Application and preparation method of torreya grandis extract

Abstract

The invention relates to the field of plant extracts, in particular to application and a preparation method of a torreya grandis extract, which can achieve the purposes of resisting oxidation, resisting wrinkles, tightening or resisting aging so as to delay the aging of a human body.

Description

Technical Field

[0001] The present invention relates to the field of plant extracts, and particularly to the application of Torreya grandis Fort. ex Lindl. extracts and a preparation method thereof. Background Art

[0002] Mitochondria are double-membrane-enclosed organelles present in most cells. They are the structures in cells that produce energy, the main sites for cells to perform aerobic respiration, and are known as the "power plants". In addition to providing energy for cells, mitochondria also participate in processes such as cell differentiation, cell information transmission, and apoptosis, and have the ability to regulate cell growth and the cell cycle. In the field of aging-related research, mitochondrial function has a complex impact on the aging process.

[0003] The mitochondrial free radical theory of aging holds that progressive mitochondrial dysfunction occurring with aging increases the production of reactive oxygen species (ROS), which in turn leads to the deterioration of mitochondrial function and overall cell damage. Multiple studies [1-3] have confirmed that mitochondrial dysfunction can accelerate mammalian aging. Aging skin keratinocytes exhibit reduced energy metabolism, increased mitochondrial oxidative stress, and mitochondrial DNA deletions, which also reflect the characteristics of the mitochondrial free radical theory of aging.

[0004] To solve the above problems, those skilled in the art urgently need to develop products with the effects of maintaining the free radical balance of cells, enhancing the mitochondrial activity of cells, anti-aging, and antioxidant to benefit the large number of people with such needs.

[0005] 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 relic 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 improving the mitochondrial function activity of Torreya grandis Fort. ex Lindl. extracts.

[0006] References:

[0007] 1. Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S, Oldfors A, Wibom R, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004; 429: 417–423. [PubMed: 15164064];

[0008] 2. Vermulst M, Wanagat J, Kujoth GC, Bielas JH, Rabinovitch PS, Prolla TA, Loeb LA. DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat Genet. 2008;40:392–394. [PubMed:18311139];

[0009] 3. Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R, Jobling WA, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005;309:481–484. [PubMed:16020738]. SUMMARY OF THE INVENTION

[0010] One object of the present invention is to provide the use of Torreya grandis Fort. ex Lindl. extract in the preparation of the following products:

[0011] 1) Products for reducing skin aging caused by environmental pollution;

[0012] 2) Products for enhancing the antioxidant capacity of skin keratinocytes;

[0013] 3) Products for enhancing ATP production in skin keratinocytes;

[0014] 4) Products for enhancing the mitochondrial functional activity of skin keratinocytes.

[0015] In some specific embodiments, the skin aging caused by environmental pollution is the skin's direct contact with particulate matter in the atmosphere with a diameter not greater than 2.5 micrometers.

[0016] In some embodiments, the concentration of the Torreya grandis Fort. ex Lindl. extract is 0.01% - 1%, for example: 0.01%, 0.03%, 0.05%, 0.07%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.

[0017] In some specific embodiments, the concentration of the Torreya grandis Fort. ex Lindl. extract is preferably 0.1%-0.5%, for example: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%.

[0018] In some specific embodiments, the concentration of the Torreya grandis Fort. ex Lindl. extract is further preferably 0.2%-0.5%, for example: 0.2%, 0.3%, 0.4%, 0.5%.

[0019] The second object of the present invention is to provide a method for preparing the above-mentioned Torreya grandis Fort. ex Lindl. extract, the method comprising cold pressing extraction, steam explosion and two rounds of steam distillation, wherein the temperature and pressure of the second round of steam distillation are both higher than those of the first round.

[0020] In some embodiments, the preparation method comprises the following steps:

[0021] 1) Cold pressing extraction: Grinding Torreya grandis Fort. ex Lindl. raw materials to obtain Torreya grandis Fort. ex Lindl. pulp and Torreya grandis Fort. ex Lindl. residue, filtering the Torreya grandis Fort. ex Lindl. pulp and then centrifuging to obtain the first part of the extract;

[0022] 2) Steam explosion: Putting the Torreya grandis Fort. ex Lindl. residue in step 1) into a steam explosion device, adding deoxygenated deionized water, and then performing steam explosion treatment;

[0023] 3) Steam distillation: Transferring the Torreya grandis Fort. ex Lindl. residue treated in step 2) to an extraction tank, conveying deoxygenated deionized water, setting the temperature and pressure and then performing distillation extraction to obtain the second part of the extract; repeating the above-mentioned steam distillation step, with the temperature and pressure of the second round of steam distillation being higher than those of the first round, to obtain the third part of the extract;

[0024] 4) Mixing the three parts of the extract to obtain the Torreya grandis Fort. ex Lindl. extract.

