Anti-aging small RNA molecules, compositions, methods of making and use

Abstract

The application discloses an anti-aging small RNA molecule, a composition and a preparation method and application, and belongs to the field of natural medicinal chemistry and molecular biology. The nucleotide sequence of the small RNA molecule is shown as SEQ ID NO:1. The composition comprises a first small RNA with the nucleotide sequence shown as SEQ ID NO:1, a second small RNA with the nucleotide sequence shown as SEQ ID NO:2, a third small RNA with the nucleotide sequence shown as SEQ ID NO:3 and a fourth small RNA with the nucleotide sequence shown as SEQ ID NO:4. The preparation method comprises the following steps: pretreatment of water washing and alcohol washing, enzyme hydrolysis of a composite enzyme and kinetic controlled hydrolysis. The composition is obtained by enriching small RNAs with the function of silencing CD38 genes, down-regulating CD38 protein expression from a post-transcriptional level, effectively improving NAD + level and playing an anti-aging role. The application can be used for medicines or cosmetics. The application realizes the transformation from extraction of ambiguous components to enrichment of precise small RNA sequences, the product quality is stable, and the anti-aging potency is remarkable.

Description

Technical Field

[0001] This invention relates to the fields of natural product chemistry and molecular biology, specifically to an anti-aging small RNA molecule, composition, preparation method, and application. Background Technology

[0002] At the molecular level, aging is manifested by the core coenzyme NAD. + A sustained decline in NAD+ levels. It is well known in the art that NAD+... + It is a core substrate for maintaining mitochondrial energy metabolism, driving DNA damage repair, and activating the Sirtuins family of longevity proteins. NAD+ in tissues + Depletion of these cells is considered a root cause of cellular senescence, metabolic disorders, and various degenerative changes.

[0003] Modern biomedical research has established that CD38 (cyclic ADP-ribohydrolase) plays a role in NAD+ metabolism. + Its central role in metabolism. CD38 is the most efficient NAD+ in mammals. + The activity of depleting enzymes is significantly positively correlated with age. With aging, the expression of CD38, regulated by chronic inflammation, is abnormally elevated, and its highly efficient hydrolysis leads to increased NAD38 levels in tissues. + A precipitous drop in reserves. In the life sciences community, blocking NAD through gene knockout or specific inhibition of CD38 is being explored. + Damage has been widely proven to significantly restore mitochondrial activity, enhance stress resistance, and extend the healthy lifespan of model organisms. This causal chain has become a key biochemical indicator in this field for evaluating the effectiveness of anti-aging interventions.

[0004] Currently improving NAD + Most attempts have focused on supplementing precursor substances (such as NMN or NR). However, due to the overexpression of CD38 in aging tissues, supplemented precursor substances are often degraded by CD38 before being utilized by cells. This "leaky bucket effect" not only limits the bioavailability of exogenous supplementation but also fails to address the pathological imbalance of the body's internal metabolic axis. Furthermore, existing chemically synthesized inhibitors still face technical bottlenecks in terms of biocompatibility and safety of long-term intake.

[0005] Turmeric (Curcuma longa L.) is a plant belonging to the genus Curcuma in the family Zingiberaceae, and is a core medicinal material in traditional East Asian medicine. It is well-known in the field that turmeric has the effects of promoting blood circulation, regulating qi, and relieving pain. Modern pharmacological studies have further confirmed that turmeric extracts have a wide range of biological activities in anti-inflammation, anti-oxidation, and regulating metabolic homeostasis. Although the biological activities of turmeric are widely known, current technologies still face the following technical obstacles when using turmeric for anti-aging interventions: 1) The effective material basis is unclear: Current technologies mainly focus on the research of macromolecular polyphenols such as curcumin. However, curcumin has drawbacks such as extremely low bioavailability and rapid metabolism. There are currently no reports on microscopic active substances (such as small RNAs) in turmeric that have cross-species gene regulatory functions and their targeting relationship with CD38.

[0006] 2) Non-specificity of extraction process: Traditional boiling, alcohol precipitation or simple ultrasonic extraction processes often cannot effectively enrich small RNA fragments that are sensitive to heat and easily digested by enzymes.

