Preparation method of chaenomeles speciosa extract and application of chaenomeles speciosa extract in preparation of products for preventing or treating Parkinson's disease

Abstract

The invention discloses a preparation method of chaenomeles speciosa extract and application of the chaenomeles speciosa extract in preparation of products for preventing or treating Parkinson's disease, an aqueous extract and an alcohol extract of chaenomeles speciosa are compounded and mixed for use, the potential of functional active substances in the chaenomeles speciosa extract is brought into full play, and the original efficacy of traditional Chinese medicine decoction is taken into consideration. And on the premise of green and environmental protection, more active substances are obtained through segmented alcohol extraction mining, and a synergistic effect is formed. The chaenomeles speciosa extract disclosed by the invention has a relatively good application prospect in the aspect of intervening neurodegenerative diseases, and is particularly applied to improvement of dyskinesia symptoms of Parkinson's disease.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a preparation method of a wrinkled papaya extract and application of the extract in preparing a product for preventing or treating Parkinson's disease. Background Art

[0002] Parkinson's disease is the second most common neurodegenerative disease. As a recognized clinical syndrome, Parkinson's disease encompasses multiple manifestations, including pre-motor, non-motor, and motor symptoms. It can also cause complications such as insomnia, depression, and constipation, and its development is becoming increasingly severe. In this context, it is imperative to explore interventions and treatments for Parkinson's disease and develop products to improve it. Traditional Chinese medicine (TCM) has potential application in the treatment of Parkinson's disease, exerting neuroprotective effects through multiple targets and pathways with a high safety profile.

[0003] Wrinkled papaya, also known as the Chinese herb Haitang (Haitang crabapple), is a traditional Chinese medicine that is both medicinal and edible. Its fruit is rich in vitamin C, vitamin A, various minerals, as well as sugars, amino acids, flavonoids, saponins, organic acids, and other nutrients. In Traditional Chinese Medicine, wrinkled papaya has been shown to relax muscles and tendons, soothe the stomach and eliminate dampness, dispel wind, relieve pain, reduce swelling, and soothe qi. It is commonly used to treat rheumatic pain, limb heaviness, back and knee pain, vomiting, diarrhea, abdominal pain, and dysentery. The polyphenols and triterpenes in wrinkled papaya can inhibit inflammation, reduce neuronal damage, and protect dopaminergic neurons through antioxidant effects.

[0004] Caenorhabditis elegans is an excellent animal model for studying neurodegenerative diseases. Its simple, transparent, and easily observable nervous system, short lifespan, and highly conserved genome make it amenable to simple genetic manipulation, screening, and low-cost experiments. The NL5901 nematode strain incorporates a fusion of the human α-synuclein (α-syn) gene with the yellow fluorescent protein (YFP) gene, driven by the unc-54 muscle-specific promoter, for expression in body wall muscle cells. Abnormal aggregation of α-syn after expression is a key pathological feature of Parkinson's disease, so observation of α-syn aggregation under a fluorescence microscope can be used to assess the severity of Parkinson's disease. Furthermore, this strain exhibits a phenotype of decreased motor function, similar to the movement disorders seen in Parkinson's patients. Anti-Parkinson's drugs can alleviate Parkinson's symptoms by improving movement disorders. The AM1246 nematode strain harbors the yellow fluorescent protein gene (YFP) and is driven by the unc-54 muscle-specific promoter, enabling expression in body wall muscle cells. It is often used as a blank control for NL5901 nematodes to eliminate the effects of YFP on muscle. The UA44 strain specifically expresses GFP in dopaminergic neurons, resulting in distinct green fluorescence under a fluorescence microscope. It is commonly used to study Parkinson's disease-related neurodegeneration and the function and pathological mechanisms of dopaminergic neurons.