[0025] In some embodiments, the Torreya grandis Fort. ex Lindl. raw materials are selected from any one or more of arils, Torreya grandis Fort. ex Lindl. leaves, and Torreya grandis Fort. ex Lindl. kernels.

[0026] In some embodiments, the particle size of the Torreya grandis Fort. ex Lindl. raw materials in step 1) is 0.1-5 mm, for example: 0.1 mm, 0.3 mm, 0.5 mm, 0.7 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm,

[0027] 3.5 mm, 4 mm, 4.5 mm, 5 mm.

[0028] In some specific embodiments, the particle size of the Torreya grandis Fort. ex Lindl. raw materials in step 1) is preferably 0.5-2 mm, for example: 0.5 mm, 0.7 mm, 1 mm, 1.5 mm, 2 mm.

[0029] In some embodiments, in step 1), the torreya grandis residue is ground multiple times, and the extraction of torreya grandis pulp is repeated; the multiple times are selected from 1 to 5 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, and preferably 4 times.

[0030] In some embodiments, the torreya grandis pulp in step 1) is a single extract obtained after grinding the torreya grandis raw material, or on the basis of the single extract, a mixture of extracts obtained by adding the torreya grandis residue ground multiple times.

[0031] In some embodiments, the centrifugation program is set to 2000 - 6000 rpm, 10 - 20 min, for example: 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm; 10 min, 13 min, 15 min, 17 min, 20 min, and preferably 4000 rpm, 15 min;

[0032] In some embodiments, in step 2), the liquid - solid mass ratio of deoxygenated deionized water to torreya grandis raw material is 1 - 2:1 - 3;

[0033] In some specific embodiments, the liquid - solid mass ratio of deoxygenated deionized water to torreya grandis raw material in step 2) is preferably 0.7:1.

[0034] In some specific embodiments, the pressure of steam explosion in step 2) is 2 - 5 Mpa, for example: 2 Mpa, 2.5 Mpa, 3 Mpa, 3.5 Mpa, 4 Mpa, 4.5 Mpa, 5 Mpa;

[0035] In some specific embodiments, the pressure of steam explosion in step 2) is preferably 3 Mpa.

[0036] In some embodiments, the treatment time of steam explosion in step 2) is 5 - 20 minutes, for example: 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 13 minutes, 15 minutes, 17 minutes, 20 minutes;

[0037] In some specific embodiments, the treatment time of steam explosion in step 2) is preferably 10 minutes.

[0038] In some embodiments, the delivery flow rate of deoxygenated deionized water in step 3) is ≤15 mL / min;

[0039] In some specific embodiments, the delivery flow rate of deoxygenated deionized water in step 3) is preferably ≤10 mL / min.

[0040] In some embodiments, the extraction temperature in step 3) is 100°C - 200°C, for example: 100°C,

[0041] 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, 160 °C, 170 °C, 180 °C, 190 °C, 200 °C, and the extraction temperature in the second round is greater than that in the first round.

[0042] In some specific embodiments, the extraction temperature in step 3) is preferably 140 °C - 175 °C, for example: 140 °C, 150 °C, 160 °C, 170 °C, 175 °C, and the extraction temperature in the second round is greater than that in the first round.

[0043] In some specific embodiments, the extraction pressure in step 3) is 5 - 10 Mpa, for example: 5 Mpa, 6 Mpa, 7 Mpa, 8 Mpa, 9 Mpa, 10 Mpa, and the extraction pressure in the second round is greater than that in the first round.

[0044] In some specific embodiments, the extraction pressure in step 3) is preferably 6 - 7 Mpa, for example: 6 Mpa, 7 Mpa, and the extraction pressure in the second round is greater than that in the first round.

[0045] In some embodiments, the extraction times in the two rounds of step 3) are the same, preferably 50 minutes - 120 minutes, for example: 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes.

[0046] In some specific embodiments, the extraction times in the two rounds of step 3) are the same, preferably 90 minutes.

