Torreya grandis total RNA (Ribonucleic Acid) extract and application thereof

Abstract

The invention relates to the field of plant extracts, in particular to a torreya grandis total RNA extract which at least comprises mRNA, tRNA, miRNA and tsRNA. The torreya grandis total RNA extract can be used as a natural active matter for an external preparation for the skin, and the effects of resisting oxidation, resisting aging or resisting wrinkles and tightening are achieved, so that the skin aging is delayed.

Description

Technical Field

[0001] The present invention relates to the field of plant extracts, and particularly to a total RNA extract of Torreya grandis Fort. cv. Merrillii and its application. Background Art

[0002] The skin is the largest organ of the human body, mainly responsible for functions such as protecting the body, sweating, sensing heat, cold and pressure, and protecting various tissues and organs in the body from physical, mechanical, chemical and pathogenic microbial invasions. Skin aging is divided into two forms: exogenous aging and endogenous aging. Exogenous aging is mainly caused by environmental factors such as ultraviolet radiation. Endogenous aging is a programmed change caused by genes over time, with a complex mechanism of occurrence. The histological manifestations may include thinning of the epidermal barrier, slow cell self-renewal, reduced synthesis of dermal matrix, fracture of elastic protein fibers, shortening of collagen fibers, etc. Therefore, studying how to improve the anti-aging ability of the skin and the expression of anti-aging components is of great significance for delaying skin aging.

[0003] Ribonucleic acid (abbreviated as RNA, i.e., Ribonucleic Acid) is a genetic information carrier present in biological cells and some viruses and viroids. According to species differences, the types of RNA are different, mainly including: messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (microRNA or miRNA), enhancer RNA (eRNA), small interfering RNA (siRNA), small activating RNA (saRNA), circular non-coding RNA (circRNA), etc.

[0004] Research has found that small RNAs (<200 nt) play a key role in various biological activities of microorganisms, plants, animals, and humans, especially in the treatment of human diseases, by regulating protein-coding genes. For example, transfer RNA (tRNA)-derived stress-induced RNAs (tiRNAs) and derived fragments (tRFs), which belong to tRNA-derived small RNAs (tsRNAs), are a new type of short-chain non-coding RNAs that play a key role in protein translation and exert various regulatory functions in important biological processes such as cell signal transduction, apoptosis, and stress response. In skin damage caused by ultraviolet irradiation, 4 tsRNAs were upregulated (tRF-Val-AAC-012, tRF-Pro-AGG-012, tRF-Val-CAC-018, tRF-Val-AAC-031), and 6 tsRNAs were downregulated (tRF-Arg-CCT-002, tRF-Trp-TCA-001, tiRNA-Ser-GCT-001, tRF-Gly-CCC-019, tRF-Ala-tgc-001, tRF-AlaTGC-002). Among them, tRF-Gly-CCC-019 plays an important role in UVB radiation-induced acute skin damage by regulating the ras-related C3 botulinum toxin substrate 1 (Rac1) gene in the WNT signaling pathway. The upregulation or downregulation of some tRFs in skin tissue caused by ultraviolet irradiation is a physiological manifestation in skin adversity, making it possible to apply tRFs in the prevention and antagonism of skin photoaging (Front Cell Dev Biol. 2021 Aug 10;9:707572.).

[0005] In addition, the research also found that plant RNAs might be an ideal RNA source. For example, in studies on rice, Rhodiola rosea, Lonicera japonica, etc., it was found that the miRNAs they contain can be used to regulate the expression of target genes to achieve therapeutic purposes; the single tRNA contained in Taxus chinensis has strong cytotoxicity to ovarian cancer cells. Through the design and screening of tRFs, it was found that the antisense product of tRF-t11(GUG) has an anti-cancer effect equivalent to that of paclitaxel on ovarian cancer cell line A2780 and its xenograft animal model (Mol Ther Nucleic Acids. 2022 Jan 3;27:718-732.).

[0006] Torreya grandis Fort. ex Lindl. belongs to the genus Torreya of the family Taxaceae and is one of the ancient species unique to China. It is a tertiary relict plant that originated in the Middle Jurassic about 170 million years ago. Torreya grandis Fort. ex Lindl. is rich in fatty acids, minerals, phenols, flavonoids, tannins, squalene and other substances, all of which have strong antioxidant functions. At present, there is no research report on the application of total RNA of Torreya grandis Fort. ex Lindl. in anti-aging. Summary of the Invention

[0007] The object of the present invention is to provide a total RNA extract of Torreya grandis, the extract has a clear action mechanism, is safe and reliable, and has the effects of anti-wrinkle and firming, and antioxidant.

