Uncaria alkaloid extract as well as preparation method and application thereof

Abstract

The invention relates to an uncaria alkaloid extract as well as a preparation method and application thereof. The method comprises the following steps: heating and leaching uncaria stem and branch powder with acidic ethanol to obtain an acid alcohol leaching solution; carrying out reduced pressure distillation and concentration on the leach liquor to obtain an uncaria total alkaloid extract; removing acid-insoluble substances from the total alkaloid extract, degreasing, adjusting alkali, extracting with chloroform, and recovering a chloroform layer to obtain uncaria crude alkaloid; and dissolving the uncaria crude alkaloid in distilled water, adding into a macroporous resin chromatographic column, adsorbing, flushing, eluting by using a 20% ethanol solution with five column volumes, and collecting the eluent to obtain the uncaria alkaloid extract. The ramulus uncariae cum uncis alkaloid extract PURA1 has the effects of improving the pathological phenotype of the Alzheimer's disease model caenorhabditis elegans and improving the oxidative stress resistance of the caenorhabditis elegans, plays a role in prolonging the service life of the caenorhabditis elegans by regulating and controlling the p38MAPK signal channel of the caenorhabditis elegans, and can be used for preparing the medicine for relieving the Alzheimer's disease and resisting aging.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to an extract of Uncaria rhynchophylla alkaloids, its preparation method, and its application. Background Technology

[0002] With the aging population, environmental pollution, life stress, and changes in dietary habits, a series of health problems have emerged, and the number of people in a sub-healthy state is increasing. Anti-aging has become a research hotspot in life sciences and the health industry. Aging is characterized by a gradual decline in physiological functions, leading to an increased risk of neurodegenerative diseases, cancer, and chronic diseases such as diabetes. Alzheimer's disease (AD) is the most common neurodegenerative disease, accounting for 60%-70% of all dementia cases. According to the World Health Organization, there are currently approximately 55 million AD patients worldwide, and this number is projected to exceed 150 million by 2050. AD not only severely impacts the quality of life of patients but also imposes a heavy economic and care burden on families and society. Therefore, developing / seeking effective drugs to treat / delay the pathological process of AD has become a major and urgent problem that needs to be solved. However, the etiology and pathological mechanisms of Alzheimer's disease (AD) are not yet fully understood, and there are currently no effective drugs for treating AD. Clinically, the main approach is "symptomatic treatment," which uses cholinesterase inhibitors (such as donepezil and rivastigmine), NMDA receptor antagonists (such as memantine), and brain metabolism activators to improve patients' cognitive function and slow the progression of the disease.

[0003] Currently, the use of natural plant components to delay aging is receiving increasing attention due to their low toxicity and significant effects. Uncaria rhynchophylla, a plant belonging to the Rubiaceae family, is a classic medicinal plant, primarily used for its hooked stems and branches. It possesses properties such as clearing heat and calming the liver, relieving wind and calming convulsions, and is mainly used to treat convulsions, hypertension, epilepsy, preeclampsia, and brain diseases. Related studies have shown that the pharmacological activity of this plant is mainly related to its alkaloid compounds, and Uncaria rhynchophylla alkaloids have potential therapeutic effects on Alzheimer's disease (AD).

[0004] Currently, the extraction of total alkaloids from Uncaria rhynchophylla mainly relies on solvent extraction. Based on the solvent used and related processing, it can be broadly classified into four types: ethanol extraction, alkalization ether extraction, alkalization benzene extraction, and alkalization alcohol extraction. However, these methods are only suitable for laboratory conditions. The chemical components of Uncaria rhynchophylla play an important role in traditional Chinese medicine and modern treatments. However, the chemical composition of Uncaria rhynchophylla is complex, low in content, and difficult to concentrate. Existing Uncaria rhynchophylla extracts still require much research and improvement. Summary of the Invention

[0005] The primary objective of this invention is to address the technical problems existing in the prior art by providing a method for preparing Uncaria rhynchophylla alkaloid extract.

[0006] Another object of the present invention is to provide an extract of Uncaria alkaloids obtained by the above method.

