Perilla frutescens extract product for activating mitochondrial autophagy and resisting aging and application thereof

Abstract

The invention belongs to the technical field of preparation and application of natural products, and particularly relates to a perilla frutescens extract product for activating mitochondrial autophagy and resisting aging and application of the perilla frutescens extract product. Perillaldehyde is obtained by purifying perilla leaves, and the purity reaches 99%. The compound has DPPH free radical scavenging capacity and tyrosinase activity inhibition capacity, and the DPPH free radical scavenging capacity is equivalent to that of vitamin E; the service life of the high-concentration perillaldehyde group is prolonged by 8%; the service life of caenorhabditis elegans under the environments of thermal stimulation, ultraviolet stimulation and oxide stimulation is remarkably prolonged, and the adversity service life prolonging rate of a high-concentration perillaldehyde group is at least 20%; sOD (superoxide dismutase) and CAT (catalase) activity of caenorhabditis elegans is remarkably improved, H2O2 accumulation is reduced, and anti-stress capability is improved; the caenorhabditis elegans mitochondrial autophagy is promoted, the mitochondrial autophagy pathway related genes are adjusted, the mitochondrial function is improved, the NAD < + > / NADH ratio is increased, the body metabolism is maintained, and the aging is delayed; the perillaldehyde capable of promoting the mitochondrial autophagy pathway can be applied to development of anti-aging drugs, health care products, food, cosmetics and other products.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product preparation and application, and particularly relates to a perilla extract product for activating mitophagy and anti-aging and its application. Background Art

[0002] Aging refers to the process in which, after the body grows, develops and matures, the physiological functions of the body gradually decline, the tissues and organs gradually undergo degenerative changes, and ultimately lead to death. The study of aging is also a frontier topic in biology and geriatrics. The mechanisms of aging include genetic and epigenetic changes, abnormal protein accumulation, oxidative stress, mitochondrial dysfunction, and cellular senescence (Y. Ratanapokasatit, Wannada Laisuan et al. “How Microbiomes Affect Skin Aging: The Updated Evidence and Current Perspectives.” Life (2022)). Mitochondria are the power source of cell energy. Healthy and efficient mitochondria can produce sufficient ATP for their own normal metabolism and can also produce antioxidants, so as to maintain free radicals in the body at an appropriate normal level. The activity of the mitochondrial respiratory chain decreases with age, the functions of mitochondrial complex I and complex IV decline, and the energy produced decreases (M. Rango, N. Bresolin. “Brain Mitochondria, Aging, and Parkinson’s Disease.” Genes (2018)). During the process of aging, ROS increases, further causing irreversible damage to DNA, proteins and biological membranes (Gang Zhong, J. Venkatesan et al. “Advances in Human Mitochondria-Based Therapies.” International Journal of Molecular Sciences (2022)).

[0003] Mitophagy is an important mechanism for clearing damaged mitochondria. However, with aging, the efficiency of autophagy declines, leading to the accumulation of damaged mitochondria (A. Terman, T. Kurz et al. “Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging..” Antioxidants & redox signaling (2010).). This accumulation further exacerbates mitochondrial dysfunction and the production of ROS, forming a vicious cycle (G. López-Lluch. “Mitochondrial activity and dynamics changes regarding metabolism in ageing and obesity.” Mechanisms of Ageing and Development (2017).). After mitochondria are damaged, the cell's self-repair mechanism is activated. By selectively clearing damaged or dysfunctional mitochondria, the balance of the quality and quantity of mitochondria within the cell is maintained, which is important for the functional integrity of the entire mitochondrial network and cell survival. Therefore, promoting mitophagy can improve mitochondrial function, rapidly and efficiently clear damaged mitochondria, increase the NAD+ / NADH ratio, and achieve the effect of delaying aging.

[0004] Perilla frutescens is an annual upright herbaceous plant of the genus Perilla in the Lamiaceae family. It is widely distributed, has a specific aroma, is warm in nature and pungent in taste, and is a traditional oil crop that can be used both as medicine and food. The 2020 edition of the Chinese Pharmacopoeia records that Perilla has the effects of relieving exterior cold, regulating qi and harmonizing the stomach, and is used for wind-cold colds, cough and nausea, pregnancy vomiting, and fish and crab poisoning. Perilla leaves can treat asthma, colds, chest tightness, vomiting, constipation, and abdominal pain, and can promote gastric function (HATJ, LEE JH, LEE MH, et al. Isolation and identification of phenolic compounds from the seeds of Perilla frutescens (L.) and their inhibitory activities against a-glucosidase and aldose reductase [J]. Food Chemistry, 2012, 135(3): 1397-1403.). Perilla fruits can reduce phlegm, moisten the intestines, and relieve asthma. The stems and stalks are often used as analgesics and have the effects of treating asthma, stroke, and preventing miscarriage. In addition to its medicinal use, the tender stems and leaves of Perilla can be eaten cold or made into soups, and can also be used as a spice to remove fishy smell, enhance flavor, and increase freshness; the seeds can be pressed for oil and used for meat preservation and food flavoring (VLKOVA M, POLESNY Z, VERNER V, et al. Ethnobotanical knowledge and agrobiodiversity in subsistence farming: case study of home gardens in Phongmy commune, central Vietnam [J]. Genetic Resources and Crop Evolution, 2011, 58(5): 629-644.).

[0005] Perilla contains a variety of chemical components, including volatile oils, flavonoids, phenolic acids, terpenoids, citronellol, etc. As a natural monoterpenoid volatile substance extracted from Perilla, perillaldehyde is one of the main active components of Perilla's pungent aroma and qi-regulating effect. Currently, there is no research on its anti-aging effect by acting on mitochondria. Summary of the Invention

[0006] The present invention discovers that by purifying Perilla leaves to obtain the volatile extract perillaldehyde of Perilla, it can promote mitophagy to exert anti-aging activity, extend the natural lifespan of Caenorhabditis elegans, improve the stress resistance of nematodes, increase the ratio of NAD+ / NADH, and can be applied to the development of anti-aging drugs, health products, foods, cosmetics and other products. Based on this, the present invention is completed.