[0008] The specific technical solution is as follows:

[0009] A total RNA extract of Torreya grandis, the extract at least includes mRNA, tRNA, miRNA, tsRNA.

[0010] In some embodiments, tsRNA includes tiRNA, tRF.

[0011] In some embodiments, the concentration of the extract is 5 ng / μL - 100 ng / μL, preferably 12.5 ng / μL - 50 ng / μL, and further preferably 25 ng / μL - 50 ng / μL.

[0012] The present invention also provides a preparation method of the above-mentioned total RNA extract of Torreya grandis, the preparation method includes the following steps:

[0013] 1) Use a lysis solution to lyse the cells of Torreya grandis, add an organic solvent and then centrifuge to extract the supernatant;

[0014] 2) Add a lower alcohol to the supernatant, transfer the solution to filter cartridge 1, centrifuge to collect the filtrate, add a lower alcohol to the obtained filtrate and then transfer it to filter cartridge 2, and discard the filtrate after centrifugation;

[0015] 3) For the two filter cartridges used in step 2), respectively use a washing solution to centrifuge and wash, and then discard the filtrate;

[0016] 4) Add ribonuclease-free water to the two filter cartridges after washing in step 3) and then centrifuge, and mix the collected filtrates to obtain the total RNA extract of Torreya grandis.

[0017] In some embodiments, the organic solvent is preferably chloroform-isoamyl alcohol.

[0018] In some embodiments, the lower alcohol is ethanol, isopropanol, n-butanol or a mixture thereof.

[0019] In some embodiments, after step 3), the filter cartridge empty column is centrifuged once to better separate impurities and improve the concentration and purity of the extract.

[0020] In some embodiments, the washing solution includes washing solution 1 and washing solution 2, washing solution 1 contains ultrapure water, NaCl, EDTA, Tris-HCl and an 80% ethanol solution, and washing solution 2 contains an 80% ethanol solution.

[0021] In some specific embodiments, the molar ratio of NaCl:EDTA:Tris-HCl is 20:0.9:2.

[0022] In some specific embodiments, in step 3), the filter element 1 is centrifugally washed once with washing solution 1, and the filter element 2 is centrifugally washed twice with washing solution 2.

[0023] In some embodiments, the filter element 1 collects macromolecular RNA, and the filter element 2 collects small molecular RNA; the macromolecular RNA is RNA with a nucleotide (NT) number ≥200, such as mRNA, etc., and RNA with NT < 200, such as miRNA, tsRNA, etc.

[0024] In some embodiments, 1) the centrifugation program in the preparation method is set as follows, the rotation speed is 8000xg - 12000xg, and the centrifugation time is 15s - 15min;

[0025] In some embodiments, the torreya grandis raw materials used in step 1) include torreya grandis fruits, seeds, leaves, seed coats, and arils;

[0026] In some embodiments, the lysis solution in step 1) is TRIzol lysis solution;

[0027] In some embodiments, the addition amount of lower alcohol in step 2) is 1 / 3 or 2 / 3 of the total volume;

[0028] In some embodiments, after adding ribonuclease-free water in step 5), it is left standing for 2 - 5 minutes, preferably 2 minutes.

[0029] The preparation method can reduce the loss of small molecular RNA during the extraction process, preferably enrich small molecular RNA, and maintain the activity of the extract.

[0030] The present invention also provides the application of the above torreya grandis total RNA extract, or its preparation method in the preparation of the following products:

[0031] 1) Anti-aging products;

[0032] 2) Products for improving sleep;

[0033] 3) Products for enhancing immunity;

[0034] 4) Anti-oxidant products.

[0035] In some embodiments, the products are external use products or internal use products, the external use products are selected from skin care products, cosmetics, and hair care products; the internal use products are selected from solid beverages, liquid beverages, dairy products, flavoring powders, nutritional products, and internal use healthcare products.

[0036] In some embodiments, the topical product is selected from emulsions, gels, creams, microneedle patches, and facial masks, preferably an emulsion.

[0037] In some embodiments, the topical product further contains excipients, and the excipients contain preservative components: selected from methyl paraben, ethyl paraben, propyl paraben, formaldehyde-releasing agents, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl ureas, isothiazolinones, phenols, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternium-15, dehydroacetic acid and its salts, or one or more thereof.

[0038] In some embodiments, the topical product further contains excipients, and the excipients contain emulsifying components: selected from polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, phospholipids, or one or more thereof.