[0047] Through the optimization of the extraction method, the effective components in the Torreya grandis raw materials are fully extracted, and at the same time, the temperature and pressure of each step are controlled within an appropriate range, which is beneficial to reducing production costs and equipment losses.

[0048] The third object of the present invention is to provide a product containing the Torreya grandis extract prepared by the above preparation method.

[0049] In some embodiments, the product is an external use product or an internal use product. The external use products are selected from skin care products, cosmetics, 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.

[0050] In some embodiments, the external use products are selected from emulsions, gels, creams, microneedle patches, masks, preferably emulsions.

[0051] In some embodiments, the topical product further contains excipients, and the excipients contain preservative components: selected from one or more of 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.

[0052] In some embodiments, the topical product further contains excipients, and the excipients contain emulsifying components: selected from one or more of polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, phospholipids.

[0053] In some embodiments, the topical product further contains excipients, and the excipients contain modifying excipients: selected from fragrances and / or pigments.

[0054] In some embodiments, the oral product further contains food-grade acceptable excipients, and the excipients contain flavoring agents: selected from one or more of fructooligosaccharide, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, edible flavor, citric acid.

[0055] In some embodiments, the oral product further contains food-grade acceptable excipients, and the excipients contain dosage form excipients: selected from one or more of maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, silicon dioxide.

[0056] The beneficial effects of the present invention are as follows:

[0057] 1. It provides the application of Torreya grandis Fort. ex Lindl. extract in improving the function of cell mitochondria. By studying the effects of external stress (such as environmental exposure) on cell viability, the production of mitochondrial reactive oxygen species (ROS), and the production of adenosine triphosphate (ATP), a more specific target of Torreya grandis Fort. ex Lindl. extract is found, which can be used to improve related aging factors and for preparing products for improving skin aging status.

[0058] 2. The preparation method of cold pressing first and then steam explosion is adopted. In the preparation process without using organic solvents, the effective nutritional components and functional components of Torreya grandis Fort. ex Lindl. can be fully extracted, verifying the effects of different preparation methods on the efficacy of the extract, screening out Torreya grandis Fort. ex Lindl. extract with low preparation conditions requirements and good effects, and the extract shows excellent properties in efficacy verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Shows the evaluation of the environmental stress resistance of Torreya grandis Fort. ex Lindl. extracts obtained by different preparation methods;

[0060] Figure 2 Shows the effect of Torreya grandis Fort. et Lindl. extract on the production of mitochondrial reactive oxygen species (ROS);

[0061] Figure 3 Shows the effect of Torreya grandis Fort. et Lindl. extract on cellular ATP production. Detailed implementation manners

[0062] 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.

[0063] Although the numerical ranges and parameter approximations shown in the broad scope of the present 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. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "0.01 to 1" recited should be considered to include any and all sub-ranges between the minimum value of 0.01 and the maximum value of 1 (including the endpoints); that is, all sub-ranges starting from the minimum value of 0.01 or greater, such as 0.01 to 0.5, and sub-ranges ending with the maximum value of 1 or less, such as 0.6 to 1. In addition, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0064] One of the objectives of the present invention is to provide a new use of Torreya grandis Fort. et Lindl. extract.

[0065] The term "Torreya grandis Fort. et Lindl." 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 Fort. et Lindl., Yangjiao Torreya, Thin Torreya, Yu Torreya, Wild Chinese hemlock, Zizyphus jujuba, etc.; Torreya taxifolia Am. produced in North America, Torreya californica Torrey; Torreya nucifera (L.) Sieb. et Zucc. produced in Japan.

[0066] In some specific implementation manners, the Torreya grandis Fort. et Lindl. is Torreya grandis Fort. et Lindl.

[0067] The Torreya grandis Fort. et Lindl. extract can be applied in the preparation of the following products:

[0068] 1) Products for reducing skin aging caused by environmental pollution;

[0069] 2) Products for enhancing the antioxidant capacity of skin keratinocytes;

[0070] 3) Products that enhance ATP production in skin keratinocytes;

[0071] 4) Products that enhance the mitochondrial functional activity of skin keratinocytes.

[0072] Among them, the skin aging caused by the environmental pollution is the skin's direct contact with particulate matter with a diameter not greater than 2.5 micrometers in the atmosphere (i.e., PM<2.5μm).