[0007] In a first aspect, the present invention provides a method for preparing perillaldehyde, and the method comprises the following steps:

[0008] S1. Put perilla leaves into an ethanol solution,

[0009] S2. Add polyethylene glycol in step S1 and then beat into a pulp, and filter to obtain perilla liquid;

[0010] S3. Add a complex enzyme to the perilla liquid obtained in step S2, carry out ultrasonic enzymolysis, heat to inactivate the enzyme, and filter to obtain an enzymolysis solution;

[0011] S4. Carry out macroporous resin adsorption and molecular distillation purification on the enzymolysis solution in step S3 to obtain perillaldehyde, and the purity of perillaldehyde is 99%.

[0012] In a second aspect, the application of perillaldehyde prepared by the method as described in the first aspect of the present invention in the preparation of anti-aging products.

[0013] Furthermore, the products include but are not limited to skin care products, foods, health products and medicines.

[0014] Furthermore, the purity of the perillaldehyde is 99%.

[0015] Furthermore, the content of perillaldehyde in the anti-aging product is selected from 0.03% - 5%, preferably 0.05% - 3%.

[0016] In a third aspect, the present invention provides a skin care product of anti-aging essence type, and the skin care product of anti-aging essence type includes an oil phase, an aqueous phase and an anti-aging component.

[0017] Furthermore, the oil phase components of the skin care product of anti-aging essence type include but are not limited to ethylhexylglycerin.

[0018] Furthermore, the mass ratio of the ethylhexylglycerin is selected from 0.1% - 2%, preferably 0.8%.

[0019] Furthermore, the aqueous phase of the skin care product of anti-aging essence type is selected from one or more of butylene glycol, glycerol, hydroxyethylurea, disodium ethylenediaminetetraacetate, hyaluronic acid, propylene glycol, ethanol and / or distilled water.

[0020] Furthermore, the mass ratio of the butylene glycol is selected from 1% - 19%, preferably 8%.

[0021] Furthermore, the mass ratio of the hydroxyethylurea is selected from 1% - 20%, preferably 8%.

[0022] Furthermore, the mass ratio of the disodium ethylenediaminetetraacetate is selected from 0.02% - 0.2%, preferably 0.05%.

[0023] Furthermore, the mass ratio of the glycerol is selected from 5%-20%, preferably 10%.

[0024] Furthermore, the mass ratio of the hyaluronic acid is selected from 0.1%-0.5%, preferably 0.4%.

[0025] Furthermore, the mass ratio of the propylene glycol is selected from 1%-10%, preferably 5%.

[0026] Furthermore, the mass ratio of the ethanol is selected from 0%-5%, preferably 4%.

[0027] Further, the anti-aging ingredients of the anti-aging essence skin care products include but are not limited to perillaldehyde; the perillaldehyde is obtained by the method described in the first aspect of the present invention; the purity of the perillaldehyde is 99%.

[0028] Furthermore, the mass ratio of the perillaldehyde is selected from 1%-5%, preferably 2%.

[0029] In the fourth aspect, the present invention provides an anti-aging emulsion skin care product, which includes an oil phase, an aqueous phase and anti-aging ingredients.

[0030] Further, the oil phase of the anti-aging emulsion skin care product is selected from one or more of stearyl alcohol, glyceryl monostearate, isopropyl myristate, squalane, propylparaben and / or vitamins.

[0031] Furthermore, the mass ratio of the stearyl alcohol is selected from 0.5%-5%, preferably 0.8%.

[0032] Furthermore, the mass ratio of the glyceryl monostearate is selected from 0.5%-5%, preferably 0.8%.

[0033] Furthermore, the mass ratio of the isopropyl myristate is selected from 1%-10%, preferably 2%.

[0034] Furthermore, the mass ratio of the squalane is selected from 2%-10%, preferably 3%.

[0035] Furthermore, the mass ratio of the propylparaben is selected from 0.1%-0.3%, preferably 0.1%.

[0036] Furthermore, the vitamin in the oil phase is vitamin A and / or vitamin E; the mass ratio of the vitamin is selected from 0.1%-2%, preferably 0.2%.

[0037] Further, the aqueous phase of the anti-aging emulsion skin care product is selected from one or more of polyvinyl alcohol, hydroxyethyl cellulose, vitamins, glycerol and / or distilled water.

[0038] Furthermore, the mass ratio of the polyvinyl alcohol is selected from 1% - 10%, preferably 8%.

[0039] Furthermore, the mass ratio of the hydroxyethyl cellulose is selected from 0.1% - 2%, preferably 0.1%.

[0040] Further, the vitamins in the aqueous phase are vitamin B and / or vitamin C.

[0041] Furthermore, the mass ratio of the vitamins is selected from 0.1% - 2%, preferably 0.2%.

[0042] Furthermore, the mass ratio of the glycerol is selected from 2% - 20%, preferably 4%.

[0043] Further, the anti-aging ingredients of the anti-aging lotion skin care products include but are not limited to perillaldehyde; the perillaldehyde is obtained by the method described in the first aspect of the present invention; the purity of the perillaldehyde is 99%.

[0044] Furthermore, the mass ratio of the perillaldehyde is selected from 1% - 5%, preferably 1%.

[0045] In the fifth aspect, the present invention provides an anti-aging fruit juice, and the ingredients of the anti-aging fruit juice include but are not limited to fruit juice, citric acid, vitamins, minerals, carbohydrates, water and / or anti-aging ingredients.