[0039] In some embodiments, the topical product further contains excipients, and the excipients contain modifying excipients:

[0040] selected from fragrances and / or pigments.

[0041] In some embodiments, the oral product further contains food-grade acceptable excipients, and the excipients contain flavoring agents: selected from fructooligosaccharide, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, edible flavor, citric acid, or one or more thereof.

[0042] In some embodiments, the oral product further contains food-grade acceptable excipients, and the excipients contain dosage form excipients: selected from maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, silicon dioxide, or one or more thereof.

[0043] The beneficial effects of the present invention are as follows:

[0044] 1) Collagen and elastin are very important for maintaining the elasticity and firmness of the skin. The reduction of such proteins will lead to skin roughness and aging phenomena (such as wrinkle formation and reduced elasticity). Through experiments at the molecular level and cell level, the present invention has confirmed that the total RNA extract of Torreya grandis can regulate the expression levels of collagen fibers and elastic fibers related to skin aging phenotypes, has antioxidant effects, and thus improves aging skin.

[0045] 2) The present invention also provides a preparation method of the total RNA extract of Torreya grandis. Compared with the existing methods, the concentration and purity of the obtained extract have been significantly improved. Description of the Drawings

[0046] Figure 1The effect of the total RNA extract of Torreya grandis on scavenging DPPH free radicals was shown.

[0047] Figure 2 The effect of the total RNA extract of Torreya grandis on promoting the synthesis of collagen I in human skin fibroblasts was shown.

[0048] Figure 3 The effect of the total RNA extract of Torreya grandis on inhibiting the synthesis of MMP-1 in human skin fibroblasts was shown. Detailed implementation manners

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] Although the numerical ranges and parameter approximations shown in the broad scope of this invention, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. Additionally, all ranges disclosed herein should be understood to encompass any and all sub-ranges contained therein. For example, the range of "5 to 100" recited should be considered to include any and all sub-ranges between the minimum value of 5 and the maximum value of 100 (including the endpoints); that is, all sub-ranges starting from 5 or greater, such as 5 to 61, and sub-ranges ending at 100 or less, such as 55 to 100. Additionally, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0051] One of the objectives of this invention is to provide a total RNA extract of Torreya grandis.

[0052] The term "Torreya grandis" refers to a perennial evergreen tree of the genus Torreya in the family Taxaceae. Common species include the wild Torreya grandis Fort. et Lindl. produced in China, with aliases such as Torreya grandis, Yangjiao Torreya, Xifei, Yufei, Yeshan, Zizi, etc.; Torreya taxifolia Am. produced in North America, Torreya californica Torrey; Torreya nucifera (L.) Sieb. et Zucc. produced in Japan.

[0053] In some specific implementation manners, the Torreya grandis is Torreya grandis Fort. et Lindl.

[0054] The term "RNA" refers to ribonucleic acid (abbreviated as RNA, i.e., Ribonucleic Acid), which is a carrier of genetic information present in biological cells, as well as in some viruses and viroids. RNA is a long-chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds. A ribonucleotide molecule consists of a phosphate, a ribose, and a base. The bases of RNA are mainly four types, namely A (adenine), G (guanine), C (cytosine), and U (uracil), among which U (uracil) replaces T (thymine) in DNA. The main role of ribonucleic acid in the body is to guide protein synthesis.

[0055] According to species differences, the types of RNA vary, mainly including: messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (microRNA or miRNA), enhancer RNA (eRNA), small interfering RNA (siRNA), small activating RNA (saRNA), circular non-coding RNA (circRNA), etc.

[0056] In some embodiments, the total Torreya grandis RNA extract includes at least mRNA, tRNA, miRNA, and tsRNA.

[0057] The term "mRNA" refers to messenger RNA (Messenger RNA) or messenger ribonucleic acid, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template and carrying genetic information to guide protein synthesis.

[0058] The term "miRNA" refers to MicroRNA (miRNA), which is a class of non-coding single-stranded RNA molecules encoded by endogenous genes and about 22 nucleotides in length. They participate in post-transcriptional gene expression regulation in animals and plants. miRNAs can regulate target mRNAs by disrupting the stability of target mRNAs and inhibiting the translation of target mRNAs.

[0059] The term "tRNA" refers to transfer RNA (Transfer RNA), also known as transfer ribonucleic acid, usually abbreviated as tRNA. It is a type of RNA composed of 76 - 90 nucleotides. Its 3' end can be attached to a specific type of amino acid under the catalysis of aminoacyl-tRNA synthetase. During the process of translation, tRNA can recognize the codon on mRNA through its own anticodon and transport the amino acid corresponding to the codon to the polypeptide chain being synthesized on the ribosome.

