Use of a perilla leaf extract combination for anti-aging

Abstract

The perilla leaf extract provided by the application exhibits excellent anti-aging comprehensive benefits in a Caenorhabditis elegans model. Experiments prove that the extract can significantly improve the heat stress resistance of the Caenorhabditis elegans through the synergistic effect of multiple targets and multiple pathways, delay the decline of physiological functions related to aging of the Caenorhabditis elegans, and enhance the average life span of the Caenorhabditis elegans. Moreover, the extract delays the aging process in multiple dimensions while not affecting the normal growth and development of the Caenorhabditis elegans, has the characteristics of high efficiency and safety, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a plant extract in anti-aging. Background Technology

[0002] Aging is the systemic decline of physiological functions that occurs in living organisms over time. This process is not only manifested in changes in external physical signs, but also serves as the common basis for the occurrence and development of many chronic diseases, such as neurodegenerative diseases, cardiovascular diseases, and metabolic syndrome. Decades of research using in vitro and in vivo models have shown that genetic, pharmaceutical, and dietary interventions can extend lifespan and improve healthy lifespan. These interventions include treatments targeting the hallmarks of aging, delaying the onset of age-related diseases, and ultimately improving health through anti-inflammatory and protein homeostasis-regulating activities.

[0003] Unlike the traditional disease-oriented medical model, the concept of "prevention of disease" is gradually moving to the forefront—especially through the use of traditional Chinese medicinal herbs, which are both food and medicine, to regulate internal physiological processes through dietary intervention, achieving a gentle, multi-pathway, and multi-target approach to slowing down aging. These substances can be used as part of food and also possess pharmacological activity. By regulating inflammation, providing antioxidant effects, maintaining metabolic balance, and improving autophagy, they can delay the overall decline of bodily functions, thus providing a feasible pathway for systemic healthy aging.

[0004] Increasing evidence supports the potential advantages of plant extracts and their products in promoting human health and addressing various pathological conditions (Impact of plant extracts upon human health: A review. Crit Rev Food Sci Nutr. 2020;60(5):873-886.). Plant extracts, rich in bioactive components such as polyphenols, flavonoids, saponins, and alkaloids, are rapidly expanding in research and application in the field of life and health due to their advantages of multi-target effects, low toxicity, and high biocompatibility, covering areas such as preventative healthcare, disease intervention, regenerative medicine, and skincare product development. Research by Gregoriou et al. showed that extracts from carob (Ceratonia siliqua L.) pods have anti-cancer activity against breast cancer cells, but have no effect on healthy cells (Anti-Cancer Activity and Phenolic Content of Extracts Derived from Cypriot Carob (Ceratonia siliqua L.) Pods Using Different Solvents. Molecules. 2021 Aug 19;26(16):5017.). Researchers used a zebrafish model to test the whitening and freckle-removing effects of the extracts and found that the compound extract of danshen, ginseng, safflower and persimmon leaf can effectively reduce the formation of melanin, lighten melanin spots, and reduce tyrosinase activity, showing good whitening and freckle-removing effects (Study on the whitening, freckle-removing and moisturizing effects of danshen compound plant extracts, [J]. Daily Chemicals Science, 2025,48(12):37-42.). With in-depth research on plant extracts, their applications are expanding beyond the medical, food, and cosmetic fields, gradually penetrating emerging fields such as environmental protection, daily chemicals, and textiles.

[0005] However, the effectiveness of plant extracts depends not only on the type of active ingredients, but also on a variety of factors such as extraction process, concentration control, raw material quality, and storage conditions, among which "concentration control" is the core point.

[0006] Perilla (Perilla frutescens) is an annual erect herb belonging to the genus Perilla in the Lamiaceae family. It is one of the first 60+ medicinal and edible plants announced by the Ministry of Health of my country. Perilla leaves are a commonly used medicinal and edible part. The 2025 edition of the Chinese Pharmacopoeia records that perilla has the effects of relieving exterior syndromes and dispelling cold, regulating qi and stomach, and is often used for colds due to wind-cold, coughs and nausea, vomiting during pregnancy, and fish and crab poisoning. Modern pharmacological studies have shown that perilla leaves are rich in volatile oils (mainly perillaldehyde, perillyl alcohol, and limonene), flavonoids (such as luteolin and apigenin), phenolic acids (such as rosmarinic acid), and triterpenoids, exhibiting various active ingredients such as antibacterial, anti-inflammatory, anti-allergic, antioxidant, and anti-anxiety activities. Perilla leaf extract refers to the active ingredients extracted from perilla leaves using water or 75% ethanol as solvents; the former is the water extract of perilla leaves, and the latter is the ethanol extract of perilla leaves.

[0007] Perilla leaf extract has a wide range of applications, showing great potential in both traditional medicinal and edible uses and modern research. Due to its natural, healthy, and multifunctional properties, it is mainly used in the food industry as a natural preservative, antioxidant, flavor improver, and nutritional fortifier. However, whether perilla extract possesses other effects and the safe concentration for its use require further investigation. Summary of the Invention

[0008] This invention discovered that both types of perilla leaf extracts possess the ability to combat oxidative stress and protect cells from free radical damage, thereby delaying aging. However, the effective component in perilla extracts is harmful to organisms when it exceeds 100 μM, and it fails to exert its anti-aging effect when it falls below 0.1 μM. Based on this, this invention was completed.