[0073] The term "ATP" refers to adenosine triphosphate, abbreviated as adenosine triphosphate, with the chemical formula C 10 H 16 N5O 13 P3, with a molecular weight of 507.18, is an unstable high-energy compound, composed of adenine, ribose, and 3 phosphate groups connected together. When hydrolyzed, it releases a relatively large amount of energy and is the most direct energy source in living organisms.

[0074] In some embodiments, the concentration of the Torreya grandis extract is 0.01% - 1%, for example: 0.01%, 0.03%, 0.05%, 0.07%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.

[0075] In some specific embodiments, the concentration of the Torreya grandis extract is preferably 0.1% - 0.5%, for example: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%.

[0076] In some specific embodiments, the concentration of the Torreya grandis extract is further preferably 0.2% - 0.5%, for example: 0.2%, 0.3%, 0.4%, 0.5%.

[0077] The second object of the present invention is to provide a preparation method of the above-mentioned Torreya grandis extract, and the method includes cold pressing extraction, steam explosion, and two rounds of steam distillation. The temperature and pressure of the second round of steam distillation are both higher than those of the first round.

[0078] The term "cold pressing" is also known as mechanical pressing. It is to put the seeds or fruits of plants into an oil press and extract the plant oil by mechanical pressing. During the cold pressing process, high-temperature heating is not required, and the natural components and fragrance of the oil can be retained to the greatest extent.

[0079] The term "steam explosion" is also called steam explosion (Steam Explosion). It is a technology for pretreating biomass by applying the principle of steam ejection explosion. The steam molecules infiltrating into the plant tissue can be instantaneously released, converting the internal energy of the steam into mechanical energy and acting on the intercellular layer of the biomass tissue, so that the raw material can be decomposed according to the purpose with less energy.

[0080] The term "steam distillation" is also known as steam extraction or stripping. During operation, steam is directly introduced so that the substance to be separated can boil at a temperature lower than its original boiling point. The generated vapor and steam escape together, and after condensation, they are separated into two liquid layers. The water can be removed by methods such as clarification and layering or centrifugal separation to obtain the product.

[0081] In some embodiments, the preparation method comprises the following steps:

[0082] 1) Cold pressing extraction: Grinding torreya grandis raw materials to obtain torreya grandis pulp and torreya grandis residue, filtering the torreya grandis pulp and then centrifuging it to obtain the first part of the extract;

[0083] 2) Steam explosion: Putting the torreya grandis residue in step 1) into a steam explosion device, adding deoxygenated deionized water, and then performing steam explosion treatment;

[0084] 3) Steam distillation: Transferring the torreya grandis residue treated in step 2) to an extraction tank, conveying deoxygenated deionized water, setting the temperature and pressure, and then performing distillation extraction to obtain the second part of the extract; Repeating the aforementioned steam distillation step, the temperature and pressure of the second-round steam distillation are both higher than those of the first round to obtain the third part of the extract;

[0085] 4) Mixing the three parts of the extract to obtain the torreya grandis extract.

[0086] In some specific embodiments, the torreya grandis raw materials are selected from any one or more of arils, torreya grandis leaves, and torreya grandis kernels.

[0087] In some embodiments, the particle size of the torreya grandis raw materials in step 1) is 0.1 - 5 mm, for example: 0.1 mm, 0.3 mm, 0.5 mm, 0.7 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm,

[0088] 3.5 mm, 4 mm, 4.5 mm, 5 mm;

[0089] In some specific embodiments, the particle size of the torreya grandis raw materials in step 1) is preferably 0.5 - 2 mm, for example: 0.5 mm, 0.7 mm, 1 mm, 1.5 mm, 2 mm.

[0090] In some embodiments, in step 1), the torreya grandis residue is ground multiple times to repeatedly extract torreya grandis pulp; The multiple times are selected from 1 - 5 times, for example 1 time, 2 times, 3 times, 4 times, 5 times, and preferably 4 times.

[0091] In some embodiments, the torreya grandis pulp is a single extract obtained by grinding torreya grandis raw materials, or a mixture of extracts obtained by adding the torreya grandis residue ground multiple times on the basis of the single extract.

[0092] In some embodiments, the torreya grandis pulp is filtered using a fine filtration device, and the filter screen preferably has a mesh size of 50 - 120 meshes, such as 50 meshes, 60 meshes, 70 meshes, 80 meshes, 90 meshes, 100 meshes, 110 meshes, 120 meshes, and is preferably 90 meshes.