[0046] Further, the fruit juice includes but is not limited to flavored fruit juice and freshly squeezed fruit juice.

[0047] Further, the mass ratio of the citric acid is selected from 0.1% - 1%, preferably 0.5%.

[0048] Further, the vitamins include but are not limited to vitamin A, vitamin E, vitamin C and / or vitamin B, preferably vitamin C.

[0049] Furthermore, the mass ratio of the vitamins is selected from 0.01% - 0.15%, preferably 0.05%.

[0050] Furthermore, the mass ratio of the minerals is selected from 0.5% - 2%, preferably 1%.

[0051] Further, the mass ratio of the carbohydrates is selected from 2% - 20%, preferably 10%.

[0052] Further, the anti-aging ingredients of the anti-aging fruit juice include but are not limited to perillaldehyde; the perillaldehyde is obtained by the method described in the first aspect of the present invention; the purity of the perillaldehyde is 99%.

[0053] Further, the mass ratio of the perillaldehyde is selected from 0.01% - 0.2%, preferably 0.03%.

[0054] In a sixth aspect, the present invention provides an anti-aging sparkling water, and the ingredients of the anti-aging sparkling water include, but are not limited to, fruit juice, citric acid, vitamins, minerals, water, infused gas, and / or anti-aging ingredients.

[0055] Further, the fruit juice includes, but is not limited to, flavored fruit juice and freshly squeezed fruit juice.

[0056] Further, the mass ratio of the citric acid is selected from 0.1% - 1%, preferably 0.5%.

[0057] Further, the vitamins include, but are not limited to, vitamin A, vitamin E, vitamin C, and / or vitamin B, preferably vitamin C.

[0058] Furthermore, the mass ratio of the vitamins is selected from 0.01% - 0.15%, preferably 0.05%.

[0059] Further, the mass ratio of the minerals is selected from 0.5% - 2%, preferably 1%.

[0060] Further, the infused gas is carbon dioxide.

[0061] Furthermore, the infusion amount of the gas is selected from 3.9 g / L - 7.8 g / L, preferably 4.8 g / L.

[0062] Further, the anti-aging ingredients of the anti-aging sparkling water include, but are not limited to, perillaldehyde; the perillaldehyde is obtained by the method described in the first aspect of the present invention; the purity of the perillaldehyde is 99%.

[0063] Further, the mass ratio of the perillaldehyde is selected from 0.01% - 0.2%, preferably 0.03%.

[0064] In a seventh aspect, the present invention provides an anti-aging candy, and the ingredients of the anti-aging candy include, but are not limited to, citric acid, vitamins, granulated sugar, glucose syrup, water, and / or anti-aging ingredients.

[0065] Further, the mass ratio of the citric acid is selected from 0.1% - 1%, preferably 0.8%.

[0066] Further, the vitamins include, but are not limited to, vitamin A, vitamin E, vitamin C, and / or vitamin B, preferably vitamin C.

[0067] Furthermore, the mass ratio of the vitamins is selected from 0.01% - 0.1%, preferably 0.1%.

[0068] Further, the mass ratio of the granulated sugar is selected from 10% - 50%, preferably 40%.

[0069] Further, the mass ratio of the glucose syrup is selected from 10%-30%, preferably 25%.

[0070] Further, the anti-aging components of the anti-aging candy include but are not limited to perillaldehyde; the perillaldehyde is obtained by the method described in the first aspect of the present invention; the purity of the perillaldehyde is 99%.

[0071] Further, the mass ratio of the perillaldehyde is selected from 0.01%-0.2%, preferably 0.05%.

[0072] Beneficial effects

[0073] It is found in this application that the perillaldehyde obtained by purifying perilla leaves has the ability of anti-aging, and its advantages include the following points:

[0074] (1) The purity of the perillaldehyde obtained by extraction in this application can reach 99%;

[0075] (2) It has the ability to scavenge DPPH free radicals and inhibit tyrosinase activity, and its ability to scavenge DPPH free radicals is equivalent to that of vitamin E;

[0076] (3) It can significantly extend the natural lifespan of Caenorhabditis elegans. Compared with the negative control group, the lifespan extension rate of the high-concentration perillaldehyde group can reach 8%;

[0077] (4) Significantly extend the lifespan of Caenorhabditis elegans in the environment of heat stimulation, ultraviolet stimulation, and oxidative stimulation, and improve the stress resistance of Caenorhabditis elegans. Compared with the negative control group, the adversity lifespan extension rate of the high-concentration perillaldehyde group is at least 20%;

[0078] (5) Significantly improve the physiological indexes of Caenorhabditis elegans, increase the activities of SOD and CAT, reduce the accumulation of H2O2, improve the stress resistance of Caenorhabditis elegans, and delay aging;

[0079] (6) Promote mitochondrial autophagy in Caenorhabditis elegans, regulate genes related to the mitochondrial autophagy pathway, improve mitochondrial function, increase the NAD+ / NADH ratio, maintain body metabolism, and play a role in delaying aging;

[0080] (7) Perillaldehyde that promotes the mitochondrial autophagy pathway can be applied to the development of products such as anti-aging drugs, health products, foods, and cosmetics.

[0081]

[0082] (7) Perillaldehyde that promotes the mitochondrial autophagy pathway can be applied to the development of products such as anti-aging drugs, health products, foods, and cosmetics. Description of the drawings

[0083] Figure 1 It is a schematic diagram of the technical process of the present invention.

[0084] Figure 2It is for detecting the purity of perillaldehyde in Example 1 of the present invention by GC-MS.

[0085] Figure 3 It is the evaluation result of the DPPH free radical scavenging ability of the perillaldehyde in Example 2 of the present invention.

