Plant extract composition as well as preparation method and application thereof

Abstract

The invention provides a plant extract composition as well as a preparation method and application thereof, and relates to the technical fields of cytobiology, molecular biology and medicine research and development. The plant extract composition comprises a torreya grandis extract, a scutellaria baicalensis root extract and a momordica grosvenori extract. The plant extract composition can significantly improve the biological effects of torreya grandis, scutellaria baicalensis and momordica grosvenori, influence a signal channel for inducing autophagy, and influence autophagy markers LC3-II and pSer90-Beclin1, so that the skin state is improved, and the plant extract composition is used for preparing cosmetics and / or food with whitening and anti-aging effects.

Description

Technical Field

[0001] The present invention relates to the technical fields of cell biology, molecular biology and drug development, and in particular to a plant nanovesicle composition, a preparation method and an application thereof. Background Art

[0002] Skin is the largest organ in the human body. The color of the skin is mainly determined by the content and distribution of skin pigments, with melanin being the most important determinant. Tyrosinase is a key enzyme in the production of melanin. In the initial stage of melanin production, it catalyzes the oxidation of tyrosine and phenylalanine to produce dopaquinone, which can become the matrix for the synthesis of eumelanin and pheomelanin.

[0003] Autophagy is a process in which cells engulf their own cytoplasmic proteins or organelles and encapsulate them into vesicles, fuse with lysosomes to form autophagolysosomes, and degrade the contents they encapsulate, thereby achieving the metabolic needs of the cell itself and the renewal of certain organelles. The different stages of autophagy are synergistically regulated by the autophagy core gene ATGs and autophagy regulatory factors. Autophagy disorders may lead to the occurrence and development of a variety of diseases. Recent studies have shown that the autophagy of keratinocytes and melanocytes on melanosomes plays an important role in pigment metabolism. Therefore, by increasing autophagy, the degradation of melanosomes in keratinocytes and melanocytes can be induced, thereby achieving a skin whitening effect.

[0004] Currently, one of the most researched and applied methods for skin whitening is to affect melanin production by regulating the activity of tyrosinase. Commercial whitening agents have certain problems with their own stability or certain safety hazards in their application. For example, arbutin, phenylethyl resorcinol, kojic acid and other ingredients that inhibit melanin synthesis are unstable under light and heat, and are easily oxidized and lose their whitening effect. Nicotinamide and other ingredients that inhibit melanin transport may cause irritation to the skin during the production process. Salicylic acid and its derivatives, vitamin C and its derivatives, and other ingredients that promote the decomposition and metabolism of melanin are irritating to a certain extent, especially not suitable for consumers with sensitive skin.

[0005] Therefore, developing whitening active ingredients with multi-pathway whitening effects, good safety and stability has become one of the important research directions in the cosmetics field.

[0006] Torreya grandis belongs to the genus Torreya of the Taxaceae family. It is one of the ancient species unique to my country and a relict plant of the Tertiary period, originating from the Jurassic period about 170 million years ago. Torreya grandis was called "Torreya grandis" in ancient times, "Torreya grandis", "Torreya grandis", "Yushan fruit" and "Red fruit". Torreya grandis fruit is rich in flavonoids, lignans, terpenes, fatty acids, vitamins, mineral elements, alkaloids and amino acids, and has a long history of medicinal and edible use. Shengzhou City is the "Hometown of Torreya grandis in China", and the history of artificial cultivation of Torreya grandis is more than 1,300 years.

[0007] Scutellaria baicalensis Georgi is a perennial herb of the genus Scutellaria in the Lamiaceae family. Compendium of Materia Medica states: "Qin is written as Qin in Shuowen, which means its color is yellow." Scutellaria baicalensis Georgi is the so-called "Qin" in ancient times and got its name because the color of the grass is yellowish. Compendium of Materia Medica records that Scutellaria baicalensis Georgi is bitter in taste, neutral in nature, and non-toxic. The root of Scutellaria baicalensis Georgi contains flavonoid compounds such as baicalein, and also contains β-sitosterol, campesterol, and stigmasterol.