[0060] The term "tsRNA" refers to tRNA-derived small RNA, a class of non-coding small RNAs discovered in recent years, which are produced by precise cleavage of different tRNAs or tRNA precursors by different enzymes, and are also called tRF, tiRNA, tRNA halves, etc. Existing studies have shown that tsRNAs have rich biological functions, including regulation of stress response, tumorigenesis, and epigenetic regulation, etc.

[0061] In some embodiments, tsRNAs include tiRNAs and tRFs.

[0062] In some embodiments, the concentration of the total Torreya grandis RNA extract is 5 ng / μL - 100 ng / μL, such as 5 ng / μL, 7.5 ng / μL, 10 ng / μL, 12.5 ng / μL, 15 ng / μL, 17.5 ng / μL, 20 ng / μL, 22.5 ng / μL, 25 ng / μL, 27.5 ng / μL, 30 ng / μL, 32.5 ng / μL, 35 ng / μL, 37.5 ng / μL, 40 ng / μL, 42.5 ng / μL, 45 ng / μL, 47.5 ng / μL, 50 ng / μL, 52.5 ng / μL, 55 ng / μL, 57.5 ng / μL, 60 ng / μL, 62.5 ng / μL, 65 ng / μL, 67.5 ng / μL, 70 ng / μL, 72.5 ng / μL, 75 ng / μL, 77.5 ng / μL, 80 ng / μL, 82.5 ng / μL, 85 ng / μL, 87.5 ng / μL, 90 ng / μL, 92.5 ng / μL, 95 ng / μL, 97.5 ng / μL, 100 ng / μL;

[0063] In some specific embodiments, the concentration of the extract is preferably 12.5 ng / μL - 50 ng / μL, such as 12.5 ng / μL, 15 ng / μL, 17.5 ng / μL, 20 ng / μL, 22.5 ng / μL, 25 ng / μL, 27.5 ng / μL, 30 ng / μL, 32.5 ng / μL, 35 ng / μL, 37.5 ng / μL, 40 ng / μL, 42.5 ng / μL, 45 ng / μL, 47.5 ng / μL, 50 ng / μL.

[0064] In some specific embodiments, the concentration of the extract is further preferably 25 ng / μL - 50 ng / μL, such as 25 ng / μL, 27.5 ng / μL, 30 ng / μL, 32.5 ng / μL, 35 ng / μL, 37.5 ng / μL, 40 ng / μL, 42.5 ng / μL, 45 ng / μL, 47.5 ng / μL, 50 ng / μL.

[0065] Another object of the present invention is to provide a method for preparing the total RNA extract of Torreya grandis, and the preparation method includes the following steps:

[0066] 1) Use a lysis solution to lyse the cells of Torreya grandis, add an organic solvent, and then centrifuge to extract the supernatant;

[0067] 2) Add a lower alcohol to the supernatant, transfer the solution to filter element 1, centrifuge to collect the filtrate, add a lower alcohol to the obtained filtrate, transfer it to filter element 2, and discard the filtrate after centrifugation;

[0068] 3) For the two filter elements used in step 2), after centrifugally washing them with a washing solution respectively, discard the filtrate;

[0069] 4) Add ribonuclease-free water to the two filter elements after washing in step 3), centrifuge, and mix the collected filtrates to obtain the total RNA extract of Torreya grandis.

[0070] In some embodiments, the organic solvent is preferably chloroform-isoamyl alcohol.

[0071] In some embodiments, the lower alcohol is an alcohol containing 1-4 carbon atoms, such as ethanol, isopropanol, n-butanol or a mixture thereof, and preferably ethanol. In some specific embodiments, the lower alcohol can be absolute ethanol.

[0072] In some embodiments, after step 3), the filter element is centrifuged once with an empty column.

[0073] In some embodiments, the washing solution includes washing solution 1 and washing solution 2. Washing solution 1 contains ultrapure water, NaCl, EDTA, Tris-HCl and an 80% ethanol solution, and washing solution 2 contains an 80% ethanol solution.

[0074] In some specific embodiments, the molar ratio of NaCl:EDTA:Tris-HCl is 20:0.9:2.

[0075] In some specific embodiments, in step 3), filter element 1 is centrifugally washed once with washing solution 1, and filter element 2 is centrifugally washed twice with washing solution 2.

[0076] In some embodiments, filter element 1 collects large molecular RNA, and filter element 2 collects small molecular RNA; the large molecular RNA is RNA with a nucleotide (NT) number > 200, such as mRNA, etc., and RNA with NT < 200, such as tRNA, miRNA, tsRNA, etc. Among them, the length of mRNA has a large variation range, so it may be present in filter element 1, and / or filter element 2.