[0005] CN119970963A invented the use of a traditional Chinese medicine composition in the preparation of a drug for Parkinson's disease. The invention uses ethyl acetate for extraction, which may result in organic solvent residues in the ointment and is not environmentally friendly. Summary of the Invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0007] As one aspect of the present invention, the present invention provides a method for preparing a wrinkled papaya extract, which comprises the following steps:

[0008] Preparation of the wrinkled papaya alcohol extract: extracting the wrinkled papaya medicinal material with a 90-95% ethanol solution, collecting the filtrate, then extracting the filter residue with 80-85% and 70-75% ethanol solutions in sequence, combining the filtrates to obtain the wrinkled papaya alcohol extract;

[0009] Preparation of the aqueous extract of the wrinkled papaya: extracting the residue obtained by alcohol extraction of the wrinkled papaya twice with hot water, and combining the filtrates to obtain the aqueous extract of the wrinkled papaya;

[0010] Combination preparation: mixing the wrinkled papaya water extract and the wrinkled papaya alcohol extract at a volume ratio of 6-8:2-4 at room temperature, and drying to obtain the wrinkled papaya extract.

[0011] As a preferred solution of the preparation method of the wrinkled papaya extract of the present invention, the wrinkled papaya medicinal material is extracted with 90-95% ethanol solution, the solid-liquid ratio is 1:8-10, the extraction temperature is 60-65°C, and the extraction time is 2-4 hours.

[0012] As a preferred solution of the method for preparing the wrinkled papaya extract of the present invention, the wrinkled papaya medicinal material is extracted with 90-95% ethanol solution, including ultrasonic extraction with an ultrasonic power of 100-200W.

[0013] As a preferred embodiment of the preparation method of the wrinkled papaya extract of the present invention, the filter residue obtained by alcohol extraction of the wrinkled papaya is extracted twice with hot water at an extraction temperature of 90-95° C. and an extraction time of 30-60 min.

[0014] As a preferred embodiment of the method for preparing the wrinkled papaya extract of the present invention, the filter residue obtained by alcohol extraction of the wrinkled papaya is extracted twice with hot water, and the solid-liquid ratio is 1:10-15.

[0015] As a preferred solution of the method for preparing the wrinkled papaya extract of the present invention, the method further comprises soaking the wrinkled papaya medicinal material in 90-95% ethanol solution for 6-12 hours before preparing the wrinkled papaya alcohol extract.

[0016] The present invention also provides the wrinkled papaya extract prepared by the preparation method of the wrinkled papaya extract: the wrinkled papaya extract comprises neochlorogenic acid, mannitol, 11-octadecenoic acid, trigonelline, malic acid, chlorogenic acid, catechin and linoleic acid components.

[0017] Beneficial effects of the present invention:

[0018] The Caenorhabditis elegans experiment of the present invention showed that 200 commonly used traditional Chinese medicines were screened through a Caenorhabditis elegans behavioral high-throughput analysis platform, and the wrinkled papaya extract showed the best intervention effect, extremely significantly improving the movement ability of nematodes with movement disorders.

[0019] In the experiment of the present invention, an ultra-high sensitivity confocal microscope was used to observe α-syn fluorescent aggregates in the NL5901 control group and the wrinkled papaya intervention group to verify whether the wrinkled papaya can improve movement disorders and alleviate Parkinson's symptoms by inhibiting the production of aggregates. Among them, in terms of quantity, the wrinkled papaya intervention group was 49.37% less than the control group. The results show that the wrinkled papaya water extract can inhibit the aggregation of α-syn in the head of NL5901 nematodes. In terms of area, the large-area (volume) aggregates in the wrinkled papaya intervention group were now less than those in the control group, inhibiting the aggregation process of α-syn and reducing neurotoxicity by hindering the synthesis of large-volume α-syn aggregates. The wrinkled papaya extract prepared by the present invention has the effect of reducing α-syn to alleviate the movement disorder symptoms of Parkinson's disease.

[0020] The present invention relies on a high-throughput analysis platform for Caenorhabditis elegans to analyze the movement status of tens of thousands of nematodes, and screens out a wrinkled papaya (traditional Chinese medicine papaya) extract that has the health care effect of improving PD.