[0086] Figure 4 It is the evaluation result of the inhibitory ability of the perillaldehyde on tyrosinase activity in Example 2 of the present invention.

[0087] Figure 5 It is the evaluation result of the natural lifespan of Caenorhabditis elegans after administration of the perillaldehyde in Example 3 of the present invention.

[0088] Figure 6 It is the evaluation result of the heat stress resistance ability of Caenorhabditis elegans after administration of the perillaldehyde in Example 4 of the present invention.

[0089] Figure 7 It is the evaluation result of the ultraviolet stress resistance ability of Caenorhabditis elegans after administration of the perillaldehyde in Example 4 of the present invention.

[0090] Figure 8 It is the evaluation result of the antioxidant stress ability of Caenorhabditis elegans after administration of the perillaldehyde in Example 4 of the present invention.

[0091] Figure 9 It is the evaluation result of the SOD activity in Caenorhabditis elegans after administration of the perillaldehyde in Example 5 of the present invention.

[0092] Figure 10 It is the evaluation result of the CAT activity in Caenorhabditis elegans after administration of the perillaldehyde in Example 5 of the present invention.

[0093] Figure 11 It is the evaluation result of the H2O2 content in Caenorhabditis elegans after administration of the perillaldehyde in Example 5 of the present invention.

[0094] Figure 12 It is the transcriptome analysis result of Caenorhabditis elegans after administration of the perillaldehyde in Example 6 of the present invention.

[0095] Note: A. Volcano plot; B. KEGG enrichment pathway map.

[0096] Figure 13 It is the schematic diagram of the anti-aging mechanism of Caenorhabditis elegans after administration of the perillaldehyde in Example 6 of the present invention.

[0097] Figure 14 It is the verification result of the gene expression of the mitophagy pathway of Caenorhabditis elegans after administration of the perillaldehyde in Example 6 of the present invention.

[0098] Figure 15 It is the product diagram of the perillaldehyde applied to the anti-aging skin care essence in Preparation Example 1 of the present invention. Detailed implementation manners

[0099] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is used to help understand the present invention, but does not limit the present invention. In addition, the technical features involved in the following described implementation manners can be combined with each other as long as they do not conflict with each other.

[0100] In the following experimental methods in the following examples, unless otherwise specified, they are all conventional methods. The test materials used in the following examples, unless otherwise specified, can all be obtained through conventional commercial channels.

[0101] Example 1 Extraction and purification of perillaldehyde

[0102] A. Test method

[0103] (1) Pretreatment: Add ethanol solution to the freshly picked perilla leaves and make a pulp, and add a dispersion promoter, continue to make a pulp, and filter to obtain perilla liquid and perilla residue;

[0104] (2) Enzymolysis: Add a complex enzyme and cysteine hydrochloride to the above-prepared perilla liquid, perform enzymolysis under ultrasonic assistance, heat to inactivate the enzyme, and filter to obtain an enzymolyzed liquid;

[0105] (3) Purification: Add macroporous adsorption resin to the above-prepared enzymolyzed liquid, stir and then let stand, filter to obtain a preliminary purified liquid; perform molecular distillation on the preliminary purified liquid, collect the fractions, and finally obtain pure perillaldehyde.

[0106] B. Test results

[0107] As Figure 2 shown, detected by GC-MS, the purity of perillaldehyde is 99%.

[0108] Example 2 In vitro efficacy verification experiment of perillaldehyde

[0109] A. Test method

[0110] Use the perillaldehyde prepared in Example 1 as the test group; set a positive control group, and the group settings are shown in Table 1:

[0111] Table 1 Group settings for in vitro efficacy verification test of perillaldehyde

[0112]

[0113] Determine according to the instructions of the DPPH free radical scavenging ability kit and the tyrosinase activity kit.

[0114] (1) The detection method of DPPH free radical scavenging ability refers to the instruction manual of the DPPH free radical scavenging ability kit (Nanjing Jiancheng). The specific steps are shown in Table 2:

[0115] Table 2 Detection steps of DPPH free radical scavenging ability of perillaldehyde

[0116]

[0117]

[0118] Result calculation formula:

[0119] DPPH free radical scavenging rate of sample (%) = (1 - (A determination - A control) ÷ A blank) × 100%

[0120] (2) The detection method of tyrosinase activity refers to the instruction manual of the tyrosinase activity detection kit. The specific steps are as follows:

[0121] Preparation of the test solution: The sample determination solution is mixed with the 2 kU / mL tyrosinase dilution solution in equal proportion.

[0122] Detection method of the kit: Adjust the wavelength to 475 nm, zero with pure water. Add Reagent 1 (the main component is the substrate that can react with tyrosinase) to the above-mentioned test solution, start timing immediately, and measure the absorbance value at 10 s (total time); continue the reaction at room temperature and measure the absorbance value at 190 s (total time).

[0123] Result calculation formula:

[0124] Inhibition rate of tyrosinase activity of sample (%) = (1 - (C - D) / (A - B)) × 100%

[0125] (A is the absorbance value at 475 m at 10 s of the blank group; B is the absorbance value at 190 s of the blank group; C is the absorbance value at 10 s of the test solution group; D is the absorbance value at 190 s of the test solution group.)

[0126] B. Test results

[0127] As Figure 3 and Figure 4 shown, with the increase of concentration, the DPPH free radical scavenging ability and tyrosinase activity inhibition ability of perillaldehyde increase. The DPPH free radical scavenging ability and tyrosinase activity of 50 mg / mL perillaldehyde can reach 80% and 40% respectively, indicating that perillaldehyde has good in vitro antioxidant activity and tyrosinase activity inhibition ability. In the scientific research practice of the present invention, it is found that the DPPH free radical scavenging ability of perillaldehyde is equivalent to that of vitamin E.