[0007] 3) Lack of quality evaluation standards: Existing turmeric extracts lack fingerprint spectral standards that can reflect their true biological efficacy in inhibiting CD38, resulting in inconsistent product activity.

[0008] Therefore, it is urgent to discover novel CD38 inhibitory active substances from turmeric and establish targeted extraction and enrichment processes to provide a new material basis and technical path for developing safe and effective anti-aging natural drugs. Summary of the Invention

[0009] Based on a deep understanding of the aforementioned shortcomings, the purpose of this invention is to provide an anti-aging small RNA molecule, composition, preparation method, and application. This invention, for the first time, inhibits CD38 through the preparation of a combination of natural small RNA sequences, representing a more physiologically safer and biologically evolutionary anti-aging pathway. By downregulating CD38 expression, NAD+ is blocked at its source. + The loss of these nutrients is of great transformative significance for maintaining the body's energy homeostasis and preventing age-related functional decline.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an anti-aging small RNA molecule, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0011] As a further optimization of the present invention, the small RNA molecule is isolated or artificially synthesized.

[0012] In a second aspect, the present invention provides an anti-aging composition comprising a first small RNA, a second small RNA, a third small RNA, and a fourth small RNA, wherein the nucleotide sequence of the first small RNA is shown in SEQ ID NO: 1, the nucleotide sequence of the second small RNA is shown in SEQ ID NO: 2, the nucleotide sequence of the third small RNA is shown in SEQ ID NO: 3, and the nucleotide sequence of the fourth small RNA is shown in SEQ ID NO: 4.

[0013] As a further optimization of the present invention, the composition is a plant extract.

[0014] As a further optimization of the present invention, the plant extract is turmeric extract.

[0015] As a further optimization of the present invention, the turmeric extract has the following sRNA fingerprint characteristics: when using SEQ ID NO: 1 as a reference, the characteristic fingerprint similarity cosθ of the sequence content ratio of SEQ ID NO: 1-4 is ≥0.94; the abundance ratios of the core functional sequence SEQ ID NO: 1 and the characteristic peak sequence SEQ ID NO: 2-4 are 1:1.5-6, 1:70-290 and 1:10-40 respectively; and the total RNA content in the turmeric extract is not less than 20 μg / g.

[0016] Thirdly, the present invention provides a method for preparing the composition, comprising the following steps: (1) The plant raw material turmeric was pretreated by water washing and alcohol washing; (2) Under pH conditions of 4.5-5.5, the pretreated raw materials were enzymatically hydrolyzed using a compound enzyme preparation; (3) Perform kinetic-controlled hydrolysis and decoction at 95-100℃ for 20-60 minutes; (4) Separate and purify to obtain the composition.

[0017] In the above scheme, the content of the target small RNA sequence changes in an inverted U-shaped curve over time; the decoction time is controlled at the peak plateau period of the curve (20-60 minutes) so that the obtained small RNA composition meets the fingerprint spectrum and content requirements.

[0018] As a further optimization of the present invention, in step 1, the water washing pretreatment is to soak the plant raw materials in water for 0.5-1 hours while keeping them in an uncrushed state, and then discard the soaking solution; the alcohol washing pretreatment is to crush the plant raw materials and then soak them in ethanol with a volume concentration of 75-85% for 0.5-1 hours, and then discard the alcohol solution. And / or, in step 2, the compound enzyme preparation comprises cellulase, pectinase, xylanase and hemicellulase; the enzymatic hydrolysis temperature is 45℃-55℃ and the enzymatic hydrolysis time is 2-4 hours; And / or, in step 4, the separation and purification includes cooling and quenching the reaction of the hydrolysis system, filtering to obtain the filtrate, concentration, and drying.

[0019] As a further optimization of the present invention, in step 2, the mass ratio of cellulase, pectinase, xylanase and hemicellulase in the compound enzyme preparation is 2:1:1:0.5.