[0077] In some embodiments, 1) the centrifugation program in the preparation method is set as follows: the rotation speed is 8000xg - 12000xg, and the centrifugation time is 15s - 15min; for example: the centrifugation program in step 1) is set to centrifuge at 12000xg for 15 minutes; the centrifugation programs in steps 2) and 3) are set to centrifuge at 8000xg for 15s; the centrifugation program in step 4) is to centrifuge at 12000xg for 1 minute.

[0078] In some specific embodiments, the lysis solution is TRIzol, a new type of total RNA extraction reagent that can directly extract total RNA from cells or tissues. It contains substances such as phenol and guanidine isothiocyanate, which can rapidly break cells and inhibit the nucleases released by cells. It can be replaced with other solvents with similar effects.

[0079] In some embodiments, the addition amount of lower alcohol in step 2) is 1 / 3 or 2 / 3 of the total volume;

[0080] In some embodiments, after adding RNase-free water in step 5), let it stand for 2 - 5 minutes, such as 2 minutes, 3 minutes, 4 minutes, 5 minutes, preferably 2 minutes.

[0081] The term "RNase-free water" refers to RNase-free water, that is, distilled water without RNase.

[0082] In some specific embodiments, the torreya grandis is any tissue of torreya grandis, including but not limited to torreya grandis fruits, seeds, leaves, seed coats, arils. Before preparation, it is pre-crushed so that the lysis solution can fully react with tissue cells.

[0083] In some embodiments, the total RNA extract of torreya grandis or its preparation method according to the present invention can be used to prepare anti-aging products;

[0084] In some embodiments, the total RNA extract of torreya grandis or its preparation method according to the present invention can be used to prepare products for improving sleep;

[0085] In some embodiments, the total RNA extract of torreya grandis or its preparation method according to the present invention can be used to prepare products for enhancing immunity;

[0086] In some embodiments, the total RNA extract of torreya grandis or its preparation method according to the present invention can be used to prepare antioxidant products.

[0087] In some specific embodiments, the products with the above-mentioned effects are external use products or oral use products.

[0088] The external use products are selected from skin care products, cosmetics, hair care products, preferably skin care products and / or cosmetics;

[0089] The oral products are selected from solid beverages, liquid beverages, dairy products, flavored powders, nutritional products, and oral healthcare products.

[0090] In some embodiments, the external products are selected from emulsions, gels, creams, microneedle patches, and facial masks, preferably emulsions.

[0091] In some embodiments, the external products further contain excipients, and the excipients contain preservative components: selected from methyl paraben, ethyl paraben, propyl paraben, formaldehyde releasers, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinones, phenols, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternium-15, dehydroacetic acid and its salts, and one or more of them.

[0092] In some embodiments, the external products further contain excipients, and the excipients contain emulsifying components: selected from polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, and phospholipids, and one or more of them.

[0093] In some embodiments, the external products further contain excipients, and the excipients contain modifying excipients:

[0094] Selected from flavors and / or pigments.

[0095] The oral products described in the present invention may include all foods in the conventional sense and may be used interchangeably with terms known in the art, such as functional foods and health functional foods.

[0096] When the Torreya grandis Fort. ex Lindl. extract described in the present invention is used as a food additive, it can be added directly or used together with other foods or food ingredients, and can be appropriately used according to conventional methods.

[0097] In some embodiments, the oral products further contain food-grade acceptable excipients, and the excipients contain flavoring agents: selected from fructooligosaccharide, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, edible flavor, and citric acid, and one or more of them.

[0098] In some embodiments, the oral products further contain food-grade acceptable excipients, and the excipients contain dosage form excipients: selected from maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, and silicon dioxide, and one or more of them.

[0099] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the embodiments described herein. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or according to the product specifications.

[0100] Example 1: Preparation of Total RNA Extract from Torreya grandis Fort. cv. Merrillii

[0101] Take 100 mg of fresh Torreya grandis Fort. cv. Merrillii, process it into fragments, divide it into two equal parts on average, and extract the total RNA from Torreya grandis Fort. cv. Merrillii using different methods respectively.