[0008] Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang is a climbing herb of the genus Siraitia in the Cucurbitaceae family. Siraitia grosvenorii is native to China, and the main production areas are in Guangdong, Guangxi, Guizhou, Hunan, Jiangxi and other regions. Among them, Guangxi is especially rich in it and it has become a local specialty of Guangxi. According to the records in Records of Traditional Chinese Medicines Collected in the Lingnan Region, Siraitia grosvenorii is cool in nature, sweet in taste, and belongs to the lung and large intestine meridians, and has the effects of clearing the lung and relieving sore throat, resolving phlegm and relieving cough, and moistening the intestines and promoting defecation. Siraitia grosvenorii contains a large amount of dietary fiber and glycosides, which has the effect of lowering blood sugar. The pulp of Siraitia grosvenorii is sweet, with a unique taste and high nutritional value. It can also be used for cooking, with a delicate fragrance, and is known as the "fruit of the immortals" by people. Summary of the Invention

[0009] The object of the present invention is to provide a combination of plant extracts, which induces autophagy in the studied models and shows a synergistic effect of promoting autophagy, especially in skin cells.

[0010] The specific technical solutions are as follows:

[0011] In a first aspect, the present invention provides a combination of plant extracts, and the plant extracts include Torreya grandis fruit extract, Scutellaria baicalensis root extract, and Siraitia grosvenorii extract.

[0012] In some specific embodiments, the extract is a nanovesicle.

[0013] In some embodiments, the particle number ratio of the Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles, and Siraitia grosvenorii nanovesicles is (0.1-2):(1-3):1, preferably (0.3-0.8):(1.5-2):1.

[0014] In some embodiments, the content of the Torreya grandis fruit nanovesicles is 1×10 9 ~1×10 11 per mL;

[0015] In some embodiments, the content of the Scutellaria baicalensis root nanovesicles is 1×10 9 ~1×10 11 indivual

[0016] / mL;

[0017] In some embodiments, the content of the monk fruit nanovesicles is 1×10 9 ~1×10 11 indivual

[0018] / mL.

[0019] In some embodiments, the Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles are all nanovesicles with a double-layer membrane vesicle structure;

[0020] In some embodiments, the particle sizes of the Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles are each independently 100 to 900 nm.

[0021] In a second aspect, the present invention provides a method for preparing the plant extract combination, the preparation method comprising the following steps:

[0022] (1) respectively breaking the wall of Torreya grandis fruit, Scutellaria baicalensis root and Momordica grosvenori to obtain Torreya grandis fruit juice, Scutellaria baicalensis root juice and Momordica grosvenori juice;

[0023] (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, Scutellaria baicalensis root juice, and Momordica grosvenori juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles, and Momordica grosvenori nanovesicles;

[0024] (3) The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) are mixed to obtain the plant nanovesicle composition.

[0025] In some embodiments, in step (1), the Torreya grandis fruit includes Torreya grandis fruit aril and / or Torreya grandis fruit kernel.

[0026] In some embodiments, in step (1), the temperature of the cell wall breaking treatment is 2-6°C.

[0027] In some embodiments, in step (2), the specific steps of centrifugation are:

[0028] The Torreya grandis fruit juice, Scutellaria baicalensis root juice and Momordica grosvenori juice obtained in step (1) are centrifuged respectively, and the supernatants are collected to obtain Torreya grandis fruit supernatant, Scutellaria baicalensis root juice supernatant and Momordica grosvenori supernatant.

[0029] In some embodiments, the centrifugal force of the centrifugation is 2000-3000×g.

[0030] In some embodiments, the centrifugation temperature is 2-6°C.

[0031] In some embodiments, the centrifugation time is 20 to 40 minutes.

[0032] In some embodiments, in step (2), the filtration is performed using a Nanshi filter.

[0033] In some embodiments, the capsule filter is a Bricap C01 capsule filter.

[0034] In some embodiments, the pore size of the Nan filter is 0.2-0.45 μm, preferably 0.45 μm.

[0035] In some embodiments, the membrane area of ​​the Nas filter is 160 to 200 cm 2 , preferably 180cm 2 .

[0036] In some embodiments, in step (2), the chromatography is performed using a Capto Core 700 chromatography column.

[0037] In some embodiments, the diameter-to-height ratio of the chromatography column is 1:(8-12).

[0038] In some embodiments, the column volume of the chromatography column is 100-200 mL.

[0039] In some embodiments, during the chromatography process, the eluent used is sterile physiological saline.

[0040] In some embodiments, during the chromatography, the flow rate of the eluent is 180-360 cm / h.

[0041] In some embodiments, during the chromatography, the loading volume is 100-500% of the column volume.

[0042] In some embodiments, in step (2), the ultrafiltration concentration is performed using a Cobetter hollow fiber column.

[0043] In some embodiments, the molecular weight cut-off range of the ultrafiltration concentration is 100-300 KD.

[0044] In some embodiments, the raw water end flow rate of the ultrafiltration concentration is 100-300 mL / min.