[0009] In a first aspect, the present invention provides a perilla extract composition, wherein the perilla extract composition contains ursolic acid, apigenin, luteolin, and farnesin, wherein: The concentration of ursolic acid is selected from 0.1-100 μM, preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of apigenin is selected from 0.1-100 μM, and preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of luteolin is selected from 0.1-100 uM, and preferably, the concentration of ursolic acid is selected from 1-50 uM; The concentration of farnesin is selected from 1-100 uM, preferably from 5-50 uM.

[0010] Furthermore, the perilla extract is an aqueous extract of perilla or an alcoholic extract of perilla.

[0011] Secondly, the present invention provides the use of the perilla extract combination as described in the first aspect of the present invention in the preparation of anti-aging products.

[0012] Furthermore, the perilla extract combination is selected from perilla water extract and / or perilla alcohol extract.

[0013] Furthermore, the perilla extract combination contains one or more of ursolic acid, apigenin, luteolin and / or acaciain.

[0014] Furthermore, in the perilla extract composition, the concentration of ursolic acid is selected from 0.1-100 μM, preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of apigenin is selected from 0.1-100 μM, and preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of luteolin is selected from 0.1-100 uM, and preferably, the concentration of ursolic acid is selected from 1-50 uM; The concentration of farnesin is selected from 1-100 uM, preferably from 5-50 uM.

[0015] Furthermore, the aging process includes skin aging and decline in bodily functions.

[0016] Thirdly, the present invention provides an anti-aging product containing a perilla extract combination, wherein the perilla extract combination contains ursolic acid, apigenin, luteolin, and farnesin, wherein: The concentration of ursolic acid is selected from 0.1-100 μM, preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of apigenin is selected from 0.1-100 μM, and preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of luteolin is selected from 0.1-100 uM, and preferably, the concentration of ursolic acid is selected from 1-50 uM; The concentration of farnesin is selected from 1-100 uM, preferably from 5-50 uM.

[0017] Furthermore, the products include health supplements, cosmetics, food additives, and pharmaceuticals.

[0018] Furthermore, the cosmetics are selected from any one of the following: facial mask liquid, lotion, cream, toner, serum, essence, facial cleanser, lotion, perfume, makeup remover, liquid foundation, foundation cream, concealer, blush, lipstick, eyeshadow, and blush.

[0019] Beneficial effects The perilla leaf extract provided by this invention exhibits remarkable comprehensive anti-aging benefits in a *C. elegans* model. Experiments have confirmed that this extract, through synergistic effects across multiple targets and pathways, can significantly improve the heat stress resistance of *C. elegans*, delay the decline of aging-related physiological functions, and enhance its average lifespan. Furthermore, while delaying the aging process in multiple dimensions, this extract does not affect the normal growth and development of the nematodes, demonstrating high efficiency and safety, and possessing promising clinical application prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the technical process of the present invention.

[0021] Figure 2 This is a photograph of the extract from Embodiment 1 of the present invention.

[0022] Figure 3 The figures show the evaluation results of the in vitro DPPH free radical scavenging effect of the perilla leaf extract in Example 2 of this invention. Figure A shows the DPPH free radical scavenging rate of the aqueous extract of vitamin C and perilla leaf; Figure B shows the DPPH free radical scavenging rate of the aqueous and then alcoholic extract of vitamin C and perilla leaf; Figure C shows the DPPH free radical scavenging rate of the alcoholic extract of vitamin C and perilla leaf; Figure D shows the DPPH free radical scavenging rate of the alcoholic and then aqueous extract of vitamin C and perilla leaf. In Figures A, B, and C, the left side represents vitamin C, and the right side represents the corresponding perilla leaf extract.

[0023] Figure 4 This is the result of heat stress in nematodes after administration of the perilla leaf extract in Example 3 of the present invention, wherein A is the water extract of perilla leaves; and B is the alcohol extract of perilla leaves.

[0024] Figure 5 This is the result of nematode reproductive toxicity after administration of the perilla leaf extract in Example 4 of the present invention, where A is the water extract of perilla leaves and B is the alcohol extract of perilla leaves.

[0025] Figure 6 This is the result of the nematode lifespan test after administration of the perilla leaf water extract in Example 5 of the present invention.

[0026] Figure 7 This is the result of the nematode lifespan test after administration of the perilla leaf ethanol extract in Example 5 of the present invention.

[0027] Figure 8 The following are the material basis evaluation results of the perilla leaf extract in Example 6 of this invention, wherein A is a circular diagram of the composition of perilla leaf metabolites; B is a Venn diagram of the differences among the groups of perilla leaf metabolites; C is a principal component analysis diagram of the groups of perilla leaf metabolites; D is an overall cluster diagram of perilla leaf metabolites; E is a correlation diagram among perilla leaf metabolite samples; and F is a volcano diagram of differential metabolites of perilla leaves.

