Application of turbellarian worm extract in preparation of anti-aging product

Abstract

The invention provides application of turbellarian worm extract in preparation of anti-aging products, products for promoting the growth and development ability of individuals or delaying decline of exercise ability, products for reducing lipofuscin in organisms and medicines or biological preparations for reducing the content of p21 protein. Wherein the extraction of the turbellarian worm extract comprises the steps of turbellarian worm cleaning, tissue crushing, alcohol extraction, centrifugal separation and concentration drying. The turbellarian worm extract has a good anti-aging effect, effectively prolongs the service life of nematodes, improves the activity of senescence mice, delays hair loss of the senescence mice, reduces the lipofuscin level in organisms and the senescence marker p21 protein level, enhances the antioxidant capacity and immunity of natural senescence organisms, and has a good anti-aging effect. The method has wide potential application in anti-aging products.

Description

Technical Field

[0001] The invention relates to application of a planarian extract in preparing an anti-aging product. Background Art

[0002] Aging is associated with an increase in age-related diseases such as cancer, cardiovascular, metabolic, renal and neurodegenerative diseases, which are the leading causes of death in humans. They also lead to a decline in the quality of life of the elderly and increase social health care expenditures, thus bringing a double burden of physical and economic burden. Currently, research in the field of aging focuses on increasing healthy life expectancy, that is, the number of years a person is in good health, rather than increasing the life expectancy of old age. A more effective strategy that is now widely recognized is to treat aging as a whole rather than a single disease, which can more effectively address age-related diseases and extend the healthy life expectancy of the elderly.

[0003] In humans, the accumulation of senescent cells in the body is the main cause of aging. Most somatic cells have a limited lifespan and, after an exhaustive round of replication or the detection of certain damage, may undergo a state of essentially irreversible growth arrest, known as cellular senescence. Cellular senescence can be triggered by exogenous cell damage, such as DNA damaging agents, ultraviolet (UV) irradiation, or endogenous cellular stressors, such as age-related telomere loss or oncogene expression. Treatments targeting cellular senescence can alleviate the aging of the body. Therefore, exploring new mechanisms for extending the healthy lifespan of organisms and thus delaying aging is a difficult problem that needs to be solved urgently in today's society.

[0004] Turbellarians are animals of the class Turbellarians in the phylum Platyhelminthes. Although their morphology is simple, they have a complex anatomical structure, including the brain, eyespots, muscle tissue, intestines, pronephros and epidermis. Turbellarians have extremely powerful tissue regeneration and organ reconstruction capabilities. After being damaged, they can regenerate any missing parts of the body. Even if they are cut into 279 pieces, each part can still grow into a complete individual. Therefore, the regenerative lifespan mechanism of turbellarians has brought new hope for people to study anti-aging and overcoming aging, but there are not many studies on this kind of thing at present. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of a planarian extract in the preparation of an anti-aging product.

[0006] First, the turbellarian extract of the present invention comprises the following steps: a. Wash the planarian with PBS solution; b. Place the planarian in a PBS solution and grind the tissue until no obvious tissue blocks remain to obtain the planarian grinding solution; c. Place the planarian grinding solution obtained in step b into an alcohol solution so that the components in the planarian grinding solution are fully dissolved in the alcohol solution; d. Centrifuge and separate the mixture in step c, collect the supernatant, and filter the supernatant; and e. Concentrate and dry the filtered supernatant to obtain the planarian extract.

[0007] Wherein: in step a and step b, the concentration of the PBS solution is 0.01 M, and in step b, the dosage ratio of planarians to the PBS solution is 1 g of planarians: (1 - 10) ml of PBS solution; Preferably: in step c, the alcohol solution is a methanol or ethanol solution, and its volume concentration is 30 - 100%.

[0008] Preferably: in step c, the volume ratio of the planarian lysate to the alcohol solution is 1: (2 - 50).

[0009] Preferably: the concentration and drying is vacuum concentration and drying.

[0010] The planarian extract obtained by the extraction method as described above in the present invention can be used for preparing anti - aging products.

[0011] In addition, the planarian extract as described above in the present invention can be used for preparing products that promote the body length development ability of an individual and / or delay the decline of motor ability.

[0012] The planarian extract as described above in the present invention can also be applied to the preparation of products for reducing lipofuscin in organisms and drugs or biological agents for reducing the content of p21 protein, etc.