[0045] In some embodiments, the transmembrane pressure difference at the reflux end of the ultrafiltration concentration is 1 to 5 psi.

[0046] In a third aspect, the present invention provides a use of the plant extract combination as described in the first aspect in preparing any of the following products:

[0047] (1) Products with whitening effect;

[0048] (2) Products that promote the decomposition of melanin;

[0049] (3) Products that delay the production of melanin.

[0050] In some embodiments, the product is an external product or an internal product, the external product is selected from skin care products, cosmetics, and hair care products; the internal product is selected from solid beverages, liquid beverages, dairy products, seasoning powders, nutritional supplements, and internal health care products.

[0051] In some embodiments, the topical product is selected from the group consisting of an emulsion, a gel, a cream, a microneedle patch, and a facial mask.

[0052] In some embodiments, the topical product further contains an auxiliary material, and the auxiliary material contains a preservative ingredient: one or more selected from methylparaben, ethylparaben, propylparaben, formaldehyde releasers, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinone, phenols, benzoic acid, benzyl alcohol, potassium sorbate, bromopol, parahydroxyacetophenone, chlorphenesin, triclosan, quaternary ammonium salt-15, dehydroacetic acid and its salts.

[0053] In some embodiments, the topical product further contains excipients, and the excipients contain emulsifying ingredients: one or more selected from polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, and phospholipids.

[0054] In some embodiments, the topical product further contains excipients, wherein the excipients contain modifying excipients selected from flavors and / or pigments.

[0055] In some embodiments, the oral product further contains food-acceptable excipients, wherein the excipients contain flavoring agents selected from one or more of oligofructose, lactose, stevioside, xylitol, honey, syrup, mint oil, instant tea powder, fruit juice powder, edible flavors, and citric acid.

[0056] In some embodiments, the oral product further contains food-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.

[0057] The beneficial effects of the present invention are as follows:

[0058] The present invention demonstrates for the first time a new use of the Torreya grandis extract: the Torreya grandis extract can affect the signal pathway that induces autophagy and has an effect on the autophagy markers LC3-II and pSer90-Beclin1, thereby improving the skin condition, and is used for preparing cosmetics and / or foods with whitening and anti-aging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Transmission electron micrographs of Torreya grandis fruit nanovesicle-A, Scutellaria baicalensis root nanovesicle-B and Momordica grosvenori nanovesicle-C.

[0060] Figure 2 Shows the effect of each sample on the expression level of autophagy active protein LC3-II.

[0061] Figure 3 The figure shows the effect of each sample on the phosphorylation level of Beclin-1 protein at Ser15 site.

[0062] Figure 4 This is the effect of each sample on the phosphorylation level of Beclin-1 protein at the Ser90 site.

[0063] Figure 5 The effect of each sample on melanin accumulation in cells. DETAILED DESCRIPTION

[0064] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0065] Unless defined otherwise, 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 disclosure belongs.

[0066] In a first aspect, the present invention provides a plant extract combination, wherein the plant extract comprises Torreya grandis fruit extract, Scutellaria baicalensis root extract and Momordica grosvenori extract.

[0067] In some specific embodiments, the extract is a nanovesicle, which is a nanoscale lipid bilayer vesicle, including exosomes and microvesicles. Exosomes / microvesicles are a mechanism of intercellular communication, which carry a variety of bioactive substances including DNA, RNA, proteins, etc. into effector cells, effectively regulate the body's cell signal transduction, and play anti-inflammatory, anti-oxidation, and promote the proliferation of effector cells.

[0068] As an optional embodiment, the particle number ratio of the Torreya grandis fruit nanovesicles, the Scutellaria baicalensis root nanovesicles and the Momordica grosvenori nanovesicles is (0.1-2):(1-3):1.

[0069] Here, “0.1-2” can be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, etc.;

[0070] Here, “1-3” can be, for example, 1, 1.5, 2, 2.5, 3, etc.

[0071] In some specific embodiments, the particle number ratio of the Torreya grandis fruit nanovesicles, the Scutellaria baicalensis root nanovesicles and the Momordica grosvenori nanovesicles is preferably (0.3-0.8):(1.5-2):1;

[0072] As an optional embodiment, the content of the Torreya grandis fruit nanovesicles is 1×10 9 ~1×10 11 / mL, for example, 1×10 9 / mL, 2×10 9 / mL, 3×10 9 / mL, 4×10 9 indivual

[0073] / mL, 5×10 9 / mL, 6×10 9 / mL, 7×10 9 / mL, 8×10 9 / mL, 9×10 9 indivual

[0074] / mL, 1×10 10 / mL, 2×10 10 / mL, 3×10 10 / mL, 4×10 10 / mL, 5×10 10 / mL, 6×10 10 / mL, 7×10 10 / mL, 8×10 10 / mL, 9×10 10 / mL, 1×10 11 Pieces / mL, etc.