[0020] Fourthly, the present invention provides the application of the small RNA molecule or the composition described herein in the preparation of an anti-aging product, wherein the product is capable of inhibiting CD38 protein expression and increasing coenzyme NAD. + The product is a pharmaceutical or cosmetic product.

[0021] The present invention has the following beneficial effects: 1. This invention breaks through the technical bias in the field that only focuses on secondary metabolites such as curcumin as the active components of turmeric. It is the first to discover and verify that SEQ ID NO:1 (sRNA-1120.34) is the core functional sequence, which specifically silences the CD38 gene at the post-transcriptional level through RNA interference, thus blocking NAD at its source. + In contrast to chemically synthesized inhibitors (such as 78c), which can only competitively bind to the CD38 enzyme active site but cannot eliminate the CD38 protein itself, the small RNA strategy of this invention can truly downregulate CD38 protein expression, significantly reducing the CD38 positivity rate in cell experiments (P<0.001), while greatly increasing NAD+ expression. + The level of action and the mechanism of action are more fundamental.

[0022] 2. This invention addresses the pain point of large batch-to-batch activity variations in traditional turmeric extracts by proposing for the first time to use the abundance ratio of the four sequences in SEQ ID NO:1-4 as a fingerprint feature. It specifies that, using SEQ ID NO:1 as the baseline, the fingerprint similarity cosθ ≥ 0.94, and the abundance ratio of the core sequence to the peak sequence falls within a fixed range. This standard upgrades quality control from a crude indicator like total RNA content or polyphenol content to a precise digital criterion for sequence composition consistency, ensuring the stability and reproducibility of the bioactivity of each batch of product and laying a technological foundation for pharmaceutical production.

[0023] 3. This invention addresses the sensitivity of small RNAs to heat and nucleases by designing an original extraction process: The water washing step utilizes the intact cell wall to protect the internal RNA from exogenous enzyme degradation; the alcohol washing step utilizes the insolubility of RNA in ethanol to remove lipid-soluble impurities; combined enzymatic hydrolysis at pH 4.5-5.5 efficiently disrupts cell walls while maintaining low activity of endogenous RNases; finally, kinetically controlled hydrolysis is performed at 95-100℃ under gentle boiling conditions. By precisely controlling the decoction time (20-60 minutes), the generation rate of the target small RNA exceeds the degradation rate, resulting in an inverted U-shaped content peak. This process allows the total RNA content of turmeric extract to reach over 70 μg / g (Example 4), while conventional water or alcohol extraction processes can hardly detect small RNA, fully verifying the superiority of this invention.

[0024] 4. In vitro cell experiments (Example 6) showed that the total RNA from the chemically synthesized product of SEQ ID NO:1 and the turmeric extract could significantly downregulate CD38 expression and increase NAD in H9c2 cells. + The levels were significantly lower than normal (P<0.001), and the extract after RNase pretreatment completely lost its activity, clearly demonstrating that the efficacy originated from the small RNA component. Animal experiments (Example 7) further confirmed that after gavage administration of the turmeric extract of this invention to aged mice, the expression of CD38 protein in the liver was significantly reduced, and NAD50 expression was significantly decreased. + The concentration increased significantly, while the RNase treatment group had no effect, further confirming that small RNA is the core material basis. The above results systematically verify that the composition of this invention utilizes CD38-NAD... + It has the effect of alleviating aging through pathways, and the effective dose is much lower than that of traditional curcumin-like ingredients.

[0025] 5. This invention is the first to identify a family of small RNA sequences with CD38 gene silencing function from turmeric, breaking the previous pattern that CD38 inhibitors were limited to chemically synthesized or antibody-based macromolecules, and providing a source of natural small RNA drugs derived from traditional Chinese medicine with good biocompatibility. This discovery not only enriches the CD38-NAD... + The toolkit for intervention in pathways has also opened up new avenues for the development of precision anti-aging drugs based on small RNAs. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a diagram showing the relative distribution characteristics of each small RNA in Example 2.

[0028] Figure 2 The following are standard curves for each small RNA in Example 5; wherein: A is the standard curve for SEQ ID NO: 1, B is the standard curve for SEQ ID NO: 2, C is the standard curve for SEQ ID NO: 3, and D is the standard curve for SEQ ID NO: 4.