[0028] Figure 9These are the network pharmacology results of the perilla leaf water extract in Example 7 of the present invention, wherein A is the intersection target map of the perilla leaf water extract; B is the component-target PPI map of the perilla leaf water extract; C is the GO enrichment map of the perilla leaf water extract; and D is the KEGG enrichment map of the perilla leaf water extract.

[0029] Figure 10 These are the network pharmacology results of the perilla leaf ethanol extract in Example 7 of the present invention, wherein A is the intersection target map of the perilla leaf ethanol extract; B is the component-target PPI map of the perilla leaf ethanol extract; C is the GO enrichment map of the perilla leaf ethanol extract; and D is the KEGG enrichment map of the perilla leaf ethanol extract.

[0030] Figure 11 This is the result of heat stress in nematodes after administration of the four monomers in Example 8 of the present invention. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the test methods in the following embodiments are conventional methods, and the test materials used in the following embodiments are all available through conventional commercial channels.

[0033] The model organism used in this invention to verify the efficacy of perilla leaf extract is the N2 wild-type *C. elegans*. *C. elegans* has an average lifespan of 2–3 weeks, and its genome contains approximately 60%–80% of the homologous genes found in the human genome; it is also convenient for genetic manipulation and has a short observation period; therefore, it has been widely used in the study of gene function and substance function screening for human diseases.

[0034] Heat stress is a common environmental stress that accelerates the aging process of organisms. Under heat stress, the physiological functions of *C. elegans* change, such as reduced heat tolerance, weakened motility, and decreased reproductive capacity, thus accelerating aging. Observing the survival rate of *C. elegans* under heat stress can indirectly reflect its aging state. Furthermore, the lifespan of *C. elegans* is the most direct reflection of its aging process.

[0035] Vitamin C has a well-defined antioxidant mechanism, capable of scavenging excess reactive oxygen species produced by metabolism in nematodes, reducing oxidative stress damage to biomolecules, and thus delaying aging. Vitamin C not only significantly improves aging-related phenotypes in nematodes, such as extending lifespan and enhancing motility and reproductive capacity, but also exhibits good stability, is not easily decomposed under experimental conditions, ensuring reproducibility of results; it is highly safe, with low toxicity, meaning it does not severely interfere with normal physiological activities in nematodes; it is easy to use, as it can be directly added to the culture medium for nematodes to ingest; furthermore, it is inexpensive and widely available, effectively reducing experimental costs.

[0036] Astaxanthin is a powerful antioxidant, exhibiting outstanding antioxidant capacity among carotenoids. It can scavenge free radicals in the body, reducing oxidative stress damage to cells and tissues. By protecting cells from oxidative damage, astaxanthin helps maintain normal cellular function and structure, thus playing an antioxidant role. Numerous studies have demonstrated that astaxanthin can delay cellular aging and the body's aging process by reducing oxidative damage, thereby extending the lifespan of *C. elegans*.

[0037] Therefore, the application test of this invention uses vitamin C and astaxanthin as positive control groups to ensure the authenticity and certainty of the test results of this invention.

[0038] Example 1: Preparation method and extraction yield calculation of perilla leaf extract Pure water and 75% ethanol were selected as the extraction solutions. After a single extraction with pure water, the product was extracted a second time with 75% ethanol; after a single extraction with 75% ethanol, the product was extracted a second time with water to recover as much of the effective substances from perilla as possible.

[0039] The specific extraction process includes: adding perilla leaf powder and the aforementioned extract at a material-to-liquid ratio of 1:10 into a round-bottom flask; extracting by heating and reflux at room temperature, and then combining the filtrates; filtering the filtrate through a Buchner funnel, separating the solid and liquid, obtaining the supernatant and filter residue, removing the filter residue and retaining the supernatant for later use; concentrating the obtained supernatant under reduced pressure (conditions: 67 hpa, t=30~40min, rotary evaporation temperature 55±3℃); after rotary evaporation to 1 / 10 of the original volume, freeze-drying at low temperature to obtain dark brown perilla leaf water extract powder, which is stored at 4℃ for later use.

[0040] Following the extraction protocol described above, 70 g of dried perilla leaf powder was prepared, yielding 24.1 g of brown perilla leaf water extract powder, with an extraction yield of 34.4%. Figure 2 A); Secondary extraction with 75% ethanol yielded 4.3%, resulting in a low product yield ( Figure 2 B). A single extraction with 75% ethanol yielded 16.8 g of perilla leaf ethanol extract powder, with an extraction yield of 24.0%. Figure 2 C), secondary water extraction 12.6%, product color is darker ( Figure 2 D).

[0041] Example 2 Evaluation of the in vitro free radical scavenging effect of perilla leaf extract on DPPH DPPH is a stable free radical scavenger. Its stability mainly comes from the resonance stabilization of the three benzene rings and steric hindrance, which prevents the unpaired electrons on the nitrogen atom in the middle from playing their proper electron pairing role. DPPH is often used to evaluate the antioxidant properties of extracts. Perilla leaf aqueous extract and perilla leaf ethanol extract powder were dissolved in pure water to prepare solutions of 5, 50, and 500 μg / mL, respectively. A vitamin C solution of the same concentration was used as a control. Following the instructions of the Nanjing Jiancheng reagent kit, the DPPH scavenging rate of perilla leaf aqueous extract, perilla leaf ethanol extract, and vitamin C at each concentration was measured using a microplate reader.