[0102] 1.1 Preparation method of the present invention

[0103] 1) Transfer the tissue fragments to a 2 mL centrifuge tube, add 1.5 mL of TRIzol (Invitrogen) lysis solution, homogenize until there are no particles and it becomes a slightly viscous and uniform solution state, vortex for 2 min, and let it stand at room temperature for 15 min to fully lyse the tissue cells;

[0104] 2) Add 300 μL of chloroform - isoamyl alcohol, tightly cap the centrifuge tube, vortex for 2 min, let it stand for 10 min, then centrifuge at 12000 x g at 4 °C for 15 minutes; aspirate the supernatant into a new EP tube, and add anhydrous ethanol with a volume 1 / 3 times the total volume of the liquid at this time;

[0105] 3) After dissolving the visible precipitate in the centrifuge tube, transfer the solution to Filter Cartridge 1 (Filter Cartridge 1, Invitrogen), centrifuge at 8000 x g for 15 s, collect the filtrate, label the used Filter Cartridge 1 as largeRNA, mainly collecting RNA with a molecular weight greater than 200 nt; repeat the operation until all the solution is filtered; add anhydrous ethanol with a volume 2 / 3 times the total volume of the liquid at this time to the filtrate, vortex and mix well, then transfer it to a new Filter Cartridge 2, centrifuge at 8000 x g for 15 s, discard the filtrate, and label the used Filter Cartridge 2 as small RNA, mainly collecting RNA with a molecular weight less than 200 nt;

[0106] 4) Add 700 μL of Wash Solution 1 (prepared by mixing 4.83 mL of ultrapure water, 0.6 mL of 5 M NaCl, 0.27 mL of 0.5 M EDTA, 0.3 mL of 1 M Tris-HCl solution, and 24 mL of absolute ethanol) to the Filter Cartridge in Step 3), centrifuge at 8000 x g for 15 s, and discard the filtrate; then add 700 μL of Wash Solution 2 (80% ethanol solution), centrifuge at 8000 x g for 15 s, and discard the filtrate; add 700 μL of Wash Solution 2 again, centrifuge at 8000 x g for 15 s, and discard the filtrate. Centrifuge the empty column at 12000 x g for 1 min; replace with a new collection tube, add 100 μL of RNase-free water (Invitrogen), let stand for 2 min, centrifuge at 12000 x g for 30 s, and mix the collected filtrate to obtain the total RNA extract of Torreya grandis.

[0107] 1.2 TRIzol preparation method

[0108] 1) Transfer the tissue fragments to a 2 mL centrifuge tube, add 1.5 mL of TRIzol (Invitrogen) lysis solution, homogenize until there are no particles and the solution is slightly viscous and homogeneous, vortex for 2 min, and let stand at room temperature for 15 min to allow sufficient lysis of tissue cells;

[0109] 2) Add 300 μL of chloroform, tighten the centrifuge tube cap, vortex for 2 min, let stand for 3 min, then centrifuge at 12000 x g at 4 °C for 15 minutes; aspirate the supernatant into a new EP tube;

[0110] 3) Add 750 μL of isopropanol, incubate at 4 °C for 10 minutes, centrifuge at 12000 x g at 4 °C for 10 minutes, and discard the supernatant;

[0111] 4) Resuspend the precipitate with 1 mL of 75% ethanol, briefly vortex the sample, and then centrifuge at 7500 x g at 4 °C for 5 minutes;

[0112] 5) Discard the supernatant, vacuum or air-dry the RNA precipitate for 5 - 10 minutes;

[0113] 6) Resuspend the precipitate with 20 - 50 μL of RNase-free water and incubate at 55 - 60 °C for 10 - 15 minutes.

[0114] 1.3 Identification of the extract

[0115] Use Nanodrop (ThermoFisher) to detect the total RNA extracted by the two preparation methods. Table 1 shows the purity and 260 / 280 ratio of the total RNA of Torreya grandis. The closer the 260 / 280 ratio is to 2.0, the better.

[0116] The results showed that the total RNA extract of Torreya grandis obtained by the preparation method of the present invention had significantly better concentration and purity than those prepared by the TRIzol method.

[0117]

[0118] Example 2: Efficacy verification of the total RNA extract of Torreya grandis

[0119] 2.1 Antioxidant capacity: in vitro DPPH free radical scavenging assay

[0120] 1) Experimental setup: Dilute the total RNA extract of Torreya grandis extracted in Example 1 with water to 12.5 ng / μl, 25 ng / μl, and 50 ng / μl respectively.

[0121] 2) Experimental steps:

[0122] Use an enzyme-linked immunosorbent assay (ELISA) plate. For each group of detections, set up T - sample wells, T0 - sample background wells, P - positive control wells, P0 - positive control background wells, C - DPPH wells, and C0 - solvent background wells.