[0013] The planarian extract obtained by the extraction of the present invention has a good anti - aging effect. It can significantly increase the lifespan and vitality of nematodes, improve the activity of aging mice, delay hair loss in aging mice, reduce the lipofuscin level and the level of the aging marker p21 protein in organisms, and enhance the antioxidant capacity and immunity of naturally aging organisms, providing a new option for the preparation of anti - aging products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shows the analysis of the effect of the planarian extract on the lifespan of nematodes; Figure 2 Shows the analysis of the effect of the planarian extract on promoting the body length development of nematodes; Figure 3 Shows the analysis of the effect of the planarian extract on promoting the motor ability of nematodes; Figure 4 Shows the analysis of the effect of the planarian extract on the lipofuscin content in nematodes; Figure 5 Analysis of the effect of planarian extract on improving the individual behavioral ability of aged mice is shown; Figure 6 Analysis of the effect of planarian extract on reducing the content of senescence marker p21 protein in aged mice is shown; and Figure 7 Analysis of the effect of planarian extract on alleviating senile physiological hair loss is shown. Detailed implementation manners

[0015] First, for the planarian extract of the present invention, its extraction process includes the following steps: a. Wash planarians with 0.01M PBS solution, wherein the planarians are Dugesia japonica Dugesia japonica (), which can be obtained by direct market purchase or self-cultivation in the laboratory; b. Place the washed planarians in step a into a 0.01M PBS solution according to the ratio of 1 g of planarians: (1 - 10) ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue blocks, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into an alcohol solution according to a volume ratio of 1: (2 - 50), and use a rotary mixer to incubate by turning at 4°C for 48 hours to fully dissolve the components in the planarian grinding solution in the alcohol solution; in the present invention, the alcohol solution can be an ethanol solution or a methanol solution, and its volume concentration is 30 - 100%; d. Place the incubated mixture in step c into a centrifuge and centrifuge at a speed of 4000 rpm for 20 minutes, then collect the supernatant, and filter the supernatant with a needle-type sterilizing filter with a filtration accuracy of 0.22 μm - 0.45 μm; e. Concentrate and dry the filtered supernatant in step d using, for example, a vacuum centrifugal concentrator to obtain the solid planarian extract of the present invention.

[0016] The present invention also provides research on the anti-aging aspect of the planarian extract obtained by the above method, which mainly includes the following research contents: (1)Culturing and treating nematodes: Synchronize the nematodes that have been normally cultured to the L4 stage. Collect the obtained nematode eggs in a 2 ml centrifuge tube, resuspend them in M9 buffer, and incubate them in an incubator at 20 °C for 48 hours. When the time is up, centrifuge them at 4000 g for 5 minutes, transfer the obtained precipitate onto NGM medium containing OP50 Escherichia coli, and after culturing for 48 hours, pick an appropriate amount of nematodes onto NGM medium containing 40 mM / L FUDR (fluorodeoxyuridine) for subsequent experimental arrangements. In the present invention, for the treatment required for the lifespan experiment, the feeding method is adopted, that is, add 50 mg / ml planarian extract solution to 1 mL of OP50 Escherichia coli concentrated 5 times, and then take 50 μL of the bacterial solution and spread it on the NGM medium for the nematodes to feed on.

[0017] Among them, the NGM medium (calculated based on a 400 mL medium system) is prepared according to the composition in Table 1 below: Table 1 Composition of NGM medium

[0018] The M9 buffer (calculated based on a 500 mL solution system) is prepared according to the composition in Table 2 below: Table 2 Composition of M9 buffer

[0019] (2)Evaluate the anti-aging effect of the planarian extract. The efficacy evaluation mainly includes differences in nematode lifespan, body length, body swing frequency, head swing frequency, and lipofuscin content, etc.: For the statistics of lifespan, use the nematodes treated with the planarian extract solution in the above experiment (1). In the first 10 days, count the number of surviving nematodes every two days. After 10 days of treatment with the extract solution, count the number of surviving nematodes every day and draw a survival curve, and at the same time draw a survival curve for the control group; for the statistics of nematode body length, use an upright microscope to separately count and compare the body lengths of the nematodes in the control group and those treated with the planarian extract solution for 5 days and 10 days; and, statistically analyze the body swing frequency, head swing frequency, and lipofuscin level of the nematodes in the control group and those treated with the planarian extract solution for 5 days and 10 days.