[0042] The results are as follows Figure 3 As shown, perilla leaf water extract ( Figure 3 A) Perilla leaf alcohol extract ( Figure 3 C) The ability to scavenge DPPH free radicals is comparable to that of vitamin C, and the scavenging effect improves with increasing concentration. This indicates that both types of perilla leaf extracts possess in vitro antioxidant capacity. In comparison, the alcohol and water extracts from the secondary extraction (…) Figure 3 B, Figure 3 D) The DPPH scavenging rate was slightly lower in the medium-dose group than in the first extraction, but the high-dose group still showed a high DPPH scavenging rate, indicating that the perilla extract has a good antioxidant effect. Considering that the second 75% ethanol extraction yielded less product and the second water extraction yielded a darker-colored 12.6% product, this invention used the first water extract and the 75% ethanol extract to evaluate the anti-aging effects of nematodes.

[0043] Example 3 Evaluation of the heat stress effect of Perilla leaf extract on Caenorhabditis elegans 1. Preparation for Nematode Heat Stress Experiment 100 mg of perilla leaf aqueous extract and perilla leaf ethanol extract powder were dissolved in 10 ml of distilled water to prepare a 10 mg / ml stock solution for later use. The stock solution was diluted to 0.5, 1, and 2 mg / ml perilla leaf aqueous extract solutions as low (PS-L), medium (PS-M), and high (PS-H) dose test groups, respectively. The 0.5, 1, and 2 mg / ml perilla leaf ethanol extract solutions were used as low (PC-L), medium (PC-M), and high (PC-H) dose test groups, respectively. Distilled water was used as the negative control group. 0.5 mg / ml vitamin C was used as the positive control group.

[0044] Using Escherichia coli OP50 as food for nematodes and 60 mm NGM solid medium as a growth medium, the following plates were prepared: O plate: Free of fluorouridine (FUdR), add 100 μL LOP50, air dry, and incubate upside down at 37°C for 12 h.

[0045] F-plate: Contains FUdR, add 100 μL LOP50, air dry, and then incubate upside down at 37°C for 12 hours. (FUdR is used to inhibit nematode oviposition and avoid interference with offspring.) Drug-containing O-plates: Add 50 μL of OP50 + 50 μL of drug solution, air dry, and then invert the plate at 37℃ for 12 h.

[0046] Drug-containing F-plate: Add 50 μL of OP50 + 50 μL of drug solution, air dry, and then invert the plate at 37℃ for 12 h.

[0047] 2. Heat stress test process *Caenorhabditis elegans* was cultured in a 20°C incubator. After obtaining L4-stage larvae through synchronization, they were randomly divided into experimental and control groups and placed onto drug-containing F-plates (each group containing 35–40 nematodes, with 3 independent replicates, for a total of 15 groups). The nematodes in each group were treated at 20°C for 72 hours, with continuous drug administration for 3 days. New drug-containing F-plates were used daily, and on the last day, the number of nematodes in each group was standardized to 25 to ensure sufficient action of the test substance. After the treatment period, the nematode plates were rapidly transferred to a 37°C incubator for stress. After 10 hours, the number of surviving nematodes was observed and counted under a stereomicroscope, and the nematode survival rate was calculated.

[0048] 3. Results of heat stress test The results are as follows Figure 4 As shown, under high-temperature stress, compared with distilled water, low, medium, and high doses of vitamin C, perilla leaf water extract, and perilla leaf alcohol extract significantly enhanced the stress resistance of nematodes and effectively improved their survival rate. Among them, the low, medium, and high doses of perilla leaf water extract showed better anti-heat stress effects than the vitamin C group.

[0049] Example 4: Evaluation of the reproductive toxicity of Perilla leaf extract to Caenorhabditis elegans. 1. Preparation for Nematode Reproduction Experiment 100 mg of perilla leaf aqueous extract and perilla leaf ethanol extract powder were dissolved in 10 ml of distilled water to prepare a 10 mg / ml stock solution for later use. The stock solution was diluted to 0.5, 1, and 2 mg / ml perilla leaf aqueous extract solutions as low (PS-L), medium (PS-M), and high (PS-H) dose test groups, respectively; 0.5, 1, and 2 mg / ml perilla leaf ethanol extract solutions were used as low (PC-L), medium (PC-M), and high (PC-H) dose test groups, respectively; distilled water was used as the negative control group; and 0.5 mg / ml vitamin C was used as the positive control group. Using Escherichia coli OP50 as nematode food and 60 mm NGM solid medium as the growth medium, the following plates were prepared: O plate: Free of fluorouridine (FUdR), add 100 μL LOP50, air dry, and incubate upside down at 37°C for 12 hours.

[0050] F-plate: Contains FUdR, add 100 μL LOP50, air dry, and then incubate upside down at 37°C for 12 hours. (FUdR is used to inhibit nematode oviposition and avoid interference with offspring.) Drug-containing O-plates: Add 50 μL of OP50 + 50 μL of drug solution, air dry, and then invert the plate at 37°C for 12 hours.