[0029] Figure 3 The graph shows the CD38 cell positivity rate of each group in Example 6. P<0.05, P<0.01, P<0.001).

[0030] Figure 4 NAD in H9c2 cells of each group in Example 6 + Content chart ( P<0.05, P<0.01, P<0.001).

[0031] Figure 5 This is a graph showing the expression of CD38 protein in the liver tissues of mice in each group in Example 7. P<0.05, P<0.01, P<0.001).

[0032] Figure 6 NAD3 concentration in mouse liver tissues from each group in Example 7 + Concentration level chart ( P<0.05, P<0.01, P<0.001). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0034] This invention reveals that the core material basis for CD38 inhibition in turmeric is not the traditionally recognized small molecule compounds, but rather small RNA sequences that downregulate CD38 levels. These small RNA sequences are extremely rare in their natural state, but under the specific kinetic-controlled hydrolysis process of this invention, a regular and efficient combination of specific small RNA sequences with clear biological activity can be generated, and their characteristics can be used as a fingerprint for quality control.

[0035] (a) The core small RNA sequence combination regulating CD38 This invention is the first to discover and validate a set of core small RNA sequences detectable in turmeric, including: The core sequence that provides core CD38 inhibition: SEQ ID NO: 1 (sRNA-1120.34): The small RNA sequence is: [cggaagagcgucgcaugu]; At least three characteristic kurtosis sequences, which are as follows: SEQ ID NO: 2 (sRNA-117.1) Its small RNA sequence: [cacaacuccgcgucacg]; SEQ ID NO: 3 (sRNA-206.1) has the following small RNA sequence: [ccugcuugggcgucaug]; SEQ ID NO: 4 (sRNA-226.34) Its small RNA sequence: [ccaaaugccucgucaucu].

[0036] In the sequence list corresponding to the sequences shown in SEQ ID NO: 1-4, T is used to replace U.

[0037] (II) Description of each plant material The raw material turmeric (CURCUMAE LONGAE RHIZOMA) mentioned in this invention is the dried rhizome of turmeric (Curcumalonga L.), a plant of the ginger family.

[0038] (III) Core Mechanism of Preparation Process This invention proposes a preparation process for extracting small RNA from the above-mentioned plants, the core physicochemical mechanism of which is as follows: (1) Physical barrier protection and degreasing: The medicinal materials are soaked in water without being crushed. The natural physical barrier of the plant cell wall is used to prevent the internal RNA from dissolving prematurely and being degraded by exogenous enzymes, and to remove inorganic salts to the maximum extent. The crushed medicinal materials are soaked in alcohol. The characteristic that RNA is insoluble in ethanol is used to remove alcohol-soluble fats and other components to the maximum extent, so as to provide a pure system for subsequent reactions.

[0039] (2) pH-enzyme dual-control targeted release: A specific complex enzyme (cellulase, pectinase, xylanase, hemicellulase) is added to a slightly acidic buffer solution with a pH of 4.5-5.5. This specific pH range not only works with the complex enzyme to efficiently break down the cell wall, but more importantly, it inactivates or reduces the activity of endogenous ribonuclease (RNase has higher activity at neutral pH), thereby protecting the released RNA from unintended degradation.

[0040] (3) Precise control of thermodynamic selectivity to achieve targeted hydrolysis: Under heating conditions of 95-100℃, the slightly acidic environment promotes the thermal hydrolysis of RNA molecules. This invention confirms that by controlling the hydrolysis time to 20-60 minutes, the generation rate of the target small RNA sequence is greater than its degradation rate, forming a kinetic peak, thereby specifically extracting the combination of the small RNA sequences.

[0041] The turmeric extract and its core sequence obtained by any of the methods described in this invention can be applied to regulate CD38-NAD. + Its application in drugs that maintain energy homeostasis and prevent age-related functional decline.