[0093] In some embodiments, the centrifugation program is set to 2000 - 6000 rpm for 10 - 20 min, such as: 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm; 10 min, 13 min, 15 min, 17 min, 20 min.

[0094] In some specific embodiments, the centrifugation program is preferably set to 4000 rpm for 15 min.

[0095] In some embodiments, in step 2), the liquid - solid mass ratio of deoxygenated deionized water to torreya grandis raw material is 1 - 2:1 - 3;

[0096] In some specific embodiments, in step 2), the liquid - solid mass ratio of deoxygenated deionized water to torreya grandis raw material is preferably 0.7:1.

[0097] In some specific embodiments, in step 2), the pressure of steam explosion is 2 - 5 Mpa, such as: 2 Mpa, 2.5 Mpa, 3 Mpa, 3.5 Mpa, 4 Mpa, 4.5 Mpa, 5 Mpa;

[0098] In some specific embodiments, in step 2), the pressure of steam explosion is preferably 3 Mpa.

[0099] In some embodiments, in step 2), the treatment time of steam explosion is 5 - 20 minutes, such as: 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 13 minutes, 15 minutes, 17 minutes, 20 minutes;

[0100] In some specific embodiments, in step 2), the treatment time of steam explosion is preferably 10 minutes.

[0101] In some embodiments, in step 3), the delivery flow rate of deoxygenated deionized water is ≤15 mL / min;

[0102] In some specific embodiments, in step 3), the delivery flow rate of deoxygenated deionized water is preferably ≤10 mL / min.

[0103] In some embodiments, in step 3), the extraction temperature is 100°C - 200°C, such as: 100°C,

[0104] 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, 160 °C, 170 °C, 180 °C, 190 °C, 200 °C, and the extraction temperature in the second round is greater than that in the first round.

[0105] In some specific embodiments, the extraction temperature in step 3) is preferably 140 °C - 175 °C, for example: 140 °C, 150 °C, 160 °C, 170 °C, 175 °C, and the extraction temperature in the second round is greater than that in the first round.

[0106] In some specific embodiments, the extraction pressure in step 3) is 5 - 10 Mpa, for example: 5 Mpa, 6 Mpa, 7 Mpa, 8 Mpa, 9 Mpa, 10 Mpa, and the extraction pressure in the second round is greater than that in the first round.

[0107] In some specific embodiments, the extraction pressure in step 3) is preferably 6 - 7 Mpa, for example: 6 Mpa, 7 Mpa, and the extraction pressure in the second round is greater than that in the first round.

[0108] In some embodiments, the extraction times in the two rounds of step 3) are the same, preferably 50 minutes - 120 minutes, for example: 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes.

[0109] In some specific embodiments, the extraction times in the two rounds of step 3) are the same, preferably 90 minutes.

[0110] The third object of the present invention is to provide a product containing the Torreya grandis extract prepared by the above preparation method.

[0111] In some embodiments, the product is an external-use product or an internal-use product. The external-use products include but are not limited to skin care products, cosmetics, hair care products; the internal-use products include but are not limited to solid beverages, liquid beverages, dairy products, flavoring powders, nutritional products, and internal-use healthcare products.

[0112] In some embodiments, the external-use product is selected from emulsions, gels, creams, microneedle patches, and facial masks, preferably an emulsion.

[0113] In some embodiments, the external-use 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 urea, isothiazolinones, phenols, benzoic acid, benzyl alcohol, potassium sorbate, bronopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternium-15, dehydroacetic acid and its salts, etc.

[0114] In some embodiments, the topical product further contains excipients, and the excipients contain an emulsifying component: one or more selected from polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, and phospholipids.

[0115] In some embodiments, the topical product further contains excipients, and the excipients contain modified excipients:

[0116] selected from essence and / or pigment.

[0117] The oral products of the present invention can include all foods in the conventional sense and can be used interchangeably with terms known in the art, such as functional foods and health functional foods.

[0118] When the Torreya grandis Fort. ex Lindl. extract of the present invention is used as a food additive, it can be directly added or used together with other foods or food ingredients, and can be appropriately used according to conventional methods.

[0119] In some embodiments, the oral product further contains food-grade acceptable excipients, and the excipients contain a flavoring agent: one or more selected from fructooligosaccharide, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, edible essence, and citric acid.