[0128] Example 3 Evaluation of the natural lifespan of Caenorhabditis elegans after administration of perillaldehyde

[0129] A. Test method

[0130] Wild-type Caenorhabditis elegans was selected and cultured at a temperature of 18 - 22 °C. After synchronous culture to the L4 larval stage, drugs were administered to each group. A negative control group, a positive control group, and experimental groups were set up, and the group settings are as follows:

[0131] Negative control group (DMSO): The solvent of perillaldehyde (0.1% concentration DMSO aqueous solution) was mixed with Escherichia coli OP50 for drug administration;

[0132] Positive control group (ASTA): 0.382 mg / mL astaxanthin was mixed with Escherichia coli OP50 for drug administration;

[0133] Low-dose experimental group (PA-L): 0.05 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for drug administration;

[0134] Medium-dose experimental group (PA-M): 0.1 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for drug administration;

[0135] High-dose experimental group (PA-H): 0.2 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for drug administration.

[0136] Using the method of controlling variables, drugs were administered to the above groups every day, and the survival and death of the nematodes were recorded. The situation was observed and recorded until all the nematodes died.

[0137] B. Test results

[0138] The results are as Figure 5 shown in Table 3. Compared with the DMSO group, the natural life curve of the experimental groups shifted significantly to the right, and the natural life extension rate increased with the increase in concentration. The natural life extension rate of the PA-H group reached 7.87%, approaching the ASTA group, indicating that perillaldehyde can significantly extend the natural life of Caenorhabditis elegans and has the effect of anti-aging.

[0139] Table 3 Effect of perillaldehyde on the natural life of Caenorhabditis elegans

[0140]

[0141] Example 4 Effect of perillaldehyde on the stress resistance of Caenorhabditis elegans

[0142] A. Test method

[0143] Wild-type Caenorhabditis elegans was selected and cultured at a temperature of 18 - 22 °C. After synchronous culture to the L4 larval stage, drugs were administered to each group. A negative control group, a positive control group, and experimental groups were set up, and the group settings are as follows:

[0144] Negative control group (DMSO): The solvent of perillaldehyde (aqueous solution of 0.1% DMSO) was mixed with Escherichia coli OP50 for administration;

[0145] Positive control group (ASTA): 0.382 mg / mL astaxanthin was mixed with Escherichia coli OP50 for administration;

[0146] Low-dose test group (PA-L): 0.05 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for administration;

[0147] Medium-dose test group (PA-M): 0.1 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for administration;

[0148] High-dose test group (PA-H): 0.2 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for administration.

[0149] Caenorhabditis elegans was cultured, and a stress test was performed after continuous administration for 5 days.

[0150] (1) Heat stress

[0151] The nematodes were transferred to a 37 °C incubator for high-temperature culture (the rest of the culture environment remained unchanged). The survival and death of the nematodes were recorded every 1 hour of heat culture until all nematodes died.

[0152] (2) UV stress

[0153] The nematodes were transferred to an incubator irradiated with 8W UVB for UV irradiation culture (the rest of the culture environment remained unchanged). The survival and death of the nematodes were recorded every 12 hours of UV irradiation culture until all nematodes died.

[0154] (3) Oxidative stress

[0155] The nematodes were transferred to a culture medium containing 5 mM hydrogen peroxide for peroxidation culture (the rest of the culture environment remained unchanged). The survival and death of the nematodes were recorded every 2 hours of culture until all nematodes died.

[0156] B. Test results

[0157] Adverse environments include heat stress, UV stress, and oxidative stress. Among them, heat stress can produce a large amount of ROS in the body, cause damage to the body and lead to a shortened lifespan. Long-term UV irradiation can accelerate aging. Hydrogen peroxide treatment can cause acute oxidative damage to the body, resulting in the accumulation of reactive oxygen species in the body and leading to rapid aging.

[0158] (1) Heat stress

[0159] The results are as Figure 6As shown in Table 4, compared with the DMSO group, the thermal environment life curve of the experimental group shifted significantly to the right, and the life extension rate of the PA-H group in the thermal environment reached 19.92%, indicating that perillaldehyde can significantly extend the lifespan of Caenorhabditis elegans in a thermal environment, improve its ability to resist heat stress, and thus play an anti-aging effect.

[0160] (2) Ultraviolet stress

[0161] As Figure 7 shown in Table 4, compared with the DMSO group, the ultraviolet irradiation environment life curve of the experimental group shifted significantly to the right, and the life extension rate of the PA-H group in the ultraviolet irradiation environment reached 43.07%, indicating that perillaldehyde can significantly extend the lifespan of Caenorhabditis elegans in an ultraviolet irradiation environment, improve its ability to resist ultraviolet stress, and thus play an anti-aging effect.

[0162] (3) Oxidative stress

[0163] As Figure 8 shown in Table 4, compared with the DMSO group, the oxidative stress environment life curve of the experimental group shifted significantly to the right, and the life extension rate of the PA-H group in the ultraviolet irradiation environment reached 27.79%, indicating that perillaldehyde can significantly extend the lifespan of Caenorhabditis elegans in an oxidative stress environment, improve its antioxidant stress ability, and thus play an anti-aging effect.

[0164] In summary, perillaldehyde can improve the stress resistance of the body and extend the lifespan in adverse environments. In scientific research practice, it was found that perillaldehyde can significantly extend the lifespan of Caenorhabditis elegans under thermal stimulation, ultraviolet stimulation, and oxidative stimulation environments, improve the stress resistance of Caenorhabditis elegans, and compared with the negative control group, the adverse environment life extension rate of the high-concentration perillaldehyde group reached at least 20%.