[0020] (3) Cultivation and treatment of aged mice: SPF-grade 12-month-old C57BL / 6 male mice weighing 25±2g were selected (the experimental mice of the present invention were purchased from Liaoning Changsheng Biotechnology Co., Ltd., animal production license number: SCXK (Liao) 2020-0001, experimental unit use license number: SYXK (Yu) 2024-0010, and the entire experimental operation process complies with the "Animal Experiment Management Regulations"). The mice were raised in an SPF-grade experimental animal room, fed and cared for uniformly, the ambient temperature was maintained at 20~25℃, the relative humidity was 45~65%, and the day and night cycle was 12h. The sterile bedding was replaced on time every 3 days. Before the start of the experiment, the mice were free to eat and drink. After one week of adaptive feeding, the mice were weighed using a balance. After the mice were normal, the experiment was formally carried out. The experiment adopted the method of intraperitoneal injection. Because there was no significant difference in the weight of the mice, each mouse in the experimental treatment group was injected with 100μL of 1mg / mL planarian extract solution, and the control group was injected with an equal amount of physiological saline. Inject twice a week for 3 months without interruption.

[0021] (4) Evaluate the effect of planarian extract on improving the behavioral ability of aged mice: The open field test, the plus maze test and the rotating rod test were used to detect the behavioral ability of the mice treated in the above experiment (3). The experimental group and the control group mice were taken 3 months after modeling for the corresponding experiments, and the experimental results were recorded and analyzed.

[0022] (5) Evaluation of the effect of planarian extract on reducing the level of p21 protein, a marker of aging, in aged mice: The control group in the above experiment (3) and the mice treated with planarian extract solution for 3 months were dissected, and the liver tissue was extracted for protein extraction. The obtained mouse liver protein was subjected to Western Blot experiment to test the p21 protein content. ImageJ software was used to quantitatively analyze the grayscale value of the target protein, and the data were normalized with the target protein / GAPDH ratio.

[0023] (6) Evaluate the effect of planarian extract on alleviating physiological hair loss in mice: The grayscale analysis method was used to detect the hair condition of mice. The experimental group and the control group mice were photographed and sampled 3 months after modeling, and the images were analyzed using ImageJ software.

[0024] It should be noted that the planarian extract solutions involved in the present invention are prepared by dissolving the planarian extract with sterile deionized water to the required concentration for the experiment, and filtering and sterilizing with a filter with a filtration accuracy of 0.22μm-0.45μm, such as a syringe-type sterilizing filter, and then set aside.

[0025] Example 1 A method for extracting a planarian extract, the extraction step comprising: a. Wash the planarians twice with PBS solution at a concentration of 0.01M; b. Place the washed planarians from step a into PBS solution at a concentration of 0.01M according to the ratio of 1g of planarians: 1ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue chunks left, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into an ethanol solution with a volume concentration of 40% according to a volume ratio of 1:50, and use a rotary mixer to incubate with inversion at 4°C for 48 hours to fully dissolve the components in the planarian grinding solution in the ethanol solution; d. Place the incubated mixture in step c into a centrifuge and centrifuge at a speed of 4000rpm for 20 minutes, then collect the supernatant and filter the supernatant with a 0.22μm needle - type sterilizing filter; e. Use a vacuum centrifugal concentrator to concentrate and dry the filtered supernatant in step d at room temperature and a vacuum degree of 20hPa to obtain a solid - state planarian extract.

[0026] Example 2 A method for extracting a planarian extract, the extraction steps of which include: a. Wash the planarians twice with PBS solution at a concentration of 0.01M; b. Place the washed planarians from step a into PBS solution at a concentration of 0.01M according to the ratio of 1g of planarians: 1ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue chunks left, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into a methanol solution with a volume concentration of 75% according to a volume ratio of 1:20, and use a rotary mixer to incubate with inversion at 4°C for 48 hours to fully dissolve the components in the planarian grinding solution in the methanol solution; d. Place the incubated mixture in step c into a centrifuge and centrifuge at a speed of 4000rpm for 20 minutes, then collect the supernatant and filter the supernatant with a 0.22μm needle - type sterilizing filter; e. Use a vacuum centrifugal concentrator to concentrate and dry the filtered supernatant in step d at room temperature and a vacuum degree of 30hPa to obtain a solid - state planarian extract.