[0007] Another object of the present invention is to provide the use of the above-mentioned Uncaria alkaloid extract in the preparation of anti-Alzheimer's disease and / or anti-aging drugs.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing an extract of Uncaria rhynchophylla alkaloids, comprising the following steps: (1) The powder of Uncaria rhynchophylla stems and branches was extracted by heating with ethanol adjusted to acidity to obtain an acid-ethanol extract; the extract was concentrated by vacuum distillation to obtain a total alkaloid extract of Uncaria rhynchophylla. (2) After removing acid-insoluble matter, defatting, adjusting alkali, and extracting with chloroform, the total alkaloid extract was concentrated under reduced pressure by recovering the chloroform layer to obtain crude Uncaria rhynchophylla alkaloids. (3) Dissolve the crude alkaloids of Uncaria rhynchophylla in distilled water, add it to a macroporous resin chromatography column for adsorption, wash the macroporous resin with distilled water, elute with 5 column volumes of 20% ethanol solution, collect the eluent, distill under reduced pressure and dry to obtain Uncaria rhynchophylla alkaloid extract.

[0009] Furthermore, the Uncaria rhynchophylla stem and branch powder mentioned in step (1) is obtained by drying and pulverizing Uncaria rhynchophylla stems and branches.

[0010] Further, the step (1) of extracting Uncaria rhynchophylla stem and branch powder by heating with ethanol adjusted to acidity includes the following steps: adding 20 mL of 80% ethanol solution with pH adjusted to 3 to every 1 g of Uncaria rhynchophylla stem and branch powder, extracting at 70°C, filtering, and obtaining acid-ethanol extract.

[0011] Furthermore, the Uncaria rhynchophylla stem and branch powder extracted in step (1) is extracted 3-5 times.

[0012] Further, the removal of acid-insoluble matter in step (2) includes the following steps: dissolving the total alkaloid extract in hot water, adjusting the pH of the solution to 2 with hydrochloric acid, filtering, and removing acid-insoluble matter.

[0013] Further, the defatting described in step (2) includes the following steps: extracting with petroleum ether, discarding the petroleum ether layer, and repeating 3-7 times.

[0014] Furthermore, the alkali adjustment mentioned in step (2) involves adjusting the pH of the solution to 10 using ammonia.

[0015] Furthermore, the chloroform extraction described in step (2) involves 5-7 extractions.

[0016] Furthermore, the macroporous resin chromatography column in step (3) is AB-8 macroporous resin with a column size of 16mm × 40mm; and even further, the packing height is 1 / 2 column volume.

[0017] Further, in step (3), after adding to the macroporous resin chromatography column, the crude alkaloid solution of Uncaria rhynchophylla is mixed with AB-8 macroporous resin and allowed to stand for 12~18h.

[0018] Furthermore, in step (3), the purpose of rinsing the macroporous resin with distilled water is to remove the unadsorbed crude alkaloid solution from Uncaria rhynchophylla.

[0019] An extract of Uncaria alkaloids was prepared by the above method.

[0020] Furthermore, the Uncaria alkaloid extract contains rhynchophylline A, cardanine, yohimbine, dehydrorhynchophylline, isodehydrorhynchophylline, konosine, amarisoxine, isorhynchophylline, rhynchophylline, rhynchophylline, rhynchophylline, and rhynchophylline.

[0021] The application of the above-mentioned Uncaria alkaloid extract in the preparation of anti-Alzheimer's disease and / or anti-aging drugs.

[0022] An anti-Alzheimer's disease and / or anti-aging drug containing the above-mentioned Uncaria rhynchophylla alkaloid extract.

[0023] Compared with the prior art, the present invention has the following advantages: The Uncaria alkaloid extract (PURA1) of this invention can improve pathological indicators of Alternaria alternata (AD nematode).

[0024] The Uncaria alkaloid extract (PURA1) of the present invention serves as a promoter for enhancing the activity of intercellular antioxidant enzyme systems.

[0025] The principle of this invention: The Uncaria alkaloid extract of this invention can inhibit the p38MAPK signaling pathway, significantly improve pathological indicators of AD nematodes, delay the paralysis process of nematodes, reduce β-amyloid protein deposition, increase the activity of antioxidant enzymes in vivo, reduce ROS levels, and prolong the lifespan of nematodes, thereby exerting anti-AD efficacy.

