the composition, the ROS content in the colon of the experimental group A1~A4 mice decreased (p<0.05), and the activities of SOD and GSH were significantly enhanced (p<0.05). Compared with the model group, the GSH of the experimental group A1~A4 increased by 98.90%, the SOD increased by 86.27%, and the ROS level decreased by 27.00%.

[0156] It can be seen from experimental groups B1 to B3 that when no probiotics were added or probiotics were used as a single component, the activities of SOD and GSH were significantly reduced compared with those of experimental groups A1 to A4, and the level of ROS was only 3.11 to 12.41% lower than that of the model group. It can be seen from experimental group B9 that when probiotics were replaced with animal Bifidobacterium, the results were roughly the same as those of experimental group B1 without the addition of probiotics, indicating that animal Bifidobacterium could not improve the antioxidant activity of mouse colon. It can be seen from experimental groups B4 to B6 that when plant extracts were not added or their components were reduced, the antioxidant activity of mouse colon was also significantly reduced, among which cranberry extract had a greater effect on antioxidant activity. It can be seen from experimental group B10 that when different plant extracts, such as lily extracts, were added, not only did the antioxidant activity not improve, but it also affected the probiotics, and the ROS level was only 4.03% lower than that of the model group. From experimental groups B7-B8, we can see that the extraction process also has a great impact on plant extracts, and their GSH and SOD activities are only increased by 5.10% and 1.34% at most compared with experimental group B6 without cranberry extract. From experimental groups B11-B13, we can see that the difference in the proportion of different components in the composition will also affect the antioxidant activity of mouse colon.

[0157] Table 4 Antioxidant detection results of colon tissue of mice in each group

[0158]

[0159] 4. Evaluation of non-motor symptoms during the prodromal period

[0160] Selection criteria for testers: A total of 220 subjects were selected, regardless of gender, aged between 35 and 65 years old, with an average age of 58 years old. The subjects had non-motor symptoms of Parkinson's disease, such as hyposmia, sleep disorders, constipation, anxiety and depression, cognitive dysfunction, autonomic dysfunction, and one or more other symptoms; and the modified Hoehn-Yahr stage of Parkinson's disease was less than stage 1, or did not meet the diagnosis conditions of Parkinson's disease, and stopped taking all drugs to improve the above symptoms one month before enrollment.

[0161] Experimental trial dosage and administration method: 300 cases were divided into 15 groups, each with 20 people, and they took the compositions of Examples 1 to 4, Comparative Examples 1 to 6, and Comparative Examples 9 to 13, 3.5 g per day, respectively.

[0162] Before taking the medicine and after taking it for 8 months, NMSS, HAMA, HAMD, PSQI, ESS scale evaluation, constipation and olfactory function diagnosis were performed.

[0163] Scale evaluation data

[0164] (1) Non-motor symptoms scale (NMSS): It is a commonly used scale for assessing non-motor symptoms in PD patients. It includes 30 questions with a total score of 30 points. The larger the weighted score, the more types of non-motor symptoms related to PD.

[0165] (2) Hamilton Anxiety Scale (HAMA): As one of the commonly used scales for assessing anxiety symptoms, its total score is 56 points. When the total score is ≥14 points, it is considered that anxiety exists; when the total score is ≥21 points, it is considered that there is obvious anxiety; when the total score is ≥29 points, it is considered that there is severe anxiety. The total score of the scale assessment is positively correlated with the severity of anxiety.

[0166] (3) Hamilton Depression Scale (HAMD): As one of the commonly used scales for assessing depressive symptoms, its total score is 76 points. When the total score is ≥20 points, it is considered to be depressed, and when the total score is ≥35 points, it is considered to be severe depression. The total score of the scale is positively correlated with the severity of depression.

[0167] (4) Pittsburgh Sleep Quality Index (PSQI); currently widely used in sleep assessment of PD patients, with a total score of 21 points. When the total score is ≥5 points, it is considered that sleep disorder exists. The total score is positively correlated with the degree of sleep disorder.

[0168] (5) The Epworth Sleeping Scale (ESS): It is often used to assess daytime sleepiness in PD patients. Its total score is 24 points. When the total score is greater than 6, it is considered to be sleepy, and when the total score is greater than 11, it is considered to be excessive sleepiness.

[0169] (6) The Rome III diagnostic criteria for functional constipation are used to assess the presence of constipation, and the odor test bottle is used to assist in assessing olfactory function.

[0170] Test Results

[0171] Before the typical motor symptoms of PD appear, that is, in the prodromal period, there are non-motor symptoms that are easily overlooked. When various non-motor symptoms are present, the risk of PD being subsequently diagnosed is higher. Anxiety, daytime sleepiness, loss of smell, bowel movement disorders, etc. can be considered as prodromal symptoms of PD and are associated with a significantly increased risk of developing Parkinson's disease.

[0172] Table 5 Statistics of scale evaluation scores

[0173]

[0174] According to Table 5, after taking the composition of Examples 1 to 4 for 8 months, the non-motor symptoms of the test subjects were alleviated, anxiety and depression were significantly relieved, sleep quality was improved, sleepiness was improved, NMSS score decreased by 9.18%, HAMA score decreased by 20.97%, HAMD score decreased by 19.82%, PSQI score decreased by 11.13%, and ESS score decreased by 18.59%.