[0075] As an optional embodiment, the content of the Scutellaria baicalensis root nanovesicles is 1×10 9 ~1×10 11 / mL, for example, 1×10 9 / mL, 2×10 9 / mL, 3×109 / mL, 4×10 9 / mL, 5×10 9 / mL, 6×10 9 / mL, 7×10 9 / mL, 8×10 9 / mL, 9×10 9 / mL, 1×10 10 / mL, 2×10 10 / mL, 3×10 10 / mL, 4×10 10 / mL, 5×10 10 / mL, 6×10 10 / mL, 7×10 10 / mL, 8×10 10 / mL, 9×10 10 / mL, 1×10 11 Pieces / mL, etc.

[0076] As an optional embodiment, the content of the monk fruit nanovesicles is 1×10 9 ~1×10 11 / mL, for example, 1×10 9 / mL, 2×10 9 / mL, 3×10 9 / mL, 4×10 9 / mL, 5×10 9 / mL, 6×10 9 / mL, 7×10 9 / mL, 8×10 9 / mL, 9×10 9 / mL, 1×10 10 / mL, 2×10 10 / mL, 3×10 10 / mL, 4×10 10 / mL, 5×10 10 / mL, 6×10 10 / mL, 7×10 10 / mL, 8×10 10 / mL, 9×10 10 / mL, 1×10 11 Pieces / mL, etc.

[0077] As an optional embodiment, the Torreya grandis berry nanovesicle is a nanovesicle with a double-layer membrane vesicle structure.

[0078] As an optional embodiment, the Scutellaria baicalensis root nanovesicles are nanovesicles with a double-layer membrane vesicle structure.

[0079] As an optional embodiment, the monk fruit nanovesicles are all nanovesicles with a double-layer membrane vesicle structure.

[0080] In the present invention, the characteristics of the Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles are identified by electron microscopy, particle size analysis and other methods. The results are consistent with the characteristics of nanovesicles and show a double-layer membrane vesicle structure under a transmission electron microscope.

[0081] As an optional embodiment, the particle size of the Torreya grandis fruit nanovesicles is 100-900 nm, for example, it can be 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, etc.

[0082] As an optional embodiment, the particle size of the Scutellaria baicalensis root nanovesicles is 100-900 nm, for example, it can be 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, etc.

[0083] As an optional embodiment, the particle size of the monk fruit nanovesicle is 100 to 900 nm, for example, it can be 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, etc.

[0084] In a second aspect, the present invention provides a method for preparing the plant nanovesicle composition, the preparation method comprising the following steps:

[0085] (1) respectively breaking the wall of Torreya grandis fruit, Scutellaria baicalensis root and Momordica grosvenori to obtain Torreya grandis fruit juice, Scutellaria baicalensis root juice and Momordica grosvenori juice;

[0086] (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, Scutellaria baicalensis root juice, and Momordica grosvenori juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles, and Momordica grosvenori nanovesicles;

[0087] (3) The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) are mixed to obtain the plant nanovesicle composition.

[0088] As an optional embodiment, in step (1), the Torreya grandis fruit includes Torreya grandis fruit aril and / or Torreya grandis fruit kernel.

[0089] As an optional embodiment, in step (1), the temperature of the cell wall breaking treatment is 2-6°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, etc.

[0090] As an optional embodiment, in step (1), the cell wall breaking treatment is to use a cell wall breaking machine and / or a homogenizer to squeeze out the juice.

[0091] As an optional embodiment, in step (2), the specific steps of the centrifugation are:

[0092] The Torreya grandis fruit juice, the Scutellaria baicalensis root juice and the Momordica grosvenori juice were centrifuged respectively, and the supernatants were collected to obtain Torreya grandis fruit supernatant, Scutellaria baicalensis root juice supernatant and Momordica grosvenori supernatant.

[0093] As an optional embodiment, the centrifugal force of the centrifugation is 2000-3000×g, for example, it can be 2000×g, 2100×g, 2200×g, 2300×g, 2400×g, 2500×g, 2600×g, 2700×g, 2800×g, 2900×g, 3000×g, etc.