[0021] The wrinkled papaya used in the present invention is a traditional Chinese medicine that is both medicinal and edible, with the traditional effect of relaxing muscles and activating blood circulation, and is non-toxic. Among them, the substances with the highest content include linoleic acid, neochlorogenic acid, mannitol, octadecenoic acid, malic acid, palmitic acid, trigonelline, catechin compounds, and proline. Among them, linoleic acid has been verified to be an effective neuroprotective and anti-inflammatory agent in the PD model, and can achieve antioxidant effects by stimulating the biogenesis of lipid droplets and improving autophagy / lipophagy flux. In Parkinson's disease, oxidative stress is one of the important factors leading to neuronal death. Neochlorogenic acid and chlorogenic acid belong to phenolic acids, and catechins belong to polyphenols. They all have the effect of scavenging free radicals and reducing oxidative stress, thereby helping to maintain the health of nerve cells and alleviate PD. Abnormal aggregation of α-synuclein is one of the important mechanisms leading to neurodegeneration. Mannitol, which has the third highest content, has been shown to increase the expression of 70 Dalton heat shock protein (HSP-70), and therefore may promote the correct folding of α-synuclein and the removal of misfolded forms. Trigonelline can increase NAD+ levels and mitochondrial activity, which can help improve the aging process. In Caenorhabditis elegans, trigonelline supplementation can increase mitochondrial activity, reduce age-related muscle atrophy, and extend lifespan. In mice, trigonelline supplementation can enhance muscle strength and prevent fatigue during aging. This shows that multiple active ingredients work synergistically to improve Parkinson's disease through multiple pathways by reducing oxidative stress, clearing α-synuclein, and enhancing mitochondrial function, thereby alleviating movement disorders and improving exercise capacity.

[0022] The solvents used in the present invention are water and ethanol, with the ethanol solvent being removed during the rotary evaporation and freeze-drying processes and being recyclable. This process not only avoids the potential residue problems associated with organic solvents, is highly safe, and reduces environmental pollution, conforming to the development trend of green chemistry, but also efficiently extracts water-soluble components such as saponins, flavonoid glycosides, amino acids, and polyphenols (such as chlorogenic acid and catechins) from the wrinkled papaya, thus improving Parkinson's disease dyskinesia more effectively than single components alone.

[0023] The present invention combines the water extract and the alcohol extract of wrinkled papaya to fully tap the potential of the functional active substances therein, taking into account the original efficacy of traditional Chinese medicine decoction, and extracting more active substances by segmented alcohol extraction under the premise of being green and environmentally friendly, thereby forming a synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments, wherein:

[0025] Figure 1 This is a standardized scatter plot of the swing data after the initial screening of 200 Chinese medicines;

[0026] Figure 2 A comparison of the functional activities of wrinkled papaya extracts prepared in different embodiments;

[0027] Figure 3 The intervention of Papaya water extract reduced the number of fluorescent α-synuclein aggregates in NL5901 nematodes;

[0028] Figure 4 The intervention of the aqueous extract of Papaya quince protects UA44 dopaminergic neurons from damage;

[0029] Figure 5 The results of chromatography-mass spectrometry identification and the structural formula of the new chlorogenic acid are shown;

[0030] Figure 6 The chromatographic mass spectrometry identification results and structural formula of chlorogenic acid are shown below;

[0031] Figure 7 The chromatographic mass spectrometry identification results and structural formula of mannitol are shown below;

[0032] Figure 8 The following are the results of chromatography-mass spectrometry identification and the structural formula of catechin hydrate;

[0033] Figure 9 The chromatographic mass spectrometric identification results and structural formula of 11-octadecenoic acid are shown below;

[0034] Figure 10 The following are the chromatography-mass spectrometry identification results and structural formula of trigonelline. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0036] Example 1

[0037] Screening from 200 kinds of traditional Chinese medicines revealed that wrinkled papaya has the best anti-Parkinson's disease effect:

[0038] Weigh 2g of each of 200 Chinese medicinal herbs into a 50ml centrifuge tube. Add 20ml of pure water at a material-to-liquid ratio of 1g:10ml and soak for 2h. Preheat the waterbath to 95°C and heat-extract the soaked herbs in the waterbath for 30min. After extraction, filter through four layers of gauze, collect the extract, label it, and return the herbs to the original centrifuge tube for a second hot-extraction. Filter and combine the two aqueous extracts, then rotary evaporate at 55°C at 70 rpm until the liquid level stops decreasing. Collect the boiled herbs in a sample bag, dry them, and store them. Two concentration gradients of NGM medium for Chinese herbs were set: high (H) and low (D). The high-concentration NGM was prepared by mixing 7.5ml of the Chinese herbal extract with 2.5ml of a 4x concentrated medium, while the low-concentration NGM was prepared by mixing 3.75ml of the Chinese herbal extract with 6.25ml of a 1.6x concentrated medium. After sterilization, the two were mixed evenly in a parallel device with a heated magnetic stirrer at 80°C using a rotor. 10 ml of culture medium was added to a 6 cm culture dish. After solidification, 0.2 ml of OP50 LB culture medium was added and spread flat. The culture was grown at 37°C for 24 hours to provide sufficient food for the C. elegans.

[0039] Prepare the sterilizing agent stock solution: Add 5 ml of purified water to 1 g of FUDR to obtain a stock solution with a concentration of 0.2 g / ml. Two experimental groups were set up: a control group and a papaya extract group. The control group was fed NGM medium without Chinese herbal medicine to feed AM1246 and NL5901 nematodes, while the papaya extract group was fed NL5901 nematodes. After synchronization, nematodes grown to the L1 stage were counted, 50-60 nematodes were added to each plate, and incubated at 20°C. When the nematodes reached the L4 stage, 1 ml of a 12.5 mg / L FUDR solution was added to each plate for sterilization. The plates were then air-dried in a laminar flow hood and grown at 20°C for four days. On the fifth day, the average number of wiggles per 30 seconds was measured for each group of nematodes.

[0040] Screening results: The higher the body oscillation frequency per unit time in the nematodes treated with traditional Chinese medicine, the stronger their locomotion ability and the better the anti-PD effect of the traditional Chinese medicine. Z-Score normalization is a common method for data processing. It can convert data of different magnitudes into a unified Z-Score for comparison. After screening, 78 low-concentration wrinkled papaya extracts were found to significantly improve the locomotion ability of NL5901 nematodes (p < 0.05). Therefore, wrinkled papaya was ultimately selected for subsequent research to explore the anti-PD efficacy of wrinkled papaya extracts.

[0041] Example 2

[0042] Dried slices of P. chinensis were soaked overnight in 95% ethanol at a ratio of 3 g:25 ml. Ultrasonic extraction was then performed at 60°C, 150 W, for 3 h. The filtrate was collected through a 0.22 μm filter. The residue was then ultrasonically extracted with 85% ethanol at 60°C for 3 h (3 g:25 ml), and the filtrate was collected. The residue was then ultrasonically extracted with 75% ethanol at 60°C for 3 h (3 g:25 ml), and the filtrate was combined to obtain the P. chinensis alcohol extract. The P. chinensis residue was then extracted twice with hot water (95°C) for 30 min at a ratio of 1 g:10 ml. The two aqueous extract filtrates were combined to obtain the P. chinensis aqueous extract. The P. chinensis aqueous extract and P. chinensis alcohol extract were mixed in a volume ratio of 6:4, rotary evaporation, and freeze-dried to obtain the P. chinensis extract powder.

[0043] The aforementioned papaya extract powder was dissolved in DMSO (final DMSO concentration of 0.1%) to prepare a 3g / ml solution. This extract and culture medium were mixed at a ratio of 1:999 to prepare papaya NGM medium, with a final papaya extract concentration of 3mg / ml. At the L4 stage, 1ml of 12.5mg / L FUDR solution was added to each plate for sterilization. The plates were then air-dried in a laminar flow hood and grown for four days. On the fifth day, the average number of wiggles per 30 seconds in each group of nematodes was measured to investigate the ability of papaya extract to prevent Parkinson's disease and improve movement disorders.