[0026] This invention is the first to apply Uncaria rhynchophylla alkaloid extract to an Alzheimer's disease (AD) model and demonstrate its significant neuroprotective activity. It clarifies its mechanism of action by regulating oxidative stress through increasing SOD and CAT activity and decreasing ROS levels. The nematode model used in this invention has the advantages of short cycle time, low cost, and ease of high-throughput screening, making it suitable for rapid evaluation of the anti-AD activity of natural products. This provides experimental evidence for the application of Uncaria rhynchophylla alkaloids in anti-AD drugs and has promising prospects for industrial application. Attached Figure Description

[0027] Figure 1 The figure shows the effect of PURA1 on the paralysis process of CL2006 nematodes in Example 2.

[0028] Figure 2 The figure shows the effect of PURA1 on Aβ deposition in CL2006 nematodes in Example 3.

[0029] Figure 3 The figure shows the effect of PURA1 on the ROS content in CL2006 nematodes in Example 4.

[0030] Figure 4 The figure shows the effect of PURA1 on SOD enzyme activity and MDA content in CL2006 nematodes in Example 5.

[0031] Figure 5 The figure shows the effect of PURA1 on the mRNA expression levels of genes related to the p38MAPK signaling pathway in CL2006 nematodes in Example 6.

[0032] Figure 6 The figure shows the effect of PURA1 on the lifespan of CL2006 and KU25 nematodes in Example 7. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0034] Example 1: Preparation of Uncaria alkaloids

[0035] The dried stems and branches of Uncaria rhynchophylla were dried in an electric hot air drying oven at 60℃ for 6 hours, and then coarsely powdered using a traditional Chinese medicine pulverizer. An 80% ethanol solution with pH 3 was added at a material-to-liquid ratio of 1:20 (g / mL), and the mixture was extracted in a water bath at 70℃. The extract was filtered, and the acid-ethanol extract was collected. The residue was extracted three times under the same conditions. The extracts were combined and concentrated by vacuum distillation to obtain the Uncaria rhynchophylla ethanol extract.

[0036] The above extract was dissolved in hot water, and the pH was adjusted to 2 with hydrochloric acid. The mixture was filtered to remove insoluble matter, and the filtrate was collected. The filtrate was extracted three times with an equal volume of petroleum ether. The petroleum ether layer was discarded, and the acidic aqueous layer was retained. The pH of the acidic aqueous layer was adjusted to 10 with ammonia, and the mixture was extracted six times with chloroform. The chloroform layers were combined and concentrated under reduced pressure to obtain the crude alkaloid sample.

[0037] AB-8 macroporous resin was soaked in 95% ethanol for 24 h and then washed for later use. The treated AB-8 macroporous resin was wet-packed into a column (column size: 16 mm × 40 mm), with a packing height of approximately 1 / 2 column volume. The crude alkaloid sample was dissolved in distilled water and mixed with an appropriate amount of AB-8 macroporous resin, then allowed to stand for 12 h to allow for complete adsorption. The macroporous resin containing the adsorbed sample was transferred to a chromatography column, packing to approximately 3 / 4 column volume, and the liquid level was compacted with cotton. The column was allowed to stand for 12 h. Elution was performed sequentially with distilled water and then with 5 column volumes of 20% ethanol solution. The 20% ethanol eluent was collected, concentrated under reduced pressure, and freeze-dried to obtain PURA1 sample powder.

[0038] Using rhynchophylline as a reference standard, the content of alkaloids in PURA1 samples was determined by the bromocresol green acid staining method. Specifically, this included: preparing a series of rhynchophylline reference standard solutions of varying concentrations; using bromocresol green as the colorimetric reagent and a pH 3.6 potassium hydrogen phthalate-hydrochloric acid buffer solution as the solvent to prepare the alkaloid sample solution; accurately pipetting 4 mL of the test solution into a separatory funnel containing 10 mL of the buffer solution, adding 10 mL of bromocresol green colorimetric reagent, shaking, and allowing it to stand for 30 min; then adding 6 mL of chloroform, shaking, and allowing it to stand for 10 min; separating the chloroform layer; repeating the extraction four times; combining the extracts and adjusting the volume to 25 mL; adding anhydrous sodium sulfate for dehydration for 30 min; filtering; and measuring the absorbance at 413 nm. A rhynchophylline standard solution was used as a control group instead of the test solution, and distilled water was used as a blank control following the same procedure. A standard curve was plotted with the concentration of the standard as the x-axis and the absorbance as the y-axis (y=3.05654x+0.00904, R2=0.99947). The content of alkaloids in the sample was calculated to be 15.515%±0.025%.