[0165] Table 4 Effects of perillaldehyde on the stress resistance of Caenorhabditis elegans

[0166]

[0167] Example 5 Effects of perillaldehyde on the physiological indexes of Caenorhabditis elegans

[0168] A. Test method

[0169] Wild-type Caenorhabditis elegans were selected and cultured at a temperature of 18 - 22°C. After synchronous culture to the L4 larval stage, they were grouped for drug administration; a negative control group, a positive control group, and an experimental group were set, and the group settings were as follows:

[0170] Negative control group (DMSO): The perillaldehyde solvent (0.1% concentration DMSO aqueous solution) was mixed with Escherichia coli OP50 for drug administration;

[0171] Positive control group (ASTA): Administer a mixture of 0.382 mg / mL astaxanthin and Escherichia coli OP50;

[0172] Low-dose test group (PA-L): Administer a mixture of 0.05 mg / mL perillaldehyde and Escherichia coli OP50;

[0173] Medium-dose test group (PA-M): Administer a mixture of 0.1 mg / mL perillaldehyde and Escherichia coli OP50;

[0174] High-dose test group (PA-H): Administer a mixture of 0.2 mg / mL perillaldehyde and Escherichia coli OP50.

[0175] Culture Caenorhabditis elegans, and extract the total protein of the nematodes after continuous administration for 5 days to measure their physiological indexes.

[0176] B. Test results

[0177] When the body is in a state of oxidative stress for a long time, the energy metabolism in the body will be affected, thereby promoting aging. SOD and CAT are antioxidant enzymes that catalyze the decomposition of reactive oxygen species widely present in the body. At the same time, when the body enters the peroxidation state, H2O2 will accumulate. Therefore, the determination of SOD, CAT and H2O2 is often used to evaluate antioxidant activity.

[0178] As Figure 9 and Figure 10 shown, compared with the DMSO group, the activities of SOD and CAT in the experimental groups were significantly increased and increased with the increase of the administration concentration, indicating that perillaldehyde can significantly increase the activities of SOD and CAT in Caenorhabditis elegans, improve the antioxidant ability and delay aging.

[0179] As Figure 11 shown, compared with the DMSO group, the content of H2O2 in the ASTA group, PA-M group and PA-H group was significantly decreased, and the accumulation of H2O2 in the PA experimental group decreased with the increase of the concentration, indicating that perillaldehyde can significantly reduce the content of H2O2 in Caenorhabditis elegans, reduce the accumulation of peroxides, improve the phenomenon of biological oxidation and delay aging.

[0180] In summary, perillaldehyde can improve the antioxidant ability of the body, reduce the peroxidation level and extend the healthy lifespan. In scientific research practice, it was found that perillaldehyde can significantly improve the physiological indexes of Caenorhabditis elegans, increase the activities of SOD and CAT, reduce the accumulation of H2O2, and play a role in delaying aging.

[0181] Example 6 Verification of the promotion of mitochondrial autophagy pathway in Caenorhabditis elegans by perillaldehyde

[0182] A. Test method

[0183] Wild-type Caenorhabditis elegans was selected and cultured at a temperature of 18-22 °C. After synchronous culture to the L4 larval stage, drugs were administered in groups. A negative control group, a positive control group, and an experimental group were set up, and the group settings are as follows:

[0184] Negative control group (DMSO): The perillaldehyde solvent (0.1% concentration DMSO aqueous solution) was mixed with Escherichia coli OP50 for drug administration;

[0185] Positive control group (ASTA): 0.382 mg / mL astaxanthin was mixed with Escherichia coli OP50 for drug administration;

[0186] Low-dose experimental group (PA-L): 0.05 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for drug administration;

[0187] Medium-dose experimental group (PA-M): 0.1 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for drug administration;

[0188] High-dose experimental group (PA-H): 0.2 mg / mL perillaldehyde was mixed with Escherichia coli OP50 for drug administration.

[0189] Caenorhabditis elegans was cultured, and after continuous drug administration for 5 days, transcriptome sequencing was performed and verified by qPCR.

[0190] The qPCR verification experiment includes the following steps:

[0191] (1) Total RNA was extracted using the Trizo1 method, mRNA with a polyA tail was enriched, or ribosomal RNA was removed from the total RNA to obtain mRNA;

[0192] RNA, thus obtaining mRNA;

[0193] (2) The RNA was fragmented into short fragments. Using the short fragment RNA as a template, the first-strand cDNA was synthesized with a hexamer random primer; (3) Buffer, dNTPs, and DNA polymerase 1 were added to synthesize the second-strand cDNA, and then the double-stranded cDNA was purified; the purified double-stranded cDNA was subjected to end repair, A-tailing, and ligation of sequencing adapters, and then the fragment size was selected, and the final cDNA library was enriched by PCR;

[0194] (4) After the library construction was completed, preliminary quantification was performed by the dye method, the insert size of the library was detected using a fragment analyzer, and after the insert size met the expectations, the effective concentration of the library was accurately quantified by the qPCR method (library effective concentration > 2n);

[0195] concentration > 2n);

[0196] After the library inspection is qualified, different libraries are pooled according to the target output data volume and sequenced using the Illumina platform;

[0197] The sequenced output data is filtered to obtain CleanData, and the

[0198] Mapped Data is obtained by sequence alignment with the specified reference genome;

[0199] (6) Use HISAT2 to perform sequence alignment between Clean Reads and the reference genome to obtain the position information on the reference genome or gene; use FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) as an indicator to measure the expression level of transcripts or genes; use DESeq2 to perform differential expression analysis between sample groups to obtain the differential expression gene set between two biological conditions;

[0200] (7) Use the Benjamini-Hochberg method to perform multiple hypothesis testing correction on the hypothesis testing probability (P value) to obtain the false discovery rate (FDR). Take log2FoldChange >= 1 and FDR < 0.05 as the screening conditions to screen for differentially expressed genes (DEGs). Finally, use GOSeq and KOBAS to perform GO enrichment and KEGG enrichment analysis of differential genes, with p < 0.05 after correction.