[0094] As an optional embodiment, the centrifugation temperature is 2-6°C, for example, it can be 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, etc.

[0095] As an optional embodiment, the centrifugation time is 20 to 40 min, for example, it can be 20 min, 22 min, 24 min, 25 min, 26 min, 28 min, 30 min, 32 min, 34 min, 35 min, 36 min, 38 min, 40 min, etc.

[0096] As an optional embodiment, in step (2), the filtration is performed using a Nascher filter.

[0097] As an optional embodiment, the capsule filter is a Bricap C01 capsule filter.

[0098] As an optional embodiment, the pore size of the Nas filter is 0.2-0.45 μm, for example, it can be 0.2 μm, 0.22 μm, 0.45 μm, etc., preferably 0.45 μm.

[0099] As an optional embodiment, the membrane area of ​​the Nas filter is 160 to 200 cm 2 , for example, it can be 160cm 2 、170cm 2 、180cm 2 、190cm 2 , 200cm 2 etc., preferably 180cm 2 .

[0100] As an optional embodiment, in step (2), the filtration is carried out using Bricap C01 (Cobetter, 0.45 μm, 180 cm 2 ) was performed using a capsule filter.

[0101] As an optional embodiment, in step (2), the chromatography is performed using a Capto Core 700 chromatography column.

[0102] As an optional embodiment, the diameter-to-height ratio of the chromatography column is 1:(8-12), for example, it can be 1:8, 1:9, 1:10, 1:11, 1:12, etc.

[0103] As an optional embodiment, the column volume of the chromatography column is 100-200 mL, for example, it can be 100 mL, 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, etc.

[0104] As an optional embodiment, in step (2), the chromatography is performed using a Capto Core 700 chromatography column (ID: H=26mm:27cm, 1CV=143.4mL).

[0105] As an optional embodiment, during the chromatography process, the eluent used is sterile physiological saline.

[0106] As an optional embodiment, during the chromatography process, the flow rate of the eluent is 180-360 cm / h, for example, it can be 180 cm / h, 190 cm / h, 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h, 250 cm / h, 260 cm / h, 270 cm / h, 280 cm / h, 290 cm / h, 300 cm / h, 310 cm / h, 320 cm / h, 330 cm / h, 340 cm / h, 350 cm / h, 360 cm / h, etc.

[0107] As an optional embodiment, during the chromatography, the sample loading volume is 100-500% of the column volume, for example, it can be 100%, 150%, 200%, 250%, 300%, 350%,

[0108] 400%, 450%, 500%, etc.

[0109] As an optional embodiment, in step (2), the ultrafiltration concentration is performed using a Cobetter hollow fiber column.

[0110] As an optional embodiment, the molecular weight cutoff range of the ultrafiltration concentration is 100-300KD, for example, it can be 100KD, 105KD, 110KD, 115KD, 120KD, 130KD, 140KD, 150KD, 160KD, 170KD, 180KD, 190KD, 200KD, 220KD, 240KD, 250KD, 260KD, 280KD, 300KD, etc.

[0111] As an optional embodiment, in step (2), the ultrafiltration concentration uses a Cobetter hollow fiber column (model: HFELA01001530P, 100KD, 236cm 2 )conduct.

[0112] As an optional embodiment, the flow rate of the raw water end (FEED) of the ultrafiltration concentration is 100-300 mL / min, for example, it can be 100 mL / min, 120 mL / min, 140 mL / min, 150 mL / min, 160 mL / min, 180 mL / min, 200 mL / min, 220 mL / min, 240 mL / min, 250 mL / min, 260 mL / min, 280 mL / min, 300 mL / min, etc., preferably 200 mL / min.

[0113] As an optional embodiment, the transmembrane pressure difference (TMP) at the reflux end of the ultrafiltration concentration is 1 to 5 psi, for example, it can be 1 psi, 1.5 psi, 2 psi, 2.5 psi, 3 psi, 3.5 psi, 4 psi, 4.5 psi, 5 psi, etc.

[0114] As an optional embodiment, after obtaining Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles, the method further includes any one or a combination of at least two of the following post-processing steps: filtration, sterilization, sample packaging, inspection, or long-term storage at -80°C.

[0115] In a third aspect, the present invention provides a use of the plant nanovesicle composition as described in the first aspect in the preparation of any of the following products:

[0116] (1) Products with whitening effect;

[0117] (2) Products that promote the decomposition of melanin;

[0118] (3) Products that delay the production of melanin.

[0119] In some specific embodiments, the product having the above-mentioned efficacy is an external product or an internal product.