[0039] Example 2: Compositional Analysis of Uncaria Rhizoma Alkaloid Extract The components of PURA1 were analyzed by LC-MS / MS. LC-MS was performed using an Agilent 1290 series high-performance liquid chromatograph equipped with a Bruker maXis impact mass spectrometer. The mobile phase was 0.1% formic acid in water (A) – 0.1% formic acid in acetonitrile (B), with the following elution gradients: 0–1 min, 95% A; 1–12 min, 95%–5% A; 12–13 min, 5%–95% A; 13–15 min, 95% A; injection volume was 2 μL, flow rate was 0.3 mL / min; mass spectrometry conditions: electrospray ionization mass spectrometer, ESI positive ion mode, capillary voltage 3500 V, gas flow rate 6.0 L / min, scan range 50–1000 m / z.

[0040] By comparing peak times and ion fragments and searching the spectral library, PURA1 mainly includes the following alkaloids: rhynchophylline A, cardanine, yohimbine, dehydrorhynchophylline, isodehydrorhynchophylline, conoxinine, amarixinine, isorhynchophylline, rhynchophylline, rhynchophylline, and rhynchophylline.

[0041] Example 3: Effect of PURA1 on the paralysis process of Caenorhabditis elegans CL2006

[0042] 1. Culture of Caenorhabditis elegans (1) Culture of Escherichia coli OP50: OP50 stored at -80℃ was taken out, thawed in a 37℃ water bath, and inoculated into LB solid medium in a clean bench. After incubation at 37℃ for 24 h, single colonies on the plate were picked and placed into 200 mL of LB liquid medium. The medium was shaken at 37℃ and 200 rpm for 8 h. The bacterial culture was concentrated by centrifugation in 50 mL sterile centrifuge tubes, resuspended in 5 mL of M9 buffer, and stored at 4℃ for later use.

[0043] (2) Preparation of OP50 plates: OP50 is used as food for Caenorhabditis elegans. 200 μL of bacterial solution is added to each NGM plate, spread evenly with a spreader, and incubated at 37℃ for 12 h to obtain plates coated with OP50 for the culture of Caenorhabditis elegans.

[0044] (3) Preparation of Uncaria alkaloid sample solution: Accurately weigh 100 mg of Uncaria alkaloid extract PURA1 sample, add 1 mL of DMSO to dissolve, prepare a stock solution of 100 mg / mL, then dilute with sterilized M9 buffer to 1 mg / mL, and filter through a 0.22 μm microporous membrane for sterilization. Mix this 1 mg / mL solution with inactivated OP50 bacterial solution with OD570=1 evenly, spread on NGM plates, and act on nematodes. Store the NGM plates with the sample at 4℃ for later use. When administering the drug, use 1% DMSO instead of Uncaria alkaloid sample as a control.

[0045] (4) Synchronization culture of nematodes: N2 nematodes were synchronized using chemical lysis. The specific operation was as follows: nematodes that had begun to lay eggs were collected into 1.5 mL centrifuge tubes. After natural sedimentation, the supernatant was discarded, and the tubes were washed three times with M9 buffer to remove OP50. 1 mL of nematode lysis buffer was added to the 1.5 mL centrifuge tube, and 500 μL of clean nematode fluid was added to the lysis buffer. The tubes were vortexed for about 3 min until most of the nematode bodies were observed to have split open under a microscope. The tubes were then rapidly centrifuged at 6000 rpm for 2 min, the supernatant was discarded, and the tubes were washed with M9 buffer. The tubes were centrifuged at 6000 rpm for 2 min, and this process was repeated three times. The remaining eggs were transferred to blank NGM plates and cultured overnight at 20°C. After about 16 h, the nematodes hatched, yielding L1-stage nematodes. The hatched nematodes were transferred to OP50 plates and cultured for another 45 h to obtain L4-stage nematodes. Because transgenic nematodes in the AD model are prone to losing their shape and are sensitive to temperature, their synchronization procedure is as follows: Nematodes that have entered the oviposition period are picked up with a platinum wire rod and placed on a blank NGM plate, with about 100 nematodes on each plate. After being cultured at 16°C for 8 hours, the oviposition nematodes are removed, and the remaining eggs are handled in the same way as N2 nematodes.