[0027] Example 3 A method for extracting a planarian extract, the extraction steps of which include: a. Wash the planarians twice with PBS solution at a concentration of 0.01M; b. Place the washed planarians in step a into a PBS solution with a concentration of 0.01 M according to the ratio of 1 g of planarians to 2 ml of solution, and use a tissue grinder to break the tissue at room temperature until there are no obvious tissue blocks, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b into an ethanol solution with a volume concentration of 90% according to a ratio of 1:20, and use a rotary mixer to incubate by turning at 4 °C for 48 hours to make the components in the planarian grinding solution fully dissolve in the ethanol solution; d. Place the incubated mixture in step c into a centrifuge and centrifuge at a speed of 4000 rpm for 20 minutes, then collect the supernatant, and filter the supernatant with a 0.22 μm needle-type sterilizing filter; e. Use a vacuum centrifugal concentrator to concentrate and dry the filtered supernatant in step d at room temperature and a vacuum degree of 25 hPa to obtain a solid planarian extract.

[0028] Analysis of the effect of planarian extract on the lifespan of Caenorhabditis elegans in Experimental Example 1 Transfer synchronized wild-type N2 Caenorhabditis elegans at the L4 stage to NGM medium containing FUDR (20 μmol), and then add 50 μL of Escherichia coli OP50 (containing 2 μL of normal saline) concentrated 5 times, 50 μL of Escherichia coli OP50 (containing 2 μL of a sterile deionized aqueous solution of the planarian extract in Example 1 at 50 mg / mL). Set up 2 parallel experiments, which are respectively recorded as the control group (CK) and the treatment group (DEE), and culture them at 20 °C, with 50 nematodes in each culture dish. Gently touch the nematodes with a platinum wire every day and observe the survival status of the nematodes, record the number of nematodes that survive, die, and are removed (crawl out of the medium, genital damage phenotype) until the last nematode dies, and use Prism statistical software to analyze the lifespan data to obtain the lifespan curve of the nematodes, and the results are as Figure 1 shown.

[0029] From Figure 1 the lifespan curve, it can be seen that for the nematodes treated with the planarian extract solution, their median lifespan is extended by more than 18% and their maximum lifespan is extended by more than 14%, indicating that the planarian extract has an obvious promoting effect on the lifespan of nematodes.

[0030] Analysis of the effect of planarian extract on increasing the body length of Caenorhabditis elegans in Experimental Example 2 Synchronized wild-type N2 Caenorhabditis elegans at the L4 stage were separately transferred to NGM medium containing FUDR (20 μmol) and cultured at 20 °C. Two parallel experiments were set up, denoted as the control group (CK) and the treatment group (DEE) respectively. On the 5th and 10th days of adulthood, 10 nematodes were randomly selected and transferred to a glass slide containing 20 μL of levamisole hydrochloride solution. After the nematodes were anesthetized, their bodies were straightened. The nematodes were photographed under an upright microscope. 30 nematodes were counted in each group, and the ImageJ software was used to calculate their body lengths. The results are as Figure 2 shown.

[0031] It can be seen from Figure 2 that compared with the control group, the average body length of the nematodes in the treatment group after being treated with planarian extract solution for 5 days increased by about 13.76%, and the average body length of the nematodes in the treatment group after being treated with planarian extract solution for 10 days increased by about 11.75%. This shows that the planarian extract effectively promoted the growth and development of nematodes.

[0032] Experimental Example 3 Analysis of the effect of planarian extract on the locomotor ability of nematodes Synchronized wild-type N2 Caenorhabditis elegans at the L4 stage were separately transferred to NGM medium containing FUDR (20 μmol) and cultured at 20 °C. Two parallel experiments were set up, denoted as the control group (CK) and the treatment group (DEE) respectively. On the 5th and 10th days of adulthood, 10 nematodes were randomly selected and transferred to a glass slide containing 100 μL of the M9 buffer solution described above. The nematodes were allowed to swim undisturbed for 1 minute to adapt to the new environment and remove the adhered bacteria. The number of head and body swings of the nematodes was counted under a stereomicroscope for 30 seconds. One swing of the head and body in opposite directions was counted as one swing, and all experiments were repeated three times. Figure 3 The number of swings of the head and body of the nematodes is shown respectively.