[0123] Add 50 μL of the total RNA extract of Torreya grandis at the same concentration to the sample wells (T) and sample background wells (T0) respectively, and add 50 μL of the positive control (50 ng / μl vitamin E) to the positive control wells (P) and positive control background wells (P0) respectively. Add 95% ethanol to all wells to make up to 150 μL, and mix well.

[0124] Add 50 μL of DPPH ethanol solution to the sample wells (T), DPPH wells (C), and positive control wells (P) respectively, and add 50 μL of 95% ethanol to the sample background wells (T0), solvent background wells (C0), and positive control background wells (P0) respectively. Gently shake well and let stand at room temperature for 5 minutes.

[0125] Transfer the ELISA plate into an enzyme - linked immunosorbent assay (ELISA) reader (Tecan, Spark), and measure the absorbance at 517 nm.

[0126] 1) Experimental results: The total RNA of Torreya grandis reacted with DPPH. Compared with the solvent control, the scavenging rate of 12.5 ng / μl of the total RNA of Torreya grandis on DPPH was higher than 10%, and p < 0.05 compared with the solvent control. The scavenging rate was positively correlated with the concentration. For 50 ng / μl of the total RNA of Torreya grandis, the scavenging rate was higher than 50%, which was comparable to the effect of the positive control. It showed that the total RNA of Torreya grandis had a significant ability to scavenge DPPH free radicals and had antioxidant efficacy.

[0127] 2.2 Anti-aging capacity: effect on type I collagen synthesis in human dermal fibroblasts (HDFn)

[0128] 1) Experimental setup: The total Torreya grandis RNA extracted in Example 1 was diluted to 12.5 ng / μl, 25 ng / μl, and 50 ng / μl respectively using DMEM medium.

[0129] 2) Experimental procedure: Logarithmic growth phase human dermal fibroblasts (self-made by the inventor) were collected and seeded into 12-well plates at a cell density of 1×10 5 cells / well. After culturing in an incubator (37 °C, 5% CO2) for 24 h, different concentrations of total Torreya grandis RNA extracts were added. The negative control group (NC) was added with DMEM medium; the positive control group was added with DMEM medium containing 100 ng / μl vitamin C and 7 ng / μl vitamin E; 4 replicate wells were set in each group.

[0130] After adding the drugs, the cells were continuously cultured in an incubator (37 °C, 5% CO2) for 24 h, the culture medium was collected, and the content of collagen I was detected and analyzed according to the operation manual of the ELISA detection kit (Sangon Biotech).

[0131] 2) Experimental results: The results showed that compared with the negative control group, vitamin C and vitamin E in the positive control group could significantly increase the content level of collagen I (p < 0.05), indicating that the positive control detection in this experiment was effective. Compared with the negative control group, the three concentrations of total Torreya grandis RNA (12.5 ng / uL, 25 ng / uL, and 50 ng / uL) all showed an enhancing effect on the content of collagen I in fibroblasts. Among them, the effect of the 50 ng / uL concentration was comparable to that of the positive control, but the concentration was only about 50% of the positive control.

[0132] 2.3 Anti-aging capacity: effect of inhibiting matrix metalloproteinase 1 (MMP-1) synthesis in human dermal fibroblasts

[0133] 1) Experimental setup: The total Torreya grandis RNA extracted in Example 1 was diluted to 12.5 ng / μl, 25 ng / μl, and 50 ng / μl respectively using DMEM medium.

[0134] 2) Experimental procedure: Logarithmic growth phase human dermal fibroblasts were collected and seeded into 12-well plates at a cell density of 1×10 5 cells / well. After culturing in an incubator (37 °C, 5% CO2) for 24 h, different concentrations of total Torreya grandis RNA extracts were added. Cells treated with 1 μmol / L dexamethasone were used as the positive control, and untreated cells were used as the negative control (NC). 4 replicate wells were set in each group.

[0135] After adding the drugs, the cells were continuously cultured in an incubator (37 °C, 5% CO2) for 24 h, the culture medium was removed, gently washed twice with pre-warmed PBS, the washing solution was removed, and 0.5 mL PBS was added. At 10 J / cm 2Cells were irradiated at the indicated dose for approximately 1 h. Cells treated only with irradiation were used as a model control. After reaching the irradiation dose, the PBS in the wells was removed, and the cells were gently washed once with pre-warmed PBS. Then, DMEM medium was added and the cells were cultured for another 16 - 24 h. The culture medium was collected, and the content of MMP-1 in the culture medium was detected using a human matrix metalloproteinase 1 ELISA kit (Sangon Biotech).