[0051] 2. Reproductive testing process *Caenorhabditis elegans* was cultured in a 20°C incubator. After obtaining L4-stage larvae through synchronization, they were randomly divided into experimental and control groups and placed onto drug-containing O-plates (each group contained one nematode, with 5 independent replicates, for a total of 40 groups). The number of eggs laid in each group was recorded daily, and the drug-containing O-plates were replaced until all nematodes ceased laying eggs. The total number of eggs laid by nematodes in each group should be counted.

[0052] 3. Results of reproductive tests The results are as follows Figure 5 As shown, the number of eggs laid and the hatching rate of offspring in nematodes after administration of perilla leaf water extract and perilla leaf alcohol extract were within the normal range and showed no significant difference compared with the control group. This indicates that neither type of perilla leaf extract caused significant damage to nematode reproduction.

[0053] Example 5: Evaluation of the lifespan of *C. elegans* by water extract of *Perilla frutescens* leaves. 1. Preparation for Nematode Lifespan Experiment 100 mg of perilla leaf aqueous extract and perilla leaf ethanol extract powder were dissolved in 10 ml of distilled water to prepare a 10 mg / ml stock solution for later use. The stock solution was diluted to 0.5, 1, and 2 mg / ml perilla leaf aqueous extract solutions as low (PS-L), medium (PS-M), and high (PS-H) dose test groups, respectively. The 0.5, 1, and 2 mg / ml perilla leaf ethanol extract solutions were used as low (PC-L), medium (PC-M), and high (PC-H) dose test groups, respectively. Distilled water was used as the negative control group. 3.84 mg / ml astaxanthin was used as the positive control group.

[0054] Using Escherichia coli OP50 as food for nematodes and 60 mm NGM solid medium as a growth medium, the following plates were prepared: O plate: Free of fluorouridine (FUdR), add 100 μL LOP50, air dry, and incubate upside down at 37°C for 12 hours.

[0055] F-plate: Contains FUdR, add 100 μL LOP50, air dry, and then incubate upside down at 37°C for 12 hours. (FUdR is used to inhibit nematode oviposition and avoid interference with offspring.) Drug-containing O-plates: Add 50 μL of OP50 + 50 μL of drug solution, air dry, and then invert the plate at 37°C for 12 hours.

[0056] Drug-containing F-plate: Add 50μL of OP50 + 50μL of drug solution, air dry, and then invert the plate at 37℃ for 12 hours.

[0057] 2. Life test process Caenorhabditis elegans was cultured in a 20°C incubator. After obtaining L4-stage larvae through synchronization, they were randomly divided into experimental and control groups and placed onto drug-containing F-plates (each group containing 35–40 nematodes, with 3 independent replicates, for a total of 27 groups). The nematodes in each group were treated at 20°C for 120 hours, with continuous drug administration for 5 days. A new drug-containing F-plate was used daily, and on the last day, the number of nematodes in each group was standardized to 25 to ensure sufficient action of the test substance. After standardizing the nematode count, the drug-containing O-plate was used every other day, and the survival time of each group of nematodes was recorded until all nematodes died.

[0058] 3. Life test results The results are as follows Figure 6-7 As shown in Tables 1-2, the natural lifespan of *C. elegans* is 2–3 weeks. No nematodes die within the first 10 days, and from the 11th day onwards, they begin to die off successively. *C. elegans* treated with perilla leaf water extract and perilla leaf alcohol extract showed a rightward shift in their survival time curves; compared to the negative control group, their lifespan was significantly prolonged. Compared to the positive control group (astaxanthin), the high-concentration (PS-H) perilla leaf water extract, low-concentration (PC-L) perilla leaf alcohol extract, medium-concentration (PC-M) perilla leaf alcohol extract, and high-concentration (PC-H) perilla leaf alcohol extract groups showed the most significant effect in delaying nematode lifespan. Among them, the high-concentration perilla leaf water extract group (PS-H) showed the most significant effect, extending the lifespan by 42.11% compared to the control group (p<0.001).

[0059] Table 1. Results of nematode lifespan after administration of perilla leaf water extract. Table 2. Lifespan of nematodes after administration of Perilla leaf ethanol extract Example 6: Study on the material basis of Perilla leaf extract To further investigate the material basis of perilla's anti-aging properties, this embodiment aims to systematically analyze the chemical composition spectra of perilla leaf aqueous extract and perilla leaf alcohol extract using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), identify their common and different metabolites, reveal the metabolite components of the two types of extracts from a chemical basis, and provide component evidence for subsequent anti-aging efficacy.

[0060] 1. Perilla Leaf Extract Sample Detection Procedure (1) Preparation of internal standard extraction solution: Dissolve 1 mg of standard in 1 mL of 70% methanol water to prepare a 1000 μg / mL standard stock solution, and further dilute the 1000 μg / mL stock solution with 70% methanol to prepare a 250 μg / mL internal standard solution; (2) Preparation of Perilla Leaf Extract: The sample was freeze-dried under vacuum for 63 h in a freeze dryer and then ground into powder using a grinder. 50 mg of the sample powder was weighed and 1200 μL of 70% methanol-water internal standard extract pre-cooled at -20 ℃ was added and centrifuged by vortexing. The supernatant was collected, the sample was filtered through a microporous membrane, and stored in a sample vial for UPLC-MS / MS analysis.