[0201] B. Experimental results

[0202] As Figure 12 shown, the volcano plot shows that there are 91 differential genes in the DMSO group and the PA-H group, 83 are significantly up-regulated, and 8 are significantly down-regulated. At the same time, it is found in the KEGG pathway analysis that perillaldehyde promotes the mitophagy pathway.

[0203] As Figure 13 shown, according to the regulatory information of perillaldehyde on the genes in the mitophagy pathway, a pathway diagram is made: First, perillaldehyde can up-regulate the NDUA1, ND4L, and NDUA2 genes, promote mitochondrial oxidative phosphorylation, ensure cell respiration and metabolism, increase the NAD+ / NADH ratio, and maintain mitochondrial function; then up-regulate the TOMM-7 and LGG-1 genes and regulate the remaining mitophagy genes. Among them, the TOMM-7 gene can promote mitochondrial protein transport, ensure that mitochondria can obtain enough proteins to perform basic mitochondrial functions, and LGG-1 can mark the substances to be cleared by autophagosomes, helping autophagosomes recognize, wrap, and degrade damaged organelles, misfolded proteins and other components in cells, and maintain cell homeostasis.

[0204] As Figure 14 shown, the genes in the above pathway diagram were verified by qPCR. The qPCR results were compared with the FPKM values in the transcriptome data, and the trends were consistent, indicating that perillaldehyde can regulate the genes related to the mitophagy pathway by promoting the mitophagy pathway, improve mitochondrial function, increase the NAD + / NADH ratio, maintain cell homeostasis, maintain body metabolism, and achieve the effect of delaying aging.

[0205] Preparation Example 1 Application of Perillaldehyde in Anti-aging Serum Skin Care Products

[0206] Component A is the oil phase component; Component B is the aqueous phase component; Component C is the anti-aging ingredient.

[0207] Referring to Table 4, heat Component A to 80°C, melt and mix it evenly, cool it to 40°C after insulation to obtain the oil phase component A; mix Component B and add distilled water to obtain the aqueous phase component B. Add the oil phase component A to the aqueous phase component B and Component C at 40°C, stir evenly, wait for the temperature to drop to 36°C, filter and bottle it to obtain the perillaldehyde anti-aging serum skin care product, and the appearance of the finished product is as Figure 14 shown.

[0208] Table 4 Ingredients List of Perillaldehyde Anti-aging Serum Skin Care Products

[0209]

[0210] Preparation Example 2 Application of Perillaldehyde in Anti-aging Emulsion Skin Care Products

[0211] Component A is the oil phase component; Component B is the aqueous phase component; Component C is the anti-aging ingredient.

[0212] Referring to Table 5, mix and heat Component A to 95°C to make the oil phase; heat Component B to 95°C to make the aqueous phase; slowly add the oil phase A to the aqueous phase B, homogenize and emulsify, mix evenly, and add Component C when cooled to about 40°C, and cool to room temperature to obtain the perillaldehyde anti-aging emulsion skin care product.

[0213] Table 5 Ingredients List of Perillaldehyde Anti-aging Emulsion Skin Care Products

[0214]

[0215] Preparation Example 3 Application of Perillaldehyde in Anti-aging Fruit Juice

[0216] Prepare according to the formula in Table 6: Mix perillaldehyde, fruit juice, citric acid, carbonated compounds, vitamin C, minerals and other ingredients, dissolve them in water, sterilize and can them to obtain perillaldehyde anti-aging fruit juice.

[0217] Table 6 Formula of Perillaldehyde Anti-aging Fruit Juice

[0218] Raw materials Ratio (mass ratio %) Perillaldehyde 0.03 Fruit juice Appropriate amount Citric acid 0.5 Vitamin C 0.05 Minerals 1 Carbohydrates 10 Water Up to 100

[0219] Preparation Example 4: Application of Perilla Aldehyde in Anti-aging Sparkling Water

[0220] Prepare according to the formula in Table 7: Mix ingredients such as perilla aldehyde, fruit juice, vitamin C, minerals, and citric acid, dissolve them in water, can and degas, sterilize and cool, then add carbonated purified water for carbonation. The gas injection amount of the carbonated purified water is 4.8 g / L to obtain perilla aldehyde anti-aging sparkling water.

[0221] Table 7 Perilla Aldehyde Anti-aging Sparkling Water

[0222] Raw materials Ratio (mass ratio %) Perillaldehyde 0.03 Fruit juice Appropriate amount Vitamin C 0.05 Citric acid 0.5 Minerals 1 Water Up to 100

[0223] Preparation Example 5: Application of Perilla Aldehyde in Anti-aging Candy

[0224] Prepare according to the formula in Table 8: Mix vitamin C, citric acid, glucose syrup, and granulated sugar, stir and boil over low heat. When the syrup keeps boiling until it becomes viscous, turn off the heat and wait until it cools to about 50 °C, then add perilla aldehyde, mix well and cool, and then pour into molds to obtain perilla aldehyde anti-aging candy.

[0225] Table 8 Perilla Aldehyde Anti-aging Candy Formula

[0226] Raw materials Ratio (mass ratio %) Perillaldehyde 0.05 Vitamin C 0.1 Citric acid 0.8 Sugar 40 Glucose syrup 25 Water Up to 100

Claims

1. A method for preparing perillaldehyde, the method comprising the following steps: S1. Put perilla leaves into an ethanol solution; S2. Add polyethylene glycol to the mixture in step S1 and then beat it into a pulp, and filter to obtain perilla liquid; S3. Add a composite enzyme to the perilla liquid obtained in step S2, perform ultrasonic enzymatic hydrolysis, heat to inactivate the enzyme, and filter to obtain an enzymatic hydrolysate; S4. Perform macroporous resin adsorption and molecular distillation purification on the enzymatic hydrolysate in step S3 to obtain perillaldehyde, and the purity of perillaldehyde is 99%.