[0046] (7) Solid culture of Caenorhabditis elegans: The nematodes were cultured on a substrate containing live OP50. If the mycelium was completely consumed, the nematodes were transferred to a new substrate. Generally, they could be transferred directly by cutting them into pieces or by rinsing them off with M9 and then transferring them to a new substrate. The nematodes were cultured in a 20°C incubator.

[0047] (5) Counting of nematodes: Use platinum wire to pick out the nematodes. When picking them out, try to avoid puncturing them and affecting their survival. If the nematodes are observed to be motionless, have no swallowing movements in their pharynx, are in a rigid state, and have no reaction when lightly touched with a nematode-picking needle, then the nematodes are dead.

[0048] (6) Preparation of nematode homogenate: An ultrasonic homogenizer with a power of 25 W was used, with an ultrasonic working time of 1 s, an interval time of 5 s, and 60 working times.

[0049] 2. CL2006 Nematode Paralysis Phenotypic Determination After being synchronized, L4-stage Caenorhabditis elegans CL2006 nematodes were transferred to NGM solid culture plates containing the sample, with a solvent-treated group without the sample serving as a control. The nematodes were cultured at 20°C. The paralysis status of the nematodes was observed under a stereomicroscope starting from the start of the drug treatment. The number of paralyzed nematodes was counted at the same time each day until all nematodes were paralyzed. The time corresponding to the paralysis of half of the AD nematodes was recorded as the half-paralysis time, denoted as PT50. The experiment was repeated three times. The determination of nematode paralysis was based on gently touching the nematode with a needle; if the nematode's body could not move as a whole or only its head moved, it was considered paralyzed. At least 30 nematodes were recorded in each group, with three replicates.

[0050] The results are as follows Figure 1 As shown.

[0051] The results showed that without PURA1 treatment, nematodes reached 50% paralysis in about 10 days, while PURA1 treatment shifted the non-paralyzed curve of nematodes to the right and delayed the paralysis process caused by Aβ. Compared with the control group, 25 μg / mL PURA1 treatment delayed the PT50 of nematodes by 34.48% (p < 0.01).

[0052] Example 4: Effect of PURA1 on Aβ deposition in Caenorhabditis elegans CL2006 cells

[0053] After being synchronized, L4-stage Caenorhabditis elegans CL2006 was transferred to NGM solid culture plates containing or without PURA1 samples and cultured at 20°C for 5 days. The nematodes were collected, and the nematodes from different treatment groups were washed into centrifuge tubes using M9 buffer. After the nematodes settled naturally, the supernatant was removed. The washing process was repeated until the supernatant was clear, and OP50 was removed. After treatment, the tubes were centrifuged at 4000 rpm for 2 min, and the supernatant was discarded. 150 μL of 4% paraformaldehyde was added to each tube, and the tubes were fixed at 4°C for 24 h. The supernatant was discarded, and 150 μL of osmotic solution (5% β-mercaptoethanol, 1% Triton X-100, 125 mM Tris-HCl, pH=7.4) was added. The tubes were incubated at 37°C for 24 h, washed three times with M9 buffer containing 0.1% Triton X-100 (pH=7.4), and then stained with 0.125% thioflavin S in the dark for 2 min. After staining, the supernatant was discarded by centrifugation, and the supernatant was destained multiple times with 50% ethanol until it became colorless. The nematode heads were photographed using a fluorescence microscope, and the deposition of Aβ was quantified using the obtained fluorescence images. At least 30 nematodes were analyzed in each experimental group.

[0054] The results are as follows Figure 2 As shown.

[0055] The experimental results show that, compared with the control group, the Aβ deposition in the head of CL2006 nematodes treated with PURA1 was significantly reduced.