[0033] According to Figure 3 the results, it can be seen that compared with the control group, the average body swing frequency of the nematodes in the treatment group on the 5th day after being treated with planarian extract solution within 30 s increased by about 11.66% compared with the control group, and the average head swing frequency increased by about 23.84%; the average body swing frequency of the nematodes in the treatment group on the 10th day after being treated with planarian extract solution within 30 s increased by about 19% compared with the control group, and the average head swing frequency increased by about 26.47%. This indicates that the locomotor ability of the nematodes treated with planarian extract is stronger and they are more young and healthy.

[0034] Experimental Example 4 Analysis of the effect of planarian extract on the level of lipofuscin Lipofuscin, also known as senile pigment, is composed of fat residues and lysosomal digest, and its formation is usually closely related to oxidative stress and cell damage. Its content increases with age and is one of the important markers of aging.

[0035] Synchronized wild-type N2 Caenorhabditis elegans at the L4 stage were transferred to NGM medium containing FUDR (20 μmol) and cultured at 20 °C, and were respectively designated as the control group (CK) and the treatment group (DEE); on the 5th and 10th days of adulthood, 10 nematodes were randomly selected and transferred to a glass slide containing 20 μL of levamisole hydrochloride solution. After the nematodes were anesthetized, their bodies were placed in the correct position; an upright fluorescence microscope was used to observe the autofluorescence intensity of lipofuscin. Photos were taken in the DAPI channel using the same exposure time. At the same time, the relative autofluorescence level of lipofuscin was measured using ImageJ software, and the results are as Figure 4 shown.

[0036] As Figure 4 can be seen, compared with the control group, the lipofuscin content in the nematodes of the treatment group on the 5th day after treatment with planarian extract solution decreased by 22.31% compared with the control group; the lipofuscin content in the nematodes of the treatment group on the 10th day after treatment with planarian extract solution decreased by 24.75% compared with the control group of the same period. It shows that planarian extract has a significant anti-aging effect on nematodes.

[0037] Experimental Example 5 Analysis of the Effect of Planarian Extract on Improving the Individual Behavioral Ability of Aged Mice One week before the end of the planarian extract treatment and sampling in the above experimental research content (3), open field, elevated plus maze and rotarod tests were performed on the mice. The process is as follows: Open field test: An open field device with a length, width and height of 45 cm × 45 cm × 35 cm was used to detect the ethological experiment of free exploration of mice. Before the experiment, the room temperature and humidity were kept stable (generally the temperature was 25 °C and the humidity was 50%). The bottom of the open field was thoroughly cleaned with 75% alcohol and water to avoid interference from the residual smell of previous mouse urine or feces on the experimental test. The experimental mice were placed in the above environment to adapt for 30 min, and then the mice were gently placed in the central area of the open field. The central area was defined as a 20 cm × 20 cm area in the center of the open field, and they were allowed to move freely for 5 min. A camera was used to track, record and analyze the movement of the mice. After each mouse experiment, it was necessary to wipe it with 75% alcohol and water again to ensure that there was no smell interfering with the subsequent mice. The time the mice stayed in the central area of the open field device and the total movement distance were calculated and analyzed.

[0038] Elevated plus-maze experiment: This behavioral experiment is used to evaluate the anxiety and memory behaviors of mice. Similar to the open-field experiment, the environment stability is maintained before the experiment. The inside of the maze is thoroughly cleaned with 75% alcohol and water. The experimental mice are placed in the above environment to adapt for 30 min, and then placed in the central area of the maze to freely move for 5 min. The residence time of the mice in the open arms of the plus-maze and the total movement distance are statistically analyzed.

[0039] Rotarod test: This behavioral experiment is used to evaluate the motor coordination ability of mice. The preparation before the experiment is the same as that of the open-field experiment. All mice are given adaptive training, starting one day before the experiment. During the training, the rotarod rotates at a speed of 20 rpm per minute for 3 min. In a normal experiment, the speed of the rotarod steadily increases from 4 rpm to 40 rpm. The longest residence time of each mouse on the rotarod is recorded, and the experiment is repeated 3 times to calculate the average value.

[0040] Figure 5 The experimental results of the above experiments are shown. As described above, during the open-field experiment, the open field is divided into a central area and a peripheral area, and the activity ability of mice is reflected by the movement of mice in the two areas. The emotional change of mice in a new environment is from tension to excitement, then to exploration and finally adaptation. Mice in a young state are more active in movement and the total distance increases. When mice are in an aging state, their movement distance will be significantly shortened. From Figure 5 it can be seen that there is no significant difference in the average movement time of mice in the central area between the treatment group (DEE) and the control group (CK) ( Figure 5 -A), but the stability of each mouse in the treatment group (DEE) is stronger than that of the control group (CK). At the same time, the average total movement distance of mice in the treatment group in the open field is higher than that of the control group ( Figure 5 -B).