[0042] The technical solution and mechanism of action of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0043] Example 1: Establishment of a Curcuma small RNA database 1) Objective: To extract total small RNA from turmeric after decoction to meet the requirements for library construction and sequencing, and to list the data summary after sequencing a specific batch of turmeric.

[0044] 2) Operating steps: (1) Take 300 μl of turmeric decoction and extract total RNA using magnetic bead method.

[0045] (2) High-throughput sequencing was performed using the Illumina Navaseq PE150 platform with a sequencing depth of 3.00 Gram data and a quality value distribution Q of 20-90%.

[0046] 3) Sequencing results: Table 1. Total RNA sequencing data from turmeric extract 4) Results Analysis The four sequences of this invention were all highly expressed in sequencing and could be detected stably by qPCR.

[0047] Example 2: Construction of sRNA fingerprinting 1) Objective: To construct a fingerprint of turmeric extract, show the distribution of characteristic sequences of the turmeric extract after sequencing, and clarify the abundance proportion of the core sequence SEQ ID NO: 1 (sRNA-1120.34).

[0048] 2) Operation steps: Total RNA was extracted from 3 batches of turmeric raw materials and sequenced to obtain RPM of SEQ ID NO: 1-4, and the proportion of each sequence was calculated.

[0049] 3) Calculation results and analysis: such as Figure 1As shown, the relative distribution map of the characteristic sequences proves that not only is the sequence SEQ ID NO: 1 of the turmeric extract consistent across multiple sequencing batches, but the internal standards SEQ ID NO: 2-4 are also highly consistent. This indicates that the turmeric extract of this invention possesses a clear biological signal. The establishment of this map can effectively prevent the counterfeiting of qualified products by adding artificially synthesized sequences to the raw materials.

[0050] Example 3: Preparation of Curcuma extract rich in specific small RNAs 1) Purpose: To demonstrate the specific operability of the extraction process.

[0051] 2) Operating steps: (1) Pretreatment: Water washing: Take 100g of sliced ​​turmeric raw material, add purified water and soak for 1 hour, then discard the soaking solution. Alcohol washing: After crushing the turmeric, add 80% (v / v) ethanol and soak for 1 hour, then discard the alcohol solution.

[0052] (2) Targeted enzymatic hydrolysis: Add compound enzyme preparation (2g cellulase, 1g pectinase, 1g xylanase, 0.5g hemicellulase), add 1.5kg of water, adjust the pH to 5.0 with citrate-sodium citrate buffer, and stir at 50°C for 3 hours for enzymatic hydrolysis.

[0053] (3) Kinetic controlled hydrolysis: After the enzymatic hydrolysis is completed, the reaction system is rapidly heated to a slight boiling state (95-100℃) and kept boiling for 40 minutes to carry out targeted thermodynamic pyrolysis.

[0054] (4) Separation and purification: After hydrolysis, the reaction was quenched by rapid cooling, and the filtrate was collected by filtration. The filtrate was concentrated under reduced pressure and then spray-dried to obtain the turmeric extract A3 containing the specific small RNA sequence combination.

[0055] Comparative Example 1: Preparation of Compositions Using Conventional Processes Operating steps: Comparative Example 1-a (conventional water extraction of total polysaccharides or water-soluble components of turmeric): Take 100g of turmeric tubers, wash and air-dry them, then pulverize them to 30-40 mesh, add 12 times the amount of water, extract at 90-95℃ for 2 hours, centrifuge and filter to remove residue, concentrate under vacuum (vacuum degree -0.06 to -0.08MPa, temperature ≤70℃) to a solid content of 20-30%, and spray dry to obtain composition B.

[0056] Comparative Example 1-b (Conventional alcohol extraction of curcumin): 100g of turmeric tubers were washed, air-dried, and pulverized to 30-40 mesh. 90-95% edible ethanol was added (solid-to-liquid ratio 1:10), and extracted at 50-60℃ for 2 hours. The residue was removed by filtration, and the extract was concentrated under vacuum (vacuum degree -0.06 to -0.08 MPa, temperature ≤65℃). Six times the volume of purified water was added to the concentrate, and the mixture was allowed to stand for 10 hours to precipitate curcumin. The wet product was centrifuged and filtered, then vacuum-dried at 60℃ to obtain composition C.