[0120] The external use product is selected from skin care products, cosmetics, and hair care products, preferably skin care products and / or cosmetics;

[0121] The oral product is selected from solid beverages, liquid beverages, dairy products, seasoning powders, nutritional supplements, and oral health care products.

[0122] In some embodiments, the topical product is selected from the group consisting of an emulsion, a gel, a cream, a microneedle patch, and a facial mask.

[0123] In some embodiments, the topical product further contains an auxiliary material, and the auxiliary material contains a preservative ingredient: one or more selected from methylparaben, ethylparaben, propylparaben, formaldehyde releasers, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinone, phenols, benzoic acid, benzyl alcohol, potassium sorbate, bromopol, parahydroxyacetophenone, chlorphenesin, triclosan, quaternary ammonium salt-15, dehydroacetic acid and its salts.

[0124] In some embodiments, the topical product further contains excipients, and the excipients contain emulsifying ingredients: one or more selected from polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, and phospholipids.

[0125] In some embodiments, the topical product further contains excipients, wherein the excipients contain modifying excipients selected from flavors and / or pigments.

[0126] The internal product of 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.

[0127] When the Torreya grandis seed oil 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 used appropriately according to conventional methods.

[0128] In some embodiments, the oral product further contains food-acceptable excipients, wherein the excipients contain flavoring agents selected from one or more of oligofructose, lactose, stevioside, xylitol, honey, syrup, mint oil, instant tea powder, fruit juice powder, edible flavors, and citric acid.

[0129] In some embodiments, the oral product further contains food-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.

[0130] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. In the following examples, if there is no special description of raw materials or processing techniques, it indicates that they are all conventional commercially available raw materials or conventional processing techniques in the art.

[0131] Example

[0132] Example 1 Preparation of plant nanovesicle composition

[0133] (1) The aril of Torreya grandis fruit, the root of Scutellaria baicalensis and Momordica grosvenori were squeezed out of juice by a homogenizer to obtain Torreya grandis fruit juice, Scutellaria baicalensis root juice and Momordica grosvenori juice.

[0134] (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, Scutellaria baicalensis root juice, and Momordica grosvenori juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles, and Momordica grosvenori nanovesicles;

[0135] The centrifugation comprises: centrifuging the juices of the three plants at medium speed (2500×g) for 30 min at 4°C to collect the supernatants; the filtration comprises: filtering the supernatants of the three plants using Bricap C01 (Cobetter, 0.45 μm, 180 cm 2 ) capsule filter to collect the filtrate; the chromatography is to use CaptoCore 700 chromatography column (ID: H = 26mm: 27cm, 1CV = 143.4mL) to chromatograph the upper filtrate of the three plants respectively (the eluent used is sterile saline eluent with a flow rate of 300cm / h, and the sample volume is 300% of the column volume), and collect the chromatographic solution; the ultrafiltration concentration is: use Cobetter hollow fiber column (model: HFELA01001530P, 100KD, 236cm 2 ) was ultrafiltration concentrated, the flow rate at the FEED end was set to 200 mL / min (152 LMH), and the TMP was controlled at 3 psi at the reflux end for concentration.

[0136] After collecting plant nanovesicles, the characteristics of the nanovesicles were identified by electron microscopy and particle size analysis. The results were consistent with the characteristics of nanovesicles: the diameter ranged from 100-900nm, and the double-layer membrane vesicle structure was observed under transmission electron microscopy, such as Figure 1 shown.

[0137] (3) Composition 1: The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) are mixed at a particle number ratio of 0.5:1:1 to obtain the plant nanovesicle composition 1; the content of the Torreya grandis fruit nanovesicles in the composition is 2.4×10 9 / mL, and the content of Scutellaria baicalensis root nanovesicles was 4.8×10 9 / mL, the content of monk fruit nanovesicles was 4.8×10 9 Pieces / mL.

[0138] (4) Composition 2: The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) are mixed at a particle number ratio of 0.3:1.5:1 to obtain the plant nanovesicle composition 2; the content of the Torreya grandis fruit nanovesicles in the composition is 1.3×10 9 / mL, and the content of Scutellaria baicalensis root nanovesicles was 6.3×10 9 / mL, the content of monk fruit nanovesicles was 4.2×10 9 Pieces / mL.

[0139] (5) Composition 3: The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) were mixed at a particle number ratio of 0.8:2:1 to obtain the plant nanovesicle composition 3; the content of the Torreya grandis fruit nanovesicles in the composition was 2.5×10 9 / mL, and the content of Scutellaria baicalensis root nanovesicles was 6.3×10 9 / mL, the content of monk fruit nanovesicles was 3.2×10 9 Pieces / mL.