[0041] The elevated plus-maze experiment utilizes the fear psychology of mice towards a strange environment to make mice have contradictory self-behaviors. By statistically analyzing the time and movement distance of mice entering the open arms and closed arms, the anxiety state and movement ability of mice are reflected. From Figure 5 it can be seen that the average movement time of mice in the treatment group (DEE) in the open arms is stronger than that of mice in the control group (CK) ( Figure 5 -C), and the average total movement distance is also farther ( Figure 5 -D).

[0042] The rotarod test is used to detect the balance and motor coordination ability of mice. The animal must maintain balance on the rotating rod. From Figure 5It can be seen that the average movement time of the mice in the treatment group (DEE) on the rotarod was longer than that of the control group (CK), indicating that the motor coordination and balance ability of the mice treated with planarian extract were stronger than those of the control group mice ( Figure 5 -E).

[0043] Generally speaking, the individual differences of the aging mice treated with planarian extract were smaller, and the overall state tended to be younger and more stable, indicating that the extract could effectively improve the motor ability of aging mice, enhance the activity of aging mice, and make them younger.

[0044] Example 6 Effect of Planarian Extract on Reducing the Content of Aging Marker p21 Protein in Aged Mice After the mice were humanely sacrificed, their limbs were fixed on a foam board, and the livers of the mice were collected. During the organ collection process, a part of the fresh liver tissue was cut and minced, placed in a 2 mL EP tube. After pre-mixing the protease inhibitor and RIPA lysis buffer at a volume ratio of 1:100 on ice, an appropriate amount of the mixed lysis buffer was added to a 2 mL pre-cooled grinding tube according to the volume of the liver tissue, and at the same time, 2 low-temperature grinding beads were placed. The pre-cooled high-speed tissue grinder (4 °C) was used to perform intermittent crushing at an oscillation frequency of 60 Hz (grinding for 30 s - pausing for 10 s, with a total of 8 cycles), and then ice-bathed for 5 min and intermittently vortexed and mixed to promote protein dissolution. Subsequently, the lysis buffer was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was aspirated into a new tube, avoiding inhaling precipitate impurities. The obtained protein extract was immediately aliquoted and stored in a -80 °C ultra-low temperature freezer for later use. The whole operation process needed to be carried out on ice to ensure protein stability.

[0045] The BCA protein detection kit from Beyotime Institute of Biotechnology was used for protein concentration determination, and the specific process referred to its instruction manual; Mix the mouse liver protein sample with protein loading buffer (5×) at a ratio of 4:1, boil at 100 °C for 10 min to complete denaturation, cool in an ice bath for 5 min, then aliquot and store at -80 °C for later use. Before the experiment, select clean glass plates with uniform thickness, wipe them with absolute ethanol and air dry, then assemble the gel rack. Refer to the instructions of the PG211 gel kit (Shanghai YaMei Biotechnology) to prepare the SDS-PAGE separating gel and stacking gel, and let it stand for 30 min until it polymerizes fully. Install the polymerized gel plate into the vertical electrophoresis tank, pour in the newly prepared 1× electrophoresis buffer (the liquid level covers the sample loading wells), and add the pre-cooled protein samples in sequence. At the initial stage, electrophorese at a constant voltage of 90 V for 30 min to compress the samples into bands, and then increase the voltage to 110 V and continue electrophoresis for 60 min until the bromophenol blue indicator migrates to the bottom of the gel plate. Before transfer, pre-cool the transfer tank and prepare 1× transfer buffer. Activate the PVDF membrane with methanol for 30 s and then immerse it in the buffer synchronously with the gel. Assemble the transfer clip according to the "sandwich" structure (filter paper - gel - PVDF membrane - filter paper), place it in an ice bath environment, and transfer at a constant voltage of 110 V for 60 min. After transfer, block the PVDF membrane with 5% skim milk (prepared with TBST) at room temperature for 60 min, and then incubate it with the primary antibody working solution diluted according to the titer overnight at 4 °C. The next day, wash the membrane 3 times with TBST buffer (10 min each time), add the corresponding species HRP-labeled secondary antibody and incubate at room temperature for 120 min, and then wash the membrane 3 times with TBST again (10 min each time).