[0057] Example 4: Relationship between kinetic parameters of controlled hydrolysis of turmeric extract and total small RNA production 1) Experimental objective: To determine the content of small RNA in turmeric extract.

[0058] 2) Experimental methods: The content of small RNA in turmeric extracts prepared by different processes (decoction times of 10 minutes, 20 minutes, 40 minutes, 60 minutes, and 80 minutes, corresponding to compositions A1, A2, A3, A4, and A5) and conventional compositions (compositions B and C) prepared in Comparative Example 1 was determined to verify the high efficiency of the extraction process of the present invention for the extraction of target small RNA. When preparing the above compositions A1-A5, except for the decoction times of kinetic controlled hydrolysis in step (3) being 10 minutes, 20 minutes, 40 minutes, 60 minutes, and 80 minutes respectively, all other conditions were the same as in Example 3. Total RNA was extracted by the Trizol method, and the total RNA content was detected by Nanodrop.

[0059] 3) Experimental results: Table 2. Detection results of small RNA content in multiple batches of turmeric extracts processed using different methods 4) Results Analysis: Composition A3 (40 minutes) from this process group had the highest total RNA content, while no bioactive small RNAs were detected in conventional processes (compositions B and C). The preparation process of this invention can effectively extract small RNAs and has significant process advantages.

[0060] Example 5: Quantitative detection of the core small RNA sequence in the composition of the present invention 1) Experimental objective: To perform RT-qPCR quantitative analysis of the core small RNA in the composition.

[0061] 2) Experimental methods: Establishment of the standard curve: Accurately weigh each synthetically produced small RNA standard and dissolve it in RNase-free water to a concentration of 0.1 μM. Perform 10-fold serial dilutions of the standards to prepare six different concentrations (0.01 μM, 1 nM, 100 pM, 10 pM, 1 pM, 0.1 pM) for the standard curve. After reverse transcription (RT) using stem-loop primers, perform real-time quantitative PCR (RT-qPCR) amplification. Plot the standard curve with the logarithm of the small RNA molar concentration on the x-axis and the Ct value on the y-axis. Calculate the linear regression equation for the standard curve, as follows: Figure 2 The standard curve shown is SEQ ID NO:1-4.

[0062] Total RNA extraction: Take 100 mg of the turmeric extract obtained in Example 3 of this invention, add 1 ml of enzyme-free water, dissolve thoroughly, take 200 μl of the solution, and extract total RNA using the Trizol method.

[0063] Quantitative detection of core small RNAs: The small RNA sequences of SEQ ID NO:1-4 were quantitatively detected using real-time quantitative PCR (RT-qPCR).

[0064] The primer sequences are as follows: Table 3 3) Experimental Results Table 4. Content of four small RNAs (pmol / g) in three batches of turmeric extract. Calculate the relative abundance ratio of SEQ ID NO:1 to SEQ ID NO:2-4 sequences using the average values ​​in Table 4: SEQ ID NO:2 / SEQ ID NO:1 = 1.12 / 0.38 = 2.95; SEQ ID NO:3 / SEQ ID NO:1 = 55.13 / 0.38 = 145.08; SEQ ID NO:4 / SEQ ID NO:1 = 7.83 / 0.38 = 20.60.

[0065] Based on the principle of RT-qPCR detection, ΔCT = ±1 is set, and the above ratios are multiplied by 2 and divided by 2 respectively to obtain the upper and lower limits: SEQ ID NO:1 / SEQ ID NO:2 = 1:1.5-6; SEQ ID NO:1 / SEQ ID NO:3 = 1:70-290; SEQ ID NO:1 / SEQ ID NO:4 = 10-40.

[0066] Similarity of characteristic small RNA profiles from different extracts, calculated using the following formula: Where: n represents the number of feature sequences; Ai is the ratio of the i-th sequence in the standard vector; Bi is the ratio of the i-th sequence in the vector to be tested.