[0044] Comparative Example 1

[0045] Reference Example 2 Extraction Method: The dried slices of P. chinensis were soaked overnight in a 95% ethanol aqueous solution at a solid-liquid ratio of 3g:25ml, then ultrasonically extracted at 60°C for 3h, the filtrate was collected by filtration, the filter residue was ultrasonically extracted with an 85% ethanol aqueous solution at 60°C for 3h (3g:25ml solid-liquid ratio), the filtrate was collected, and the resulting filter residue was ultrasonically extracted with a 75% ethanol aqueous solution at 60°C for 3h (3g:25ml solid-liquid ratio), the filtrate was collected, and the three alcohol extraction filtrates were combined to obtain an alcohol extract of P. chinensis. The aqueous extract of P. chinensis was rotary evaporated and freeze-dried to obtain a P. chinensis extract powder. The P. chinensis extract powder was dissolved in DMSO (final DMSO concentration of 0.1%) to obtain a solution with a concentration of 3g / ml. The extract and culture medium were mixed in a ratio of 1:999 to prepare P. chinensis NGM culture medium, with a final concentration of 3mg / ml of P. chinensis extract. When the nematodes reached the L4 stage at 20°C, 1 ml of 12.5 mg / L FUDR solution was added to each plate for sterilization, and the plates were blown dry in a clean bench. After growing for four days, the average number of swings per 30 seconds of each group of nematodes was measured on the fifth day to investigate the ability of wrinkled papaya extract to prevent Parkinson's disease and improve movement disorders.

[0046] Comparative Example 2

[0047] Referring to the extraction method of Example 2, the wrinkled papaya water extract and the wrinkled papaya ethanol extract were combined in a volume ratio of 8:2, and the wrinkled papaya extract powder was obtained by rotary evaporation and freeze-drying. The other conditions were the same as in Example 2.

[0048] Comparative Example 3

[0049] Referring to the extraction method of Example 2, the wrinkled papaya water extract and the wrinkled papaya ethanol extract were combined in a volume ratio of 2:8, and the wrinkled papaya extract powder was obtained by rotary evaporation and freeze-drying. The other conditions were the same as in Example 2.

[0050] Comparative Example 4

[0051] Referring to the extraction method in Example 2, dried quince was soaked overnight in 95% ethanol at a solid-liquid ratio of 3 g:25 ml and then subjected to ultrasonication at 60°C for 3 hours. After extraction, the extracts were then extracted again with 85% and 75% ethanol, respectively. The three extracts were combined to obtain a quince extract. The quince extract was then subjected to rotary evaporation and freeze-dried to obtain a quince extract powder.

[0052] Comparative Example 5

[0053] Referring to the extraction method of Example 2, dried quince was soaked overnight in 95% ethanol at a solid-liquid ratio of 3 g:25 ml, extracted, ultrasonicated at 60°C for 3 h, and then extracted twice with 95% ethanol. The three alcohol extracts were combined to obtain an alcohol extract of quince. The alcohol extract of quince was rotary evaporated and freeze-dried to obtain a quince extract powder.

[0054] Comparative Example 6

[0055] Referring to the extraction method of Example 2, dried quince was soaked overnight in 85% ethanol at a solid-liquid ratio of 3 g:25 ml, extracted, ultrasonicated at 60°C for 3 h, and then extracted twice with 85% ethanol. The three alcohol extracts were combined to obtain an alcohol extract of quince. The alcohol extract of quince was rotary evaporated and freeze-dried to obtain a quince extract powder.

[0056] Comparative Example 7

[0057] Referring to the extraction method of Example 2, dried quince was soaked overnight in 75% ethanol at a solid-liquid ratio of 3 g:25 ml, extracted, ultrasonicated at 60°C for 3 h, and then extracted twice with 75% ethanol. The three alcohol extracts were combined to obtain an alcohol extract of quince. The alcohol extract of quince was rotary evaporated and freeze-dried to obtain a quince extract powder.