[0136] 3) Experimental results: Activation of matrix metalloproteinases (MMPs) can lead to excessive degradation of collagen and elastin that support the skin structure in the dermis, resulting in aging symptoms such as wrinkles and decreased elasticity of the skin. Total RNA from Torreya grandis has the ability to inhibit the synthesis of MMP-1. After acting on HDFn cells at concentrations of 25 ng / μL and 50 ng / μL, it can inhibit the synthesis of MMP-1 in cells by more than 50%.

Claims

1. A Torreya grandis total RNA extract, characterized in that, The extract at least includes mRNA, tRNA, miRNA, and tsRNA.

2. The extract according to claim 1, wherein The concentration of the extract is 5 ng / μL - 100 ng / μL, preferably 12.5 ng / μL - 50 ng / μL, and more preferably 25 ng / μL - 50 ng / μL.

3. The extract according to claim 1 or 2, characterized in that, The preparation method of the extract includes the following steps: 1) Use a lysis solution to lyse the Torreya grandis cells, add an organic solvent, and then centrifuge to extract the supernatant. 2) Add a lower alcohol to the supernatant, transfer the solution to filter cartridge 1, centrifuge to collect the filtrate, add a lower alcohol to the obtained filtrate, transfer it to filter cartridge 2, and discard the filtrate after centrifugation. 3) For the two filter cartridges used in step 2), centrifuge and wash them respectively with a washing solution, and then discard the filtrate. 4) Add ribonuclease-free water to the two filter cartridges after washing in step 3), centrifuge, and mix the collected filtrates to obtain the total RNA extract of Torreya grandis.

4. The extract according to claim 3, wherein After step 3), centrifuge the empty filter cartridge once.

5. The extract according to claim 3, characterized in that, The washing solution includes washing solution 1 and washing solution 2. Washing solution 1 contains ultrapure water, NaCl, EDTA, Tris-HCl, and an 80% ethanol solution, and washing solution 2 contains an 80% ethanol solution.

6. The extract according to claim 5, characterized in that, The molar concentration ratio of NaCl, EDTA, and Tris-HCl in washing solution 1 is 20:0.9:

2.

7. The extract according to any one of claims 3 - 6, characterized in that, The preparation method includes any one or more of the following features: 1) Filter cartridge 1 collects large molecular RNA, and filter cartridge 2 collects small molecular RNA. 2) The centrifugation program in the preparation method is set as follows: the rotation speed is 8000 xg - 12000 xg, and the centrifugation time is 15 s - 15 min. 3) The lysis solution in step 1) is TRIzol lysis solution. 4) The organic solvent in step 1) is preferably chloroform - isoamyl alcohol. 5) The lower alcohol in step 2) is ethanol, isopropanol, n-butanol, or a mixture thereof. 6) The addition amount of the lower alcohol in step 2) is 1 / 3 or 2 / 3 of the total volume. 7) After adding ribonuclease-free water in step 4), let it stand for 2 - 5 minutes, preferably 2 minutes.

8. The application of the total RNA extract of Torreya grandis according to any one of claims 1 - 7 in the preparation of the following products: 1) Anti-aging products; 2) Products for improving sleep; 3) Products for enhancing immunity; 4) Antioxidant products.

9. The application according to claim 8, characterized in that, The products are external use products or internal use products. The external use products are selected from skin care products, cosmetics, and hair care products; the internal use products are selected from solid beverages, liquid beverages, dairy products, flavoring powders, nutritional products, and internal use healthcare products.

10. The product according to claim 9, characterized in that, The external use products are selected from emulsions, gels, creams, microneedle patches, and facial masks, preferably emulsions.

11. The product according to any one of claims 9-10, characterized in that, The external use products also contain excipients, and the excipients have any one or more of the following features: 1) Containing preservative components: The preservative components are selected from one or more of methyl paraben, ethyl paraben, propyl paraben, formaldehyde-releasing agents, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinones, phenols, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternium-15, dehydroacetic acid and its salts; 2) Containing emulsifying components: The emulsifying components are selected from one or more of polyethylene glycols, glycerol esters, sugar derivatives, anionic emulsifiers, cationic emulsifiers, phospholipids; 3) Containing modifying excipients: The modifying components are selected from flavors and / or pigments.

12. The product according to claim 9, wherein, The oral product further contains food-grade acceptable excipients, and the excipients have any one or more of the following characteristics: 1) Containing flavoring agents, and the flavoring agents are selected from one or more of fructooligosaccharides, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, edible flavors, citric acid; 2) Containing dosage form excipients, and the dosage form excipients are selected from one or more of maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, silicon dioxide.

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