[0067] Table 5. Calculation of similarity of characteristic sequences in different batches of turmeric extracts In Table 5, turmeric extract B and turmeric extract C are prepared according to the process in Example 3 using different batches of turmeric raw materials.

[0068] 4) Results Analysis: Characteristic small RNA sequences (SEQ ID NO: 1-4) were all detected in turmeric. Given the enzymatic reaction bias during sequencing library preparation, the RPM value reflects the relative distribution characteristics of the sequences, rather than the absolute physical molar quantity. The absolute quantification technology of qPCR and sequence characteristic mapping complement each other; qPCR ensures the titer of the core sequence, while the high cosine similarity of the sequencing fingerprint ensures the overall consistency of complex biological small RNA compositions.

[0069] Example 6: In vitro cell experiments 1) Experimental Objective: To investigate the core sequence SEQ ID NO:1, the total RNA of the composition, and the effects of the composition on intracellular CD38 protein and NAD. + Its regulatory role.

[0070] 2) Experimental methods: (1) Synthesis of SEQ ID NO:1 (1120.34) standard by chemical solid-phase synthesis: This invention uses RNA solid-phase synthesis technology based on phosphoramidite chemistry. This technology is currently the most mature and widely used chemical synthesis method for small RNAs internationally. It was first created by Marvin Caruthers et al. in 1981 and has been highly standardized after more than 40 years of development. It is suitable for synthesizing small RNAs of any sequence length within 100 nt.

[0071] (2) Total RNA was extracted from turmeric extract using the Trizol method.

[0072] (3) H9c2 cells were incubated in 6-well plates. After D-Gal modeling, the cells were transfected with Lipo3000 with the core small RNA sequence (SEQ ID NO:1), total RNA from turmeric extract, and positive control drug 78c for 72 hours.

[0073] Dosage: SEQ ID NO:1: 1 nmol / 6-well plate; Total RNA from turmeric extract: 600 mg extract (total RNA extracted by Trizol method) / 6-well plate.

[0074] (4) The positive rate of CD38 cells was detected by flow cytometry, and NAD in H9c2 cells was detected by colorimetric method. + content.

[0075] 3) Experimental Results Table 6. Positive rate of CD38 cells in H9c2 cell experiments Table 7. NAD in H9c2 cells + Concentration level 4) Results Analysis: such as Figure 3 and Figure 4 As shown, compared with the model group (D-Gal), the chemically synthesized standard of the core sequence SEQ ID NO:1 of this invention, by downregulating CD38 protein expression, fundamentally eliminates the enzymatic activity of CD38, and not only enhances NAD... + The concentration also disrupted the CD38-mediated pro-senescence signaling pathway, thereby reducing the positive rate of senescent cells. The positive control drug 78c is a high-affinity competitive inhibitor of CD38. It prevents NAD by binding to the extracellular domain of CD38. + Hydrolysis, causing NAD + The concentration increased significantly. However, the bound CD38 protein remained intact on the H9c2 cell membrane, so the cell positivity rate did not improve significantly. This experiment confirms that SEQ ID NO:1 of this invention downregulates CD38 expression at the gene level, and the benefit of this structural deletion is superior to the simple activity inhibition of 78c.

[0076] Meanwhile, the experimental results showed that both total RNA obtained from turmeric extract using the Trizol method and turmeric extract significantly upregulated NAD. + This confirms that the core substance in turmeric that affects CD38 anti-aging is small RNA.

[0077] Example 7: Animal experiments with core small RNA and the composition CD38 1) Experimental objective: To verify that the composition inhibits CD38 and enhances NAD. + Does the drug's efficacy originate from small RNA?

[0078] 2) Experimental methods: Animal species: 18-month-old mouse (C57BL / 6J).

[0079] Grouping: blank model group (aged mice), SEQ ID NO: 1, turmeric extract + RNase, turmeric extract, positive drug 78c, 6 mice in each group, 5 groups in total.

[0080] Dosing regimen: The blank model group was given 2 mL of physiological saline by gavage daily, while the other experimental groups were given the drug twice daily at 8:00 AM and 4:00 PM. The dosing period was 8 weeks, and the dosing regimen is shown in Table 8.