[0120] In some embodiments, the oral product further contains food-grade acceptable excipients, and the excipients contain dosage form excipients: one or more selected from maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, and silicon dioxide.

[0121] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0122] Example 1 Preparation of Torreya grandis Fort. ex Lindl. Extract

[0123] Take Torreya grandis Fort. ex Lindl. raw materials (including Torreya grandis Fort. ex Lindl. sarcotesta, Torreya grandis Fort. ex Lindl. leaves, and Torreya grandis Fort. ex Lindl. kernels), wash them with water, remove surface dust and impurities, and crush them to a particle size of 0.5 - 2 mm. Divide the crushed raw materials into six equal parts, set the parameters of each step according to the conditions in Table 1.1, and obtain each group of Torreya grandis Fort. ex Lindl. extracts respectively.

[0124] Taking Experimental Group 2 as an example, the specific preparation steps are as follows: Grind the crushed particles of Torreya grandis raw materials with water at a ratio of 1:1 for 2 minutes, filter and separate the Torreya grandis pulp and Torreya grandis residue obtained by grinding. Repeat the grinding of the Torreya grandis residue 4 times. Mix the Torreya grandis pulp obtained 5 times and then conduct fine filtration. The filter screen of the fine filtration equipment is 90 mesh. Place the obtained oil-water substance into a centrifuge for high-speed centrifugation (4000 rpm, 15 min). After centrifugation, let it stand at a low temperature of 15 °C for 60 minutes to remove water and impurities, obtaining the first part of the extract, Torreya grandis oil. Put the treated Torreya grandis residue into a steam explosion device (Peterford Technology, BTF-088), add deoxygenated deionized water according to the liquid-solid mass ratio of 0.7:1, control the steam pressure at 3 Mpa, and conduct steam explosion treatment for 10 min. Then transfer the Torreya grandis raw materials treated by steam explosion to a stainless-steel extraction tank, and transport deoxygenated deionized water to the extraction tank at a flow rate less than or equal to 10 mL / min. Under the condition of 6 Mpa, raise the temperature to 140 °C, and the extraction time is 90 min. Release the pressure and collect the second part of the extract; maintain the pressure at 7 Mpa, continue to raise the temperature to 175 °C, extract for 90 min, release the pressure, and collect the third part of the extract. Mix the three parts of the extract to obtain the Torreya grandis extract.

[0125] Table 1.1 Torreya grandis extracts under different conditions

[0126]

[0127]

[0128] Evaluation of the resistance of Torreya grandis extract in Example 2 to environmental stress

[0129] Environmental stress is caused by polluted particulate matter (PM < 2.5 μm). Cell viability is measured by the dose of lactate dehydrogenase (LDH) activity. LDH is a class of NAD-dependent kinases and an oxidoreductase that catalyzes the conversion of pyruvate in lactate. Its activity is related to the presence of damage and toxicity in tissues and cells.

[0130] (1) Experimental method: Divide healthy human keratinocytes (self-made by the inventor) into four groups, with a cell density of 1×10 4 cells / cm 2 , inoculate them in a glass-bottom culture dish, In (NEST), keratinocytes were cultured in KSFM medium (Gibco) containing 50 μg / ml bovine pituitary extract (Gibco, Auckland, NZ) and 5 ng / ml human recombinant EGF (Gibco). The extracts of Torreya grandis Fort. ex Lindl. prepared in each group in Example 1 were used for verification respectively, and the specific components were added according to Table 2.1. Among them, the extracts of Torreya grandis Fort. ex Lindl. were added twice a day, and PM<2.5μm was added 24 hours before the end of the culture, and the total culture time was 48 hours. The LDH activity was measured using a lactate dehydrogenase activity assay kit (SIGMA-ALDRICH MAK066).

[0131] Table 2.1

[0132]

[0133] (2) Experimental results: As Figure 1 shown, the 0.5% extract of Torreya grandis Fort. ex Lindl. had no effect on the LDH activity of normal human keratinocytes and no effect on cell viability. 70 μg / ml of PM<2.5μm could cause a significant increase in the LDH activity of normal human keratinocytes, resulting in a significant decrease in cell viability. Pretreating normal human keratinocytes with 0.5% extract of Torreya grandis Fort. ex Lindl. in parallel with PM<2.5μm could significantly reduce the LDH increase induced by PM<2.5μm and prevent the decrease in cell viability. It shows that the extract of Torreya grandis Fort. ex Lindl. described in the invention can reduce the influence of environmental stress on the viability of keratinocytes and improve the resistance of cells to environmental stress.