[0056] Example 5: Effect of PURA1 on ROS content in Caenorhabditis elegans CL2006 cells

[0057] L4-stage nematodes obtained through synchronization were picked onto NGM plates containing or without PURA1 and cultured for 5 days. The nematodes were washed into 1.5 mL conical centrifuge tubes using an M9 centrifuge, allowed to settle naturally, and the supernatant was discarded. This process was repeated three times until the supernatant was clear, then discarded. 100 μL of DCFH-DA fluorescent probe working solution was added, and the tubes were stained at 100 rpm and 37°C in the dark for 2 h. After staining, the probe was washed several times with an M9 centrifuge to remove the probe. The nematodes were then anesthetized with levamisole hydrochloride solution and transferred to agarose gel-coated slides. At least 30 nematodes were photographed per group. The green fluorescence within the nematodes was captured using a fluorescence microscope, and the relative fluorescence intensity of ROS in the nematodes was analyzed using ImageJ.

[0058] The results are as follows Figure 3 As shown.

[0059] The experimental results showed that, compared with the control group, the ROS level in nematodes treated with 25 μg / mL PURA1 was reduced by 46.80%.

[0060] Example 6: Effects of PURA1 on SOD activity and MDA content in *C. elegans*

[0061] The synchronized L4 stage CL2006 nematodes were placed on NGM plates with or without PURA1 and cultured for 5 days. The nematodes were washed into 1.5 mL conical centrifuge tubes using an M9 centrifuge, allowed to settle naturally, and the supernatant was discarded. The washing process was repeated three times until the supernatant was clear. The nematodes were then homogenized using an ultrasonic homogenizer to prepare a nematode homogenate. The SOD activity and MDA content of the nematodes were determined using the Nanjing Jiancheng Biotechnology Research Institute's superoxide dismutase (SOD) assay kit and malondialdehyde (MDA) assay kit, following the kit's operating procedures.

[0062] The results are as follows Figure 4 As shown.

[0063] The experimental results showed that, compared with the control group, treatment with PURA1 at concentrations of 12.5 μg / mL, 25 μg / mL and 50 μg / mL increased SOD activity by 21.18%, 34.42% (p<0.05) and 34.84% (p<0.05), respectively, and decreased MDA content in nematodes by 22.73% (p<0.001), 22.06% (p<0.001) and 31.58% (p<0.0001), respectively.

[0064] Example 7: Effect of PURA1 on mRNA expression levels of the p38MAPK signaling pathway in Caenorhabditis elegans

[0065] L4-stage nematodes obtained through synchronization were transferred to NGM plates containing or without PURA1 and cultured for 5 days. Total RNA was extracted from the nematodes using the rapid RNA extraction kit from Beijing Adley Biotechnology Co., Ltd., and the RNA was reverse transcribed to obtain cDNA products, which were then stored at -20℃. Using the reverse-transcribed cDNA as a template, targeting the target gene... nsy-1 , sek-1 , pmk-1 and internal reference genes act-1 Specific primers were designed (primer sequences are shown in Table 1) for qPCR. The reaction program was set as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, for 40 cycles; the melting curve acquisition program was: 65℃-95℃, increasing by 0.5℃ every 5 s. Fluorescence data were collected using 2... - The method calculates the relative expression of relevant gene mRNAs.

[0066] Table 1 Primer Sequences

[0067] The results are as follows Figure 5 As shown.

[0068] Compared with the control group, PURA1 treatment significantly reduced the levels of nematodes in vivo. nsy-1 , sek-1 , pmk-1 mRNA expression. Treatment with PURA1 at concentrations of 12.5, 25, and 50 μg / mL resulted in... nsy-1 The expression of [specific substance] decreased to 61.16% (p < 0.0001), 60.02% (p < 0.0001), and 40.45% (p < 0.0001) of the control group; [further details needed]. sek-1 The expression of [specific substance] decreased to 52.10% (p < 0.0001), 48.94% (p < 0.0001), and 60.68% (p < 0.0001) of the control group; making [specific substance] expression [lower / ... pmk-1 The expression of [a substance] decreased to 82.05% (p<0.001), 58.99% (p<0.0001), and 62.47% (p<0.0001) of the control group.

[0069] Example 8: PURA1 against CL2006 nematodes and pmk-1 The impact of defective nematode KU25 on survival rate The L4-stage nematodes CL2006 and KU25 obtained through synchronization were transferred to 90 mm NGM plates with or without PURA1 for culture, which was recorded as day 0. On day 3, they were picked and transferred to 60 mm plates containing the corresponding samples, with three replicates for each drug group. Each plate contained 60 nematodes and was placed in an incubator at 20°C for further culture. Every two days, the surviving nematodes were transferred to a new NGM plate, and the number of nematodes that died normally was recorded. The counting was stopped when all nematodes died. This process was repeated three times, and the nematode lifespan curve was plotted to calculate the average lifespan.