[0140] (6) Composition 4: The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) are mixed at a particle number ratio of 2:3:1 to obtain the plant nanovesicle composition 4; the content of the Torreya grandis fruit nanovesicles in the composition is 4×10 9 / mL, and the content of Scutellaria root nanovesicles was 6×10 9 / mL, the content of monk fruit nanovesicles is 2×10 9 Pieces / mL.

[0141] Comparison of cytotoxicity of the composition of Example 2

[0142] By placing zebrafish embryos in test substance solutions of different concentrations, the development and death of the zebrafish embryos in the test substance are observed. When starting from a certain concentration, no death or other toxic effects such as teratogenicity are observed in the zebrafish embryos. This concentration is the maximum tolerable concentration of the test substance.

[0143] Zebrafish embryos at the second day of development were selected as experimental organisms, and a blank control group and a sample group (composition 1) (3.125%, 6.25%, 12.5%, 25.0%, 50.0%) were set up respectively, with a geometric dilution factor of 2, and 20 embryos were set up in each group, and placed in a 96-well plate (1 / well); each 96-well plate to be tested was placed in a constant temperature incubator at 28.5±0.5°C and incubated for 24 hours, and then the death and deformity were counted.

[0144] The results are shown in Table 1. Under the experimental conditions, the maximum tolerable concentration tested was 12.5%, and no death or deformity was observed in this concentration group.

[0145] Table 1 Mortality and deformity rate of zebrafish embryos in each group (24h)

[0146]

[0147] Example 3 Analysis of the expression level of autophagy active protein LC3-II and the phosphorylation level of Beclin-1 protein at Ser15 and Ser90 sites after treatment with the composition

[0148] To investigate the signaling pathways involved in autophagy induction, the experiment monitored the phosphorylation levels of Beclin-1 protein at Ser15 and Ser90, which are phosphorylation sites regulated by the mTOR pathway or the AMPK pathway, respectively. [1,2] .

[0149] Human epidermal melanocytes from dark-colored donors (HEMn-DP cells) were cultured in medium 254 supplemented with human melanocyte growth supplement (Gibco, ThermoFisher).

[0150] Before harvesting for western blotting, cells were treated with different concentrations of composition 1-4 for 48 hours. After treatment, cells were washed with PBS and lysed in ice-cold RIPA buffer (ThermoFFisher) supplemented with Halt protease inhibitor (ThermoFisher). The protein concentration of the lysate was measured using a MicroBCA detection kit (ThermoFisher). 30 micrograms of protein were separated by SDS-PAGE using 4%-12% bis-tris gel and transferred to a nitrocellulose membrane (Bio-Rad; Hercules, CA, USA). The membrane was blocked in TBS blocking solution (Li-Cor; Lincoln, NE, USA) for 1 hour and then incubated overnight in the primary antibody. The following primary antibodies were used: mouse anti-α-tubulin (dilution 1:5000, cat no. ab7291; Abcam), Phospho-Beclin-1 Ser90 (dilution 1:500, cat no. 86455; Cell Signaling Technology, Danvers, USA), Phospho-Beclin-1 Ser15 rabbit monoclonal antibody (dilution 1:500, cat no. 84966; Cell Signaling Technology), and LC3B (dilution 1:650 cat no. 2775; Cell Signaling Technology). All primary antibodies were diluted in TBS blocking buffer containing 0.2% Tween 20 (Li-Cor). After washing three times with 0.1% TBS-Tween (ThermoFisher), the membrane was incubated with IR dye-labeled secondary antibody (Li-Cor) at a dilution of 1:20 000 for 1 hour at room temperature. The membrane was washed three times with 0.1% TBS-Tween and scanned using the Odyssey infrared imaging system (Li-Cor).

[0151] The results are shown in Table 2. LC3-II protein participates in the formation of autophagosomes and activates autophagy. The composition increases the level of LC3-II, which is proportional to the dose. The composition treatment has no significant effect on the phosphorylation level of Ser15, but significantly increases the phosphorylation level of Ser90, which is proportional to the dose. This reflects that the composition provided by the invention can induce activation of autophagy-related signaling pathways ( Figure 2-Figure 4 ).