[0046] During the detection stage, prepare the working solution by mixing ECL luminescent reagent A and B in a ratio of 1:1 in the dark (refer to the instructions of the PG211 gel kit (Shanghai YaMei Biotechnology)), evenly drop it onto the target band area, and collect the signal with a chemiluminescence imaging system. Use ImageJ software to quantitatively analyze the gray value of the target protein, and normalize the data with the ratio of the target protein to GAPDH. The results are as Figure 6 shown.

[0047] The cell cycle-related factor p21 accumulates in senescent cells. This abnormal accumulation leads to the continuous activation of proteins in the Rb family, thereby inhibiting the trans-activation of E2F, and further causing an irreversible arrest of the cell cycle. In this invention, the aging level of mice was detected by sampling the mouse liver and performing WB experiments to detect the p21 protein content. From Figure 6 the detection results, it can be seen that the p21 level in the mouse liver treated with planarian extract is significantly lower than that in the control group (CK), indicating that planarian extract can effectively improve the activity of senescent cells in mice.

[0048] Experimental Example 7 Analysis of the effect of planarian extract on alleviating senile physiological hair loss Male C57BL / 6 strain mice at 12 months of age were intraperitoneally injected with the planarian extract solution of Example 1 of the present invention at a dosage of 100 μL per mouse, twice a week for 3 months. After 3 months, the hair condition on the backs of the mice was examined under a microscope to observe the hair condition on the backs of the mice. The results are as Figure 7 shown.

[0049] As can be seen Figure 7 from Figure 7 , during the treatment with the planarian extract, differences in the hair of the two groups of mice were found. Physiological hair loss and whitening of hair color occur during the normal aging process of mice. However, after 3 months of treatment with the planarian extract, the number and gray scale of the hair on the backs of the mice in the treatment group (DEE) were significantly higher than those in the control group (CK), indicating that the planarian extract can effectively delay physiological hair loss and graying of hair in aging mice.

[0050] Through the above experimental studies of the present invention, it can be known that the planarian extract has the functions of improving the healthy lifespan, promoting individual growth and development and behavioral ability, reducing the level of lipofuscin, reducing the content of the senescence marker p21 protein in elderly individuals, and alleviating senile physiological hair loss.

[0051] The present invention has been described in detail through preferred embodiments. However, through the study of the foregoing, changes and additions to each embodiment are also obvious to those of ordinary skill in the art. The intention of the applicant is that all such changes and additions fall within the scope of protection of the claims of the present invention. The terms used herein are only used to illustrate specific embodiments, and they are not intended to limit the present invention. Unless otherwise defined, all terms used herein (including technical terms and scientific terms) are the same as those understood by those of ordinary skill in the art to which the present invention belongs. Any modification and improvement of this product, and the substitution and use of similar or similar substances within the scope of the patent, fall within the scope of protection of the present invention.

Claims

1. Use of planarian extract in the preparation of anti-aging products.

2. Use of planarian extract in the preparation of products for promoting the body length development ability of individuals and / or delaying the decline of motor ability.

3. Use of planarian extract in the preparation of products for reducing lipofuscin in organisms.

4. Use of planarian extract in the preparation of drugs or biological agents for reducing the content of p21 protein.

5. The application according to any one of claims 1-4, characterized in that: The extraction steps of the planarian extract include: a. Wash planarians with PBS solution; b. Place planarians in PBS solution and grind the tissue until there are no obvious tissue chunks, obtaining a planarian grinding solution; c. Place the planarian grinding solution obtained in step b in an alcohol solution so that the components in the planarian grinding solution are fully dissolved in the alcohol solution; d. Centrifuge the mixture in step c, collect the supernatant, and filter the supernatant; and e. Concentrate and dry the filtered supernatant to obtain the planarian extract.

6. The application according to claim 5, characterized in that: In step a and step b, the concentration of the PBS solution is 0.01 M, and in step b, the dosage ratio of planarians to PBS solution is 1 g of planarians: (1 - 10) ml of PBS solution.

7. The application according to claim 5, characterized in that: In step c, the alcohol solution is methanol or ethanol solution, and its volume concentration is 30 - 100%.

8. The application according to claim 5, wherein: In step c, the volume ratio of the planarian crushing solution to the alcohol solution is 1: (2 - 50).

9. The application according to claim 5, characterized in that: The concentration and drying are vacuum concentration and drying.

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