[0134] The experimental results also showed that there were differences in the effects of extracts obtained by different preparation methods. High temperature and high pressure were helpful for extracting active ingredients, but too high experimental conditions had high requirements for equipment and were not conducive to actual production. The extract of experimental group 2 was used in the following experiments.

[0135] Example 3 Effect of Torreya grandis Fort. ex Lindl. extract on the production of mitochondrial reactive oxygen species ROS

[0136] (1) Experimental method: Healthy human keratinocytes were divided into four groups, and the cell density of each group was 1×10 4 cells / cm 2 , and they were inoculated in a glass-bottom culture dish. In (NEST), keratinocytes were cultured in KSFM medium (Gibco) containing 50 μg / ml bovine pituitary extract (Gibco, Auckland, NZ) and 5 ng / ml human recombinant EGF (Gibco). Except for the control group, the other three groups were pretreated with 0.2%, 0.5% and 1% extracts of Torreya grandis Fort. ex Lindl. respectively, added twice a day, and the total culture time was 48 hours.

[0137] Replace the medium with fresh KSFM and continue culturing for 1 hour. Then add 5 μM (LifeTechnologies, Carlsbad, CA, USA), and incubate the cells in an incubator at 37 °C with 5% CO2 for 20 minutes. Wash the cells three times in Hank's Balanced Salt Solution (Gibco), fix them with 4% paraformaldehyde (Beyotime) for 20 minutes, and stain them with 0.3 μM DAPI solution for 10 minutes to visualize the cell nuclei. Images were obtained using an inverted fluorescence microscope (Nikon, Ts2) and Imageview software. All values are expressed as mean ± SEM.

[0138] (2) Experimental results: As Figure 2 shown, compared with untreated keratinocytes, the production of ROS in keratinocytes treated with 0.2% Torreya nucifera extract decreased by 10%, the production of ROS in keratinocytes treated with 0.5% Torreya nucifera extract decreased significantly by 42%, and the production of ROS in keratinocytes treated with 1% Torreya nucifera extract decreased significantly by 58%. It shows that Torreya nucifera extract can significantly reduce the mitochondrial ROS level in skin keratinocytes, and shows a certain concentration-dependent relationship.

[0139] Example 4 Effect of Torreya nucifera extract on the generation of cellular ATP.

[0140] (1) Experimental method: Seed healthy human keratinocytes on a 6-well plate and divide them into four groups, with a cell density of 1×10 4 cells / cm 2 . Among them, three groups were treated with 0.1%, 0.5%, and 1% Torreya nucifera extract for 48 hours respectively, and the control group was treated with KSFM medium for 48 hours. Aspirate the cell culture medium, wash once with PBS, add 200 μL of cell lysate to each well, and lyse the cells on ice for 30 minutes. After lysis, centrifuge at 13000 rpm at 4 °C for 15 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube, adjust the cell concentration, and use it for subsequent ATP concentration determination.

[0141] Use an ATP detection kit (Beyotime) to detect the ATP level in cells. Dissolve the ATP detection reagent on ice, dilute the ATP standard concentration solution to 0.1 μM, 1 μM, 10 μM, 50 μM, and 100 μM with the ATP detection lysate, and measure the standard curve of ATP concentration.

[0142] Prepare a 96-well plate with light-impermeable holes between the wells. Add 100 μL of the ATP detection working solution to each well according to the required number of wells. After standing at room temperature for 5 min, add 10 μL of the sample to each detection well, mix well with an interval of 3 seconds, and measure the RLU value using a liquid scintillation counter (Tecan, Spark). Calculate the absolute value of the ATP concentration in the sample according to the standard sample curve.

[0143] (2) Experimental results: As Figure 3 shown, after normal human keratinocytes were treated with Torreya nucifera extract for 48 hours, the ATP concentration increased significantly, indicating that Torreya nucifera extract can promote the production of cellular ATP.

Claims

1. Application of Torreya grandis Fort. ex Lindl. extract in preparing the following products: 1) Products for reducing skin aging caused by environmental pollution; 2) Products for enhancing the antioxidant capacity of skin keratinocytes; 3) Products for enhancing ATP production in skin keratinocytes; 4) Products for enhancing the mitochondrial functional activity of skin keratinocytes.