[0058] Comparative Example 8

[0059] Referring to the extraction method of Example 2, dried quince was soaked overnight in 65% ethanol at a solid-liquid ratio of 3 g:25 ml, extracted, ultrasonicated at 60°C for 3 h, and then extracted twice with 65% ethanol. The three alcohol extracts were combined to obtain an alcohol extract of quince. The alcohol extract of quince was rotary evaporated and freeze-dried to obtain a quince extract powder.

[0060] Comparative Example 9

[0061] Referring to the extraction method of Example 2, dried quince was soaked overnight in 55% ethanol at a solid-liquid ratio of 3 g:25 ml, extracted, ultrasonicated at 60°C for 3 h, and then extracted twice with 55% ethanol. The three alcohol extracts were combined to obtain an alcohol extract of quince. The alcohol extract of quince was rotary evaporated and freeze-dried to obtain a quince extract powder.

[0062] Comparative Example 10

[0063] Referring to the extraction method in Example 2, dried quince was soaked overnight in 75% ethanol at a solid-liquid ratio of 3 g:25 ml and then subjected to ultrasonication at 60°C for 3 hours. After extraction, the extracts were then extracted again with 65% and then 55% ethanol, respectively. The three extracts were combined to obtain a quince extract. The extract was then rotary evaporated and freeze-dried to obtain a quince extract powder.

[0064] Comparison results of the functional activities of the wrinkled papaya extracts in various examples:

[0065] Table 1 and Figure 2 The results show that the wrinkled papaya extracts prepared in each example improve the motility of NL5901 Caenorhabditis elegans. In Comparative Examples 1-10, the number of worm body swings within 30 seconds increased by 71.6%, 89.8%, 43.8%, 40.0%, 19.7%, 20.9%, 23.6%, 20.1%, 20.2%, and 30.6%, respectively, demonstrating varying degrees of improvement in motility.

[0066] In Example 2, after the NL5901 nematode was treated with the extract of the wrinkled papaya water extract: alcohol extract in a ratio of 6:4, the number of body swings within 30 seconds increased by 106.3% compared with the NL5901 nematode without wrinkled papaya treatment, which significantly improved its movement ability ( ＊＊＊＊ P<0.0001).

[0067] Table 1 Effects of wrinkled papaya extracts prepared in various examples on the motor ability of NL5901 Caenorhabditis elegans

[0068]

[0069] The Bliss independence model assumes that the two drugs have independent mechanisms of action and the combined effect is the product of their independent effects. A ), Comparative Example 4 is the efficacy verification of using alcohol extract alone (E B ), Example 2 is the efficacy verification using a 6:4 combination of water extract and alcohol extract (EA B ):

[0070] ΔE=E AB -(EA+E B -E A ·E B )=0.2334>0, which proves that the combined use of ethanol extract and water extract has a synergistic effect.

[0071] Example 3

[0072] Verification of the anti-Parkinson's disease function of wrinkled papaya water extract:

[0073] Add 0.5g agarose and 25ml M9 to a 50ml beaker, place it on an electric stove and heat until boiling. Take a glass slide, add 30μL agarose solution (2%), take another glass slide and cover it on top, and remove it after 30s when the agarose pad solidifies. Add 10μL 20mM M9\levamisole buffer to the agarose pad to anesthetize the nematodes, pick 12 NL5901 Caenorhabditis elegans that have been intervened with the wrinkled papaya water extract obtained in Example 1 and put them into the droplet, cover with a coverslip and seal. Turn on the mercury lamp power of the fluorescence microscope. Turn off the fluorescence shutter and determine the observation angle under bright field. Turn on the fluorescence shutter, resolution 1024×1024, select the GFP-uv filter block, voltage 509V, adjust the brightness and focal length appropriately, and shoot the fluorescence aggregation of the nematode head in Z-axis mode. Image J was used to analyze the number and size of fluorescent aggregates on the nematode head. The 8-bit type and invert LUT were used to invert the color. The thresholds were 33 and 255, respectively. The number and size of the particles were then analyzed.