[0061] (3) Qualitative analysis of Perilla Leaf Extract: Based on the MWDB database, the substance was qualitatively analyzed according to the secondary spectrum information. Isotope signals were removed during the analysis. It contains K + Na + NH4 + The repeating signals, as well as the repeating signals of fragment ions that are themselves other substances with larger molecular weights; (4) Quantification of Perilla Leaf Extract: After obtaining the metabolite mass spectrometry data of different samples, the peak area of ​​all substances was integrated, and the mass spectrometry peaks of the same metabolite in different samples were integrated and corrected (Fraga et al. 2010).

[0062] 2. Results Analysis Total metabolite analysis: UPLC-MS / MS was used to analyze the aqueous and ethanolic extracts of perilla leaves. A total of 3579 metabolites were detected from both extracts, mainly including monoterpenes, phenols, flavonoids, and polysaccharide degradation products. Among these, 55 components differed between the aqueous and ethanolic extracts, while the remainder were common components. (Details follow...) Figure 8 A and Figure 8 As shown in B.

[0063] Multivariate statistical results: Principal component analysis (PCA) was performed on the samples (including quality control samples) to preliminarily understand the overall metabolite differences among the groups and the magnitude of variability within each group. PCA results showed a trend of metabolomic separation among the groups, indicating whether there were differences in metabolomics within the sample groups. With water extract as the ST group and alcohol extract as the CT group, the PCA score plot showed a clear separation between water and alcohol extract samples on PC1, indicating a systematic difference in their chemical composition, specifically as follows... Figure 8 As shown in C.

[0064] Clustering heatmap results: The heatmap uses color and clustering to show the expression differences of various metabolites in the perilla leaf water extract group and alcohol extract group (ST-1, ST-2, CT-1, CT-2). This can help screen differentially expressed metabolites between groups and summarize metabolite expression patterns, providing a visual basis for subsequent anti-aging studies of differentially expressed metabolites. Specifically, as shown below... Figure 8 As shown in D.

[0065] Reproducibility Correlation Results: Correlation analysis between samples allows observation of biological repeatability among samples within a group. A higher correlation coefficient between samples within a group and between groups indicates more reliable differential metabolites. The Pearson correlation coefficient (r) is used as an indicator of biological repeatability. The Pearson correlation coefficient is calculated using the built-in cor function in R software; the closer |r| is to 1, the stronger the correlation between the two replicated samples. The correlation coefficients for both the water extract and the alcohol extract of Perilla frutescens leaves are 1, representing a perfect positive correlation between the variables, indicating strong repeatability. Specifically... Figure 8 As shown in E.

[0066] Volcano plot results of differential metabolites: Among the total metabolites, monoterpenes, phenols, and flavonoids were identified as the main components of interest, totaling 1393, and analyzed. Using the ethanol extract (CT) as the experimental group and the water extract (ST) as the control group, 738 significantly differentially expressed metabolites were screened based on VIP and p-values. Of these, 585 metabolites showed significant upregulation, and 153 showed significant downregulation. (Details are as follows...) Figure 8 As shown in F.

[0067] The above results systematically detected and statistically analyzed the main components and key differences between the two types of perilla leaf extracts. Chemically, they clearly confirmed a high degree of complementarity between the aqueous and alcoholic extracts of perilla leaves on a chemical basis. The main components of both the aqueous and alcoholic extracts are terpenes, phenolic acids, and flavonoids; however, the aqueous extract shows a higher proportion of polar components such as phenolic acids, while the alcoholic extract has higher levels of terpenes and flavonoids. This synergy and difference in components provides a direct chemical explanation for the potential synergistic and complementary effects of the two extracts in anti-aging applications.

[0068] Example 7: Network pharmacology and molecular docking study of the anti-aging properties of Perilla leaf extract 1. Network pharmacology and molecular docking process 1.1 Screening of potential targets for aging Using "Aging", "Senescence", and "Ageing" as keywords, we searched for age-related disease targets in disease target databases such as Genecards (https: / / www.genecards.org / ), OMIM (https: / / omim.org / ), and TTD (http: / / db.idrblab.net / ttd / ). The results from each database were merged, duplicate values ​​were removed, and a set of age-related targets was obtained.

[0069] 1.2 Screening of active ingredients and potential targets of two types of perilla leaf extracts This study broadly targeted the bioactive components of water and alcohol extracts of Perilla frutescens leaves, focusing on the top 50 most abundant components. Potential targets for each bioactive component were collected using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP https: / / old.tcmsp-e.com / tcmsp.php). The obtained target protein names were standardized and corrected using the UniProt database (https: / / www.uniprot.org / ), and uniformly converted to official gene symbols to ensure data accuracy.

[0070] 1.3 Construct an intersection target Venn diagram Search the Venn mapping website, enter drug targets and disease targets respectively, generate a map, and filter out the overlapping targets.