[0175] As can be seen from Comparative Examples 1 to 3, probiotics have a greater effect on the non-motor symptoms of the testers. When probiotics are not added or a single component probiotic is added, the NMSS, HAMA, HAMD, PSQI and ESS scores are slightly improved. After the probiotic strains are replaced with animal Bifidobacterium in Comparative Example 9, the non-motor symptom scale score results of the testers are similar to or slightly improved with Comparative Examples 1 to 3, indicating that the addition of other strains has no effect on the non-motor symptoms of Parkinson's prodromal period. As can be seen from Comparative Examples 4 to 6, the non-motor symptoms of the testers were not significantly relieved by changing the components of the plant extracts. Although the scale scores were roughly the same as when they were not taken, they were lower than the scale scores of Example 4 in which only cranberry extracts were added, and the scale scores of Example 1 in which the complete plant extracts were added were significantly lower. As can be seen from Comparative Example 10, after the plant extracts were replaced with lily extracts, the tester's scale scores were roughly the same as those of Comparative Example 4 in which no plant extracts were added, indicating that the lily extract had no significant effect on the non-motor symptoms of the testers. It can be seen from Comparative Examples 11 to 13 that the mass fractions of different components and the content of each component have a certain impact on the improvement of the non-motor symptoms of the test subjects.

[0176] Table 6 Number of people with constipation and hyposmia

[0177]

[0178] According to the survey results, 178 people with constipation appeared in the testers, accounting for 59.33% of the total samples. According to the results of Table 6, in Examples 1 to 4, the proportion of constipation in the testers decreased by 36.25%, and 48 testers with constipation were significantly improved, accounting for 60.42%. According to Comparative Examples 1 to 3, without the addition of probiotics, the proportion of constipation in the testers increased slightly, and when only one probiotic was added, the proportion of constipation decreased by 3.33%. According to Comparative Example 9, when probiotics were replaced by animal bifidobacterium, the number of constipation remained unchanged, indicating that animal bifidobacterium had no effect on the constipation caused by the prodromal stage of Parkinson's. In Comparative Examples 4 to 6, the proportion of constipation decreased by 20%, combined with the data in Table 6, indicating that the full-component probiotics play a major role in improving constipation.

[0179] According to the survey results, 170 testers had hyposmia, accounting for 56.67% of the total samples. According to the results in Table 6, in Examples 1 to 4, the proportion of testers with hyposmia decreased by 30.00%, 45 testers had hyposmia, and 53.33% of the testers had significantly improved hyposmia. According to Comparative Examples 1 to 3 and 9, when the probiotics added components were changed, the number of testers with hyposmia decreased by 60.87%. According to Comparative Examples 4 to 6 and 10, when the plant extract components were changed, the number of testers with hyposmia decreased by 15.91%. Combined with the data in Table 6, it can be seen that plant extracts have a greater impact on the tester's hyposmia.

[0180] It can be seen from Comparative Examples 11 to 13 that the mass fractions of different components and the content of each component have a certain impact on the improvement of constipation and hyposmia of the test subjects.

[0181] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A composition for preventing Parkinson's disease, characterized in that: Prepared from the following materials in parts by weight: 30~44 servings of probiotics, 5~7 parts of plant extract, Nutritional supplements 0~1 portion; The probiotics are composed of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175; the plant extracts are composed of cranberry extract, ashwagandha extract and saffron extract; The mass ratio of cranberry extract, ashwagandha extract and saffron extract in the plant extract is (100-160): (80-120): (5-7); The total live bacteria concentration of the probiotics is not less than 8×10 8 CFU / g; The extraction process of the cranberry extract comprises the following steps: (1) Select cranberries in the red fruit stage, freeze-dry and then crush; (2) adding an extracting solution prepared by mixing ethanol and acetic acid, wherein the volume concentration of ethanol in the extracting solution is 50-60%, the volume concentration of acetic acid is 0.3-0.8%, and the ratio of cranberry to the extracting solution is 1:8-15 kg / L; (3) Adjust the pH to 1-3; (4) extracting by ultrahigh pressure assisted method to obtain an extract; (5) Drying the extract at a temperature not exceeding 40° C. to obtain cranberry extract; In the extraction process of cranberry extract, the conditions of the ultrahigh pressure assisted method are: temperature 20-35°C, pressure 250-400 MPa, extraction 2-8 min; The cranberry extract includes the following components and contents: anthocyanin content is 30-50 mg / g, proanthocyanidin content is 23-35 mg / g; In the ashwagandha extract, the content of withanolide is 50-80 mg / g; in the saffron extract, the content of safranal is 2-5 mg / g, and the content of crocetin is 0.4-0.8 mg / g.

2. The composition for preventing Parkinson's disease according to claim 1, characterized in that: The nutritional supplements include one or more of dopamine, gamma-aminobutyric acid, melatonin, vitamins, zinc, and magnesium.

3. The composition for preventing Parkinson's disease according to claim 2, characterized in that: The vitamins include vitamin B1, B2, B6, B 12 , vitamin C, folic acid or more.

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