[0081] Table 8 Dosage and Administration Detection indicators: After drug administration, the expression of CD38 protein and NAD in liver tissue was detected. + Concentration level.

[0082] 3) Experimental results: Table 9. CD38 protein expression in liver tissue of aging mice Table 10 NAD in liver tissue of aging mice + Concentration level 4) Results analysis, such as Figure 5 and Figure 6 As shown, animal experiments revealed that the core sequence SEQ ID NO:1, synthesized chemically, and the turmeric extract prepared by the extraction method of this invention significantly reduced CD38 protein expression and effectively increased NAD+ expression in an aged mouse aging model. + At certain concentration levels, it has an anti-aging effect. Most importantly, when the composition is pretreated with RNase enzyme to degrade the small RNAs (turmeric extract + RNase group), it loses its ability to enhance NAD+. + This counter-evidence demonstrates that the composition of the present invention exerts the effect of downregulating CD38 protein expression and enhancing NAD+ expression. + The core material basis for the concentration level is the core small RNA extracted by this specific process, rather than other conventional small molecule metabolites in the composition.

[0083] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-aging small RNA molecule, characterized in that, The nucleotide sequence of the small RNA molecule is shown in SEQ ID NO:

1.

2. An anti-aging composition, characterized in that, The composition comprises a first small RNA, a second small RNA, a third small RNA, and a fourth small RNA, wherein the nucleotide sequence of the first small RNA is shown in SEQ ID NO: 1, the nucleotide sequence of the second small RNA is shown in SEQ ID NO: 2, the nucleotide sequence of the third small RNA is shown in SEQ ID NO: 3, and the nucleotide sequence of the fourth small RNA is shown in SEQ ID NO:

4.

3. The composition according to claim 2, characterized in that, The composition is a plant extract.

4. The composition according to claim 3, characterized in that, The plant extract is turmeric extract.

5. The composition according to claim 4, characterized in that, The turmeric extract has the following sRNA fingerprint characteristics: when using SEQ ID NO: 1 as a reference, the characteristic fingerprint similarity cosθ of the sequence content ratio of SEQ ID NO: 1-4 is ≥0.94; the abundance ratios of the core functional sequence SEQ ID NO: 1 and the characteristic peak sequence SEQ ID NO: 2-4 are 1:1.5-6, 1:70-290 and 1:10-40 respectively; the total RNA content in the turmeric extract is not less than 20 μg / g.

6. A method for preparing the composition according to any one of claims 2-5, characterized in that, Includes the following steps: Step 1: Pre-treat the plant raw material turmeric with water washing and alcohol washing; Step 2: Under pH conditions of 4.5-5.5, the pretreated raw materials are enzymatically hydrolyzed using a compound enzyme preparation; Step 3: Perform kinetic-controlled hydrolysis and decoction at 95-100℃ for 20-60 minutes; Step 4: Separate and purify to obtain the composition.

7. The preparation method according to claim 6, characterized in that, In step 1, the water washing pretreatment involves soaking the plant material in water for 0.5-1 hour while keeping it in a non-crushed state, and then discarding the soaking solution; the alcohol washing pretreatment involves crushing the plant material and then soaking it in ethanol with a volume concentration of 75-85% for 0.5-1 hour, and then discarding the alcohol solution. And / or, in step 2, the compound enzyme preparation comprises cellulase, pectinase, xylanase and hemicellulase; the enzymatic hydrolysis temperature is 45℃-55℃ and the enzymatic hydrolysis time is 2-4 hours; And / or, in step 4, the separation and purification includes cooling and quenching the reaction of the hydrolysis system, filtering to obtain the filtrate, concentration, and drying.

8. The preparation method according to claim 7, characterized in that, In step 2, the mass ratio of cellulase, pectinase, xylanase, and hemicellulase in the compound enzyme preparation is 2:1:1:0.

5.

9. The use of the small RNA molecule of claim 1 or the composition of claim 2 in the preparation of anti-aging products, characterized in that, The product in question is a pharmaceutical or cosmetic product.

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