2. Application of perillaldehyde in the preparation of anti-aging products, the perillaldehyde is prepared by the method described in claim 1; the purity of the perillaldehyde is 99%; the products include but are not limited to skin care products, foods, health products and medicines.

3. Application of perillaldehyde in the preparation of anti-aging products as described in claim 2, wherein the content of perillaldehyde in the anti-aging product is selected from 0.03% - 3%.

4. A skin care product in the form of an anti-aging essence, the anti-aging essence skin care product includes an oil phase, an aqueous phase and an anti-aging component; the oil phase components of the anti-aging essence skin care product include but are not limited to ethylhexylglycerin; the aqueous phase of the anti-aging essence skin care product is selected from one or more of butanediol, glycerol, hydroxyethylurea, disodium ethylenediaminetetraacetate, hyaluronic acid, propylene glycol, ethanol and / or distilled water; the anti-aging components of the anti-aging essence skin care product include but are not limited to perillaldehyde; the perillaldehyde is prepared by the method described in claim 1; the purity of the perillaldehyde is 99%.

5. For the anti-aging essence skin care product as described in claim 3, the mass ratio of butanediol is 8%; the mass ratio of hydroxyethylurea is 8%; the mass ratio of disodium ethylenediaminetetraacetate is 0.05%; the mass ratio of glycerol is 10%; The mass ratio of hyaluronic acid is 0.4%; the mass ratio of propylene glycol is 5%; the mass ratio of ethanol is 4%; The mass ratio of perillaldehyde is 2%.

6. A skin care product in the form of an anti-aging lotion, the anti-aging lotion skin care product includes an oil phase, an aqueous phase and an anti-aging component; the oil phase of the anti-aging lotion skin care product is selected from one or more of stearyl alcohol, glyceryl monostearate, isopropyl myristate, squalane, propylparaben and / or vitamins; the vitamins in the oil phase are vitamin A and / or vitamin E; the aqueous phase of the anti-aging lotion skin care product is selected from one or more of polyvinyl alcohol, hydroxyethyl cellulose, vitamins, glycerol and / or distilled water; the vitamins in the aqueous phase are vitamin B and / or vitamin C; the anti-aging components of the anti-aging lotion skin care product include but are not limited to perillaldehyde; the perillaldehyde is prepared by the method described in claim 1; the purity of the perillaldehyde is 99%.

7. The anti-aging emulsion skin care product as described in claim 5, wherein the mass ratio of stearyl alcohol is 0.8%; the mass ratio of glyceryl monostearate is 0.8%; the mass ratio of isopropyl myristate is 2%; the mass ratio of squalane is 3%; the mass ratio of propylparaben is 0.1%; the vitamins in the oil phase are vitamin A and / or vitamin E; the vitamins in the aqueous phase are vitamin B and / or vitamin C; the mass ratio of the vitamins is 0.2%; the mass ratio of polyvinyl alcohol is 8%; the mass ratio of hydroxyethyl cellulose is 0.1%; the mass ratio of glycerol is 4%; the mass ratio of perillaldehyde is 1%.

8. An anti-aging fruit juice, the components of the anti-aging fruit juice including but not limited to fruit juice, vitamins, citric acid, minerals, carbohydrates, water and / or anti-aging components; the fruit juice including but not limited to flavored fruit juice and freshly squeezed fruit juice; the vitamins including but not limited to vitamin A, vitamin E, vitamin C and / or vitamin B, preferably vitamin C; the mass ratio of the vitamins is 0.05%; the mass ratio of citric acid is 0.5%; the mass ratio of minerals is 1%; the mass ratio of carbohydrates is 10%; the anti-aging components of the anti-aging fruit juice including but not limited to perillaldehyde; the perillaldehyde is obtained by the method described in claim 1; the purity of the perillaldehyde is 99%; the mass ratio of the perillaldehyde is 0.03%.

9. An anti-aging sparkling water, the components of the anti-aging sparkling water including but not limited to fruit juice, vitamins, citric acid, minerals, water, infused gas and / or anti-aging components; the fruit juice including but not limited to flavored fruit juice and freshly squeezed fruit juice; the vitamins including but not limited to vitamin A, vitamin E, vitamin C and / or vitamin B, preferably vitamin C; the mass ratio of the vitamins is 0.05%; the mass ratio of citric acid is 0.5%; the mass ratio of minerals is 1%; the infused gas is carbon dioxide; the gas infusion amount is 4.8 g / L; the anti-aging components of the anti-aging sparkling water including but not limited to perillaldehyde; the perillaldehyde is obtained by the method described in claim 1; the purity of the perillaldehyde is 99%; the mass ratio of the perillaldehyde is 0.03%.

10. An anti-aging candy, the components of the anti-aging candy including but not limited to vitamins, citric acid, granulated sugar, glucose syrup, water and / or anti-aging components; the vitamins including but not limited to vitamin A, vitamin E, vitamin C and / or vitamin B, preferably vitamin C; the mass ratio of the vitamins is 0.1%; the mass ratio of citric acid is 0.8%; the mass ratio of granulated sugar is 40%; the mass ratio of glucose syrup is 25%; the anti-aging components of the anti-aging candy including but not limited to perillaldehyde; the perillaldehyde is obtained by the method described in claim 1; the purity of the perillaldehyde is 99%; the mass ratio of the perillaldehyde is 0.05%.

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