2. The application according to any one of claims 1-2, characterized in that, The concentration of the Torreya grandis Fort. ex Lindl. extract is 0.01%-1%, preferably 0.1%-0.5%, and more preferably 0.2%-0.5%.

3. A method for preparing Torreya grandis Fort. ex Lindl. extract, characterized in that, The method includes cold pressing extraction, steam explosion, and two rounds of steam distillation, and the temperature and pressure in the second round of steam distillation are higher than those in the first round.

4. The preparation method according to claim 3, wherein The preparation method includes the following steps: 1) Cold pressing extraction: Grind Torreya grandis Fort. ex Lindl. raw materials to obtain Torreya grandis Fort. ex Lindl. pulp and Torreya grandis Fort. ex Lindl. residue, filter the Torreya grandis Fort. ex Lindl. pulp and then centrifuge to obtain the first part of the extract; 2) Steam explosion: Put the Torreya grandis Fort. ex Lindl. residue in step 1) into a steam explosion device, add deoxygenated deionized water, and perform steam explosion treatment; 3) Steam distillation: Transfer the Torreya grandis Fort. ex Lindl. residue treated in step 2) to an extraction tank, convey deoxygenated deionized water, set the temperature and pressure, and then perform distillation extraction to obtain the second part of the extract; Repeat the above steam distillation step, and the temperature and pressure in the second round of steam distillation are higher than those in the first round to obtain the third part of the extract; 4) Mix the three parts of the extract to obtain the Torreya grandis Fort. ex Lindl. extract.

5. The preparation method according to claim 3 or 4, characterized in that, Step 1) has one or more of the following characteristics: 1) The Torreya grandis Fort. ex Lindl. raw materials are selected from any one or more of arils, Torreya grandis Fort. ex Lindl. leaves, and Torreya grandis Fort. ex Lindl. kernels; 2) The particle size of the Torreya grandis Fort. ex Lindl. raw materials is 0.1-5 mm, preferably 0.5-2 mm; 3) Grind the Torreya grandis Fort. ex Lindl. residue multiple times and repeat the extraction of Torreya grandis Fort. ex Lindl. pulp; The multiple times are selected from 1-5 times, preferably 4 times; 4) The Torreya grandis Fort. ex Lindl. pulp is a mixture after single extraction or multiple extractions; 5) The centrifugation program is set at 2000-6000 rpm, 10-20 min, preferably 4000 rpm, 15 min.

6. The preparation method according to claim 3 or 4, characterized in that, Step 2) has one or more of the following characteristics: 1) The liquid-solid mass ratio of deoxygenated deionized water to Torreya grandis Fort. ex Lindl. raw materials is 1-2:1-3, preferably 0.7:1; 2) The pressure of steam explosion is 2-5 Mpa, preferably 3 Mpa; 3) The treatment time of steam explosion is 5-20 minutes, preferably 10 minutes.

7. The preparation method according to claim 3 or 4, characterized in that, Step 3) has one or more of the following characteristics: 1) The conveying flow rate of deoxygenated deionized water ≤15 mL / min, preferably ≤10 mL / min; 2) The extraction temperature is 100°C-200°C, preferably 140°C-175°C, and the extraction temperature in the second round is higher than that in the first round; 3) The extraction pressure is 5-10 Mpa, preferably 6-7 Mpa, and the extraction pressure in the second round is higher than that in the first round; 4) The extraction time in the two rounds is the same, preferably 50 minutes - 120 minutes, and more preferably 90 minutes.

8. A product containing torreya grandis extract prepared by the preparation method according to any one of claims 4 to 7, characterized in that, The product is an external product or an internal product. The external products are selected from skin care products, cosmetics, and hair care products; The internal products are selected from solid beverages, liquid beverages, dairy products, flavoring powders, nutritional products, and internal medical health care products.

9. The product according to claim 8, wherein, The external products are selected from emulsions, gels, creams, microneedle patches, and facial masks, preferably emulsions.

10. The product according to any one of claims 8-9, characterized in that, The external product further contains excipients, and the excipients have any one or more of the following characteristics: 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.

11. The product according to claim 8, characterized in that, The internal 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 fructooligosaccharide, lactose, stevioside, xylitol, honey, syrup, peppermint oil, instant tea powder, fruit juice powder, edible flavor, 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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