[0070] The results are as follows Figure 6 As shown.

[0071] The average lifespan of CL2006 was 15.43 ± 1.20 days. Treatment with 25 μg / mL PURA1 extended the average lifespan of the nematodes to 17.27 ± 1.26 days, an increase of 11.92% (p < 0.0001). Compared with the untreated control group, treatment with 25 μg / mL PURA1 resulted in... pmk-1 The survival rate of the defective strains under normal culture conditions did not change significantly. This experiment shows that PURA1 intervention can significantly prolong the lifespan of CL2006, but not extend it. pmk-1 The lifespan of the defective strains indicates that the anti-AD effect of PURA1, which delays senescence, is partially dependent on... pmk−1 .

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an extract of Uncaria rhynchophylla alkaloids, characterized in that, It includes the following steps: (1) The powder of Uncaria rhynchophylla stems and branches was extracted by heating with ethanol adjusted to acidity to obtain an acid-ethanol extract; the extract was concentrated by vacuum distillation to obtain a total alkaloid extract of Uncaria rhynchophylla. (2) After removing acid-insoluble matter, defatting, adjusting alkali, and extracting with chloroform, the total alkaloid extract was concentrated under reduced pressure by recovering the chloroform layer to obtain crude Uncaria rhynchophylla alkaloids. (3) Dissolve the crude alkaloids of Uncaria rhynchophylla in distilled water, add it to a macroporous resin chromatography column for adsorption, wash the macroporous resin with distilled water, elute with 5 column volumes of 20% ethanol solution, collect the eluent, distill under reduced pressure and dry to obtain Uncaria rhynchophylla alkaloid extract.

2. The preparation method according to claim 1, characterized in that, The Uncaria rhynchophylla stem and branch powder mentioned in step (1) is obtained by drying and pulverizing Uncaria rhynchophylla stems and branches; The step (1) of extracting Uncaria rhynchophylla stem and branch powder by heating with ethanol adjusted to acidity includes the following steps: 1g of Uncaria rhynchophylla stem and branch powder is added to 20mL of 80% ethanol solution with pH adjusted to 3, extracted at 70℃, filtered, and the acid-ethanol extract is obtained. The Uncaria rhynchophylla stem and branch powder mentioned in step (1) is extracted 3-5 times.

3. The preparation method according to claim 1, characterized in that, The removal of acid-insoluble matter in step (2) includes the following steps: dissolving the total alkaloid extract in hot water, adjusting the pH of the solution to 2 with hydrochloric acid, filtering, and removing acid-insoluble matter; The defatting process described in step (2) includes the following steps: extraction with petroleum ether, discarding the petroleum ether layer, and repeating 3-7 times; The alkali adjustment mentioned in step (2) involves using ammonia to adjust the pH of the solution to 10; The chloroform extraction described in step (2) involves 5-7 extractions.

4. The preparation method according to claim 1, characterized in that, The macroporous resin chromatography column in step (3) is AB-8 macroporous resin, and the column size is 16mm×40mm; In step (3), after adding the crude alkaloid solution of Uncaria rhynchophylla to the macroporous resin chromatography column, the solution is mixed with AB-8 macroporous resin and allowed to stand for 12-18 hours.

5. An extract of Uncaria alkaloids, characterized in that, It was prepared by the above method.

6. The Uncaria alkaloid extract according to claim 4, characterized in that, The aforementioned Uncaria alkaloid extract contains rhynchophylline A, cardanine, yohimbine, dehydrorhynchophylline, isodehydrorhynchophylline, conoxin, amarixin, isorhynchophylline, rhynchophylline, rhynchophylline, and rhynchophylline.

7. The use of the Uncaria alkaloid extract according to claim 4 in the preparation of anti-Alzheimer's disease and / or anti-aging drugs.

8. An anti-Alzheimer's disease and / or anti-aging drug, characterized in that, It contains the Uncaria alkaloid extract as described in claim 4.

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