[0071] 1.4 Construction of Protein-Protein Interaction (PPI) Network and Screening of Core Targets Import the common targets obtained in step 1.3 into the STRING database (https: / / string-db.org / ), set the species to "Homo sapiens", and the confidence score > 0.900. Hide disconnected nodes in the network to obtain PPI network data. Import the data into Cytoscape software, use the CytoHubba plugin, and perform maximum clique centrality (MCC) algorithm to select the top 10 core targets in the PPI network.

[0072] 1.5 GO Function and KEGG Pathway Enrichment Analysis Common targets were imported into the DAVID database (https: / / david.ncifcrf.gov / ) or the clusterProfiler package in R was used for enrichment analysis of gene ontology (GO) biological processes (BP), cellular components (CC), molecular functions (MF), and Kyoto Encyclopedia of Genes and Genomes (KEGG). A p-value < 0.05 and a false discovery rate (FDR) < 0.05 were defined as significant enrichment. Two types of key biological processes and signaling pathways that may be involved in the anti-aging effects of perilla leaf extract were screened and visualized.

[0073] 1.6 Molecular docking verification From steps 1.3 and 1.4, the top 5 key active ingredients and top 10 core targets with the highest degree values ​​were selected. Crystal structures of the core target proteins were downloaded from the RCSB PDB database (https: / / www.rcsb.org / ), prioritizing structures with high resolution, homology to human proteins, and small ligands. Water molecules and protoligands were removed using PyMOL software, and preprocessing such as hydrogenation and charge calculation was performed using AutoDock Tools software. The 3D structures of the key active ingredients were downloaded from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) and similarly preprocessed, including energy minimization. Molecular docking was performed using AutoDock Vina software, with a docking binding energy ≤ -5.0 kcal / mol considered to have good binding activity. The docking results were visualized using PyMOL and LigPlot+ software to analyze specific interaction forces (such as hydrogen bonds and hydrophobic interactions).

[0074] 2. Results of network pharmacology and molecular docking 2.1 Network pharmacology prediction results (1) Results of water extract of perilla leaves as follows Figure 9 As shown.

[0075] There are 745 targets for aging-related diseases; 504 targets are found among the top 50 major components of perilla leaf water extract metabolites; and 109 targets are found at the intersection of perilla leaf water extract and aging-related diseases.

[0076] The top 10 key genes may include: INS, GAPDH, IL6, AKT1, TNF, TP53, ESR1, CTNNB1, CASP3, and EGFR. The top 5 key ingredients are: farnesin, rosmarinic acid, luteolin, apigenin, ursolic acid, and perillaldehyde.

[0077] GO enrichment results: Common targets were significantly enriched in biological processes such as "cellular response to oxidative stress" (GO:0034599), "positive regulation of apoptosis" (GO:0043068), "positive regulation of protein phosphorylation" (GO:0001934), "negative regulation of cell proliferation" (GO:0008285), and "senescence" (GO:0007568). Growth factor signaling pathways and the membrane structures and receptor molecular functions they depend on are core regulatory modules of anti-aging mechanisms.

[0078] KEGG enrichment results: Significantly enriched in the PI3K-Akt signaling pathway (hsa04151) and the MAPK signaling pathway (hsa04010). These signaling pathways can regulate cell proliferation, survival and metabolism, and are key targets for aging intervention.

[0079] (2) Results of perilla leaf alcohol extract as follows Figure 10 As shown.

[0080] There are 745 targets for aging-related diseases; 248 targets are found among the top 50 major components of perilla leaf alcohol extract metabolites; and 55 targets are found at the intersection of perilla leaf water extract and aging-related diseases.

[0081] The top 10 key genes may include: TNF, TP53, PTGS2, ESR1, MMP9, EGFR, MMP2, KDR, IL2, and KEAP1. The top 5 key ingredients are: farnesin, rosmarinic acid, asiatic acid, shikonin B, and cinnamic acid. GO enrichment results: Common targets were significantly enriched in biological processes such as "epithelial cell differentiation" (GO:0030855), "negative regulation of mesenchymal cell differentiation" (GO:2000698), and "response to growth factors" (GO:0070848). It promotes cell survival through the PI3K / Akt and EGFR pathways and improves vascular function by regulating NO synthesis.

[0082] KEGG enrichment results: Significantly enriched in the PI3K-Akt signaling pathway (hsa04151) and the MAPK signaling pathway (hsa04010). These signaling pathways can regulate cell proliferation, survival and metabolism, and are key targets for aging intervention.

[0083] The 10 key components screened above were molecularly docked with the proteins encoded by the genes of INS, PTGS2, GAPDH, MMP9, MMP2, KDR, IL6, AKT1, TNF, TP53, ESR1, CTNNB1, CASP3, EGFR, IL2, and KEAP1, respectively. The molecular docking results, combined with a material basis evaluation, revealed that ursolic acid, apigenin, farnesin, and luteolin in perilla extract play a major role in anti-aging.