[0074] Validation of the anti-Parkinson's disease function of a papaya extract revealed that α-syn is a protein highly expressed in presynaptic terminals in the central nervous system and is closely implicated in the pathogenesis of Parkinson's disease. α-syn gene mutations (such as A53T, A30P, and E46K) or post-translational modifications (phosphorylation and nitration) lead to misfolding and oligomerization. Excessive accumulation of α-syn leads to the death of dopaminergic neurons in the brain, inducing Parkinson's disease.

[0075] Depend on Figure 3It can be seen that after the intervention of papaya water extract, the number of α-syn protein aggregates in the head of Caenorhabditis elegans decreased and the area became smaller. It is speculated that the number of aggregates may be reduced by blocking the α-syn aggregation pathway to alleviate the movement disorders caused by Parkinson's disease, and there is a potential regulatory mechanism for movement disorders and neuroprotection. In addition, the fluorescent aggregates of 12 NL5901 nematodes in the control group and the papaya water extract intervention group were counted. The average number of α-syn protein aggregates in the blank group was 79.5±29.47, and the average number of α-syn protein aggregates in the papaya intervention group was 40.25±28.48, a decrease of 49.37%. The results show that papaya water extract can significantly reduce the aggregation of α-syn protein in NL5901 nematodes ( ＊＊ P<0.01), and improve exercise ability.

[0076] Example 4

[0077] Verification of the protective effect of wrinkled papaya water extract on dopaminergic neurons:

[0078] Add 0.5g agarose and 25ml M9 to a 50ml beaker, place it on an electric stove and heat until boiling. Take a glass slide, add 30μL agarose solution (2%), take another glass slide and cover it on top, and remove it after 30s when the agarose pad solidifies. Add 10μL 20mM M9\levamisole buffer to the agarose pad to anesthetize the nematodes, pick 12 UA44 Caenorhabditis elegans that have been intervened by the wrinkled papaya water extract in Example 1 and put them into the droplet, cover with a coverslip and seal. Turn on the mercury lamp power of the fluorescence microscope. Turn off the fluorescence shutter and determine the observation angle under bright field. Turn on the fluorescence shutter, resolution 1024×1024, select the GFP-uv filter block, voltage 509V, adjust the brightness and focal length appropriately, and shoot the fluorescence aggregation of the nematode head in Z-axis mode. ImageJ was used to analyze the number and size of fluorescent aggregates in the nematode head. The 8-bit type, invert LUT color, and threshold values ​​of 6 and 255 were used to analyze the number and size of the particles.

[0079] The results of the validation of the protective effect of wrinkled papaya water extract on dopaminergic neuron function: Figure 4 As can be seen, the GFP fluorescence of dopaminergic neurons in the UA44 blank group was disrupted and absent, indicating degeneration of its CEP and ADE neurons. However, in the UA44 wrinkled papaya treated group, the GFP fluorescence of two pairs of CEP neurons and one pair of ADE neurons was highly intact and continuous, indicating that the papaya aqueous extract has an ameliorative, repairing, and protective effect on damaged UA44 dopamine neurons. Comparing the GPF fluorescence area of ​​12 blank nematodes with that of 12 papaya treated nematodes, the papaya treated group showed a 42.70% increase in fluorescence area, significantly protecting the dopaminergic neurons in UA44 nematodes (*P < 0.05).

[0080] The components of the wrinkled papaya extract obtained in Example 2 were analyzed by liquid chromatography-mass spectrometry. The positive ion liquid chromatography test conditions were as follows: chromatographic column: waters C18 Column, 1.7 μm, 2.1 mm × 100 mm, mobile phase: A: 0.1% formic acid in water, B: acetonitrile, column temperature: 35°C, injection volume: 5 μL, elution gradient: 0 min, 95% A 5% B; 0.5 min, 95% A 5% B; 15 min, 0% A 100% B; 18 min, 0% A 100% B; 18.1 min, 95% A 5% B; 20 min, 95% A 5% B; mass spectrometry conditions: TOFMS IDA positive mode; scan range: 40–1000 Da; primary DP voltage, CE collision energy: 80, 10; secondary DP voltage, CE collision energy: 80, 35.