[0084] Example 8 Evaluation of the heat stress of four monomeric compounds on Caenorhabditis elegans 1. Preparation for Nematode Heat Stress Experiment Take 1 mg of ursolic acid, apigenin, farnesin, and luteolin respectively, dilute with dimethyl sulfoxide (DMSO) according to the specified ratio to prepare 5 mM stock solutions for later use; dilute the stock solutions according to the specified ratio to prepare 6 working solutions with concentrations of 100 uM, 50 uM, 10 uM, 5 uM, 1 uM, and 0.1 uM, which are used as experimental groups; DMSO is used as a negative control group; and 3.84 mg / ml astaxanthin is used as a positive control group.

[0085] 2. Heat stress test process Caenorhabditis elegans was cultured in a 20°C incubator. After obtaining L4-stage larvae through synchronization, they were randomly divided into experimental and control groups and placed onto drug-containing F-plates. Each group contained 35–40 nematodes, with three independent replicates, for a total of 84 groups. The nematodes in each group were treated at 20°C for 72 hours, with continuous drug administration for three days. New drug-containing F-plates were used daily, and on the last day, the number of nematodes in each group was standardized to 25 to ensure sufficient action of the test substance. After the treatment period, the nematode plates were rapidly transferred to a 37°C incubator for stress. After 10 hours, the number of surviving nematodes was observed and counted under a stereomicroscope, and the nematode survival rate was calculated.

[0086] 3. Results of heat stress test Under high temperature stress, compared with DMSO, ursolic acid, apigenin, farnesin and luteolin can significantly enhance the stress resistance of nematodes within a certain concentration range and effectively improve the survival rate of nematodes.

[0087] like Figure 11 As shown in Figure A, the effective range of apigenin in enhancing the heat stress resistance of nematodes is 1-50 μM; a concentration of 0.1 μM is too low to be effective, while a concentration of 100 μM may cause toxicity. When the concentration of apigenin is controlled at 5 μM, its effectiveness in enhancing the heat stress resistance of nematodes is stronger than that of the astaxanthin control group.

[0088] like Figure 11As shown in B, the effective range of ursolic acid in enhancing the heat stress resistance of nematodes is 1-50 μM; a concentration of 0.1 μM is too low to be effective, while a concentration of 100 μM may cause toxicity. When the concentration of ursolic acid is controlled at 1-10 μM, its effectiveness in enhancing the heat stress resistance of nematodes is stronger than that of the astaxanthin control group.

[0089] like Figure 11 As shown in C, the effective range of luteolin in enhancing the heat stress resistance of nematodes is 1-50 μM; a concentration of 0.1 μM is too low to be effective, while a concentration of 100 μM may cause toxicity. When the concentration of luteolin is controlled at 1-5 μM, its effectiveness in enhancing the heat stress resistance of nematodes is stronger than that of the astaxanthin control group.

[0090] like Figure 11 As shown in D, the effective range of acaciain in enhancing the heat stress resistance of nematodes is 5-50 μM; concentrations of 0.1 μM and 1 μM are ineffective due to their low concentrations, while concentrations of 100 μM may cause toxicity. When the concentration of acaciain is controlled within 5-50 μM, its effectiveness in enhancing the heat stress resistance of nematodes is stronger than that of the astaxanthin control group.

Claims

1. A perilla extract composition, wherein the perilla extract composition contains ursolic acid, apigenin, luteolin, and farnesin, wherein: The concentration of ursolic acid is selected from 0.1-100 μM, preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of apigenin is selected from 0.1-100 μM, and preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of luteolin is selected from 0.1-100 uM, and preferably, the concentration of ursolic acid is selected from 1-50 uM; The concentration of farnesin is selected from 1-100 uM, preferably from 5-50 uM.

2. The perilla extract combination as described in claim 1, wherein the perilla extract is perilla aqueous extract or perilla ethanol extract.

3. The use of the perilla extract combination as described in claim 1 in the preparation of anti-aging products.

4. The application as described in claim 3, wherein the perilla extract combination is selected from perilla water extract and / or perilla alcohol extract.

5. The application as described in claim 3, wherein the perilla extract combination contains one or more of ursolic acid, apigenin, luteolin and / or acaciain.

6. In the application as described in claim 3, the concentration of ursolic acid in the perilla extract combination is selected from 0.1-100 μM, preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of apigenin is selected from 0.1-100 μM, and preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of luteolin is selected from 0.1-100 uM, and preferably, the concentration of ursolic acid is selected from 1-50 uM; The concentration of farnesin is selected from 1-100 uM, preferably from 5-50 uM.

7. An anti-aging product, said product containing a perilla extract combination, said perilla extract combination containing ursolic acid, apigenin, luteolin, and farnesin, wherein: The concentration of ursolic acid is selected from 0.1-100 μM, preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of apigenin is selected from 0.1-100 μM, and preferably, the concentration of ursolic acid is selected from 1-50 μM; The concentration of luteolin is selected from 0.1-100 uM, and preferably, the concentration of ursolic acid is selected from 1-50 uM; The concentration of farnesin is selected from 1-100 uM, preferably from 5-50 uM.

8. The product as described in claim 7, wherein the product includes health products, cosmetics, food additives, and pharmaceuticals.

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