[0081] The negative ion liquid phase test conditions are as follows: chromatographic column: waters C18 Column, 1.7μm, 2.1mm X100mm, mobile phase: A: 5mM ammonium acetate aqueous solution B: acetonitrile, column temperature: 35℃, injection volume: 10μL, elution gradient: 0min, 95% A 5% B; 0.5min, 95% A 5% B; 15min, 0% A 100% B; 18min, 0% A 100% B; 18.1min, 95% A 5% B; 20min, 95% A 5% B; mass spectrometry conditions: TOF MS IDA negative mode; scanning range: 40-1000Da; primary DP voltage, CE collision energy: 80, -10; secondary DP voltage, CE collision energy: 80, -35.

[0082] Table 2 Analysis results of the components and proportions of the wrinkled papaya extract obtained in Example 2

[0083]

[0084]

[0085]

[0086] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a wrinkled papaya extract, characterized in that: The following steps are included: Preparation of the wrinkled papaya alcohol extract: extracting the wrinkled papaya medicinal material with a 90-95% ethanol solution, collecting the filtrate, then extracting the filter residue with 80-85% and 70-75% ethanol solutions in sequence, combining the filtrates to obtain the wrinkled papaya alcohol extract; Preparation of the aqueous extract of the wrinkled papaya: extracting the residue obtained by alcohol extraction of the wrinkled papaya twice with hot water, and combining the filtrates to obtain the aqueous extract of the wrinkled papaya; Combination preparation: mixing the wrinkled papaya water extract and the wrinkled papaya alcohol extract at a volume ratio of 6-8:2-4 at room temperature, and drying to obtain the wrinkled papaya extract.

2. The method for preparing the wrinkled papaya extract according to claim 1, wherein: The wrinkled papaya medicinal material is extracted with 90-95% ethanol solution, the material-liquid ratio is 1:8-10, the extraction temperature is 60-65° C., and the extraction time is 2-4 hours.

3. The method for preparing the wrinkled papaya extract according to claim 1 or 2, wherein: The wrinkled papaya medicinal material is extracted with 90-95% ethanol solution, including ultrasonic extraction, and the ultrasonic power is 100-200W.

4. The method for preparing the wrinkled papaya extract according to claim 1 or 2, wherein: The filter residue obtained by alcohol extraction of wrinkled papaya is extracted twice with hot water, the extraction temperature is 90-95° C., and the extraction time is 30-60 minutes.

5. The method for preparing the wrinkled papaya extract according to claim 1 or 2, wherein: The filter residue obtained by alcohol extraction of wrinkled papaya is extracted twice with hot water, and the solid-liquid ratio is 1:10-15.

6. The method for preparing the wrinkled papaya extract according to claim 1 or 2, wherein: The method also includes soaking the wrinkled papaya medicinal material in 90-95% ethanol solution for 6-12 hours before preparing the wrinkled papaya alcohol extract.

7. The wrinkled papaya extract prepared by the method for preparing the wrinkled papaya extract according to claim 1, characterized in that: The wrinkled papaya extract comprises neochlorogenic acid, mannitol, 11-octadecenoic acid, trigonelline, malic acid, chlorogenic acid, catechin and linoleic acid components.

8. Use of the wrinkled papaya extract prepared by the preparation method of the wrinkled papaya extract according to claim 1 in preparing products for treating Parkinson's disease.

9. The use according to claim 8, characterized in that: The invention relates to an application of the wrinkled papaya extract in preparing a product for improving movement disorder symptoms of Parkinson's disease.

10. The use according to claim 8, characterized in that: Application of the wrinkled papaya extract in preparing products for preventing Parkinson's disease.

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