[0152] Table 2

[0153] Concentration (g / L) LC3-II pSer15-Beclin1 pSer90-Beclin1 Control group 0 1 1 1 Composition 1 0.1％ 1.81 1.05 0.82 Composition 1 0.2％ 2.74 1.04 1.37 Composition 1 0.5％ 3.59 0.85 1.42 Composition 2 0.1％ 1.76 0.95 0.68 Composition 2 0.5％ 3.18 0.89 1.11 Composition 3 0.1％ 1.55 0.98 0.73 Composition 3 0.5％ 2.79 0.87 1.17 Composition 4 0.1％ 1.63 1.02 0.79 Composition 4 0.5％ 2.93 0.92 1.33

[0154] Example 4 Analysis of the Effect of Composition on Melanin Accumulation

[0155] Human epidermal melanocytes from dark-colored donors (HEMn-DP cells) were cultured in medium 254 supplemented with human melanocyte growth supplement (Gibco, ThermoFisher).

[0156] In order to quantify the melanin in the cells, the cells were treated with 2 μL of blank control or different concentrations of composition 1-4 every 3 days for a total of 7 days. After treatment, the cells were lysed in 350 μL of 1M sodium hydroxide. 50 μL of cell supernatant and a series of synthetic melanin standards were transferred to each well in the black wall detection plate. The absorbance value of the sample was read at a wavelength of 490 nm using a Spectramax 340PC microplate reader (Molecular Devices, San Jose, California, the United States), and the melanin content was calculated according to the value of the synthetic melanin standard curve.

[0157] The results are shown in Table 3 and Figure 5 As shown, the plant extract combination provided by the invention can significantly reduce the melanin content and has great potential for preparing whitening products.

[0158] Table 3

[0159] Concentration (g / L) Melanin content (%) Control group 0 100 Composition 1 0.1％ 72 Composition 1 0.2％ 65 Composition 1 0.5％ 49 Composition 2 0.1％ 81 Composition 2 0.5％ 67 Composition 3 0.1％ 78 Composition 3 0.5％ 61 Composition 4 0.1％ 76 Composition 4 0.5％ 55

[0160] References:

[0161] [1] RRC Russell, Y. Tian, ​​H. Yuan, et al., "ULK1 Induces Autophagy by PhosphorylatingBeclin-1and Activating VPS34 Lipid Kinase," Nature Cell Biology 15, no. 7 (2013): 741–750, https: / / doi.org / 10.1038 / ncb2757.

[0162] [2] J.Kim, YCKim, C.Fang, et al., “Differential Regulation of DistinctVps34Complexes by AMPK in Nutrient Stress and Autophagy,” Cell152, no.1–2(2013):290–

[0163] 303, https: / / doi.org / 10.1016 / j.cell.2012.12.016.

Claims

1. A plant extract combination, characterized in that: The plant extracts include Torreya grandis fruit extract, Scutellaria baicalensis root extract and Momordica grosvenori extract.

2. The plant extract combination according to claim 1, characterized in that The extract is nanovesicles.

3. The plant extract combination according to claim 1 or 2, characterized in that: The particle number ratio of the nanovesicles of Torreya grandis fruit extract, Scutellaria baicalensis root extract and Momordica grosvenori extract is (0.1-2):(1-3):1, preferably (0.3-0.8):(1.5-2):1; Preferably, the content of the Torreya grandis fruit nanovesicles is 1×10 9 ~1×10 11 / mL; Preferably, the content of the Scutellaria baicalensis root nanovesicles is 1×10 9 ~1×10 11 Pieces / mL; Preferably, the content of the monk fruit nanovesicles is 1×10 9 ~1×10 11 Pieces / mL.

4. The plant extract combination according to claim 2 or 3, characterized in that: The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root rice nanovesicles and Momordica grosvenori nanovesicles are all nanovesicles with a double-layer membrane vesicle structure; Preferably, the particle sizes of the Torreya grandis fruit nanovesicles, Scutellaria baicalensis root rice nanovesicles and Momordica grosvenori nanovesicles are independently 100 to 900 nm.

5. A method for preparing the plant extract combination according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) respectively breaking the wall of Torreya grandis fruit, Scutellaria baicalensis root and Momordica grosvenori to obtain Torreya grandis fruit juice, Scutellaria baicalensis root juice and Momordica grosvenori juice; (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, Scutellaria baicalensis root juice, and Momordica grosvenori juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles, and Momordica grosvenori nanovesicles; (3) The Torreya grandis fruit nanovesicles, Scutellaria baicalensis root nanovesicles and Momordica grosvenori nanovesicles obtained in step (2) are mixed to obtain the plant nanovesicle composition.

6. The method for preparing the plant extract combination according to claim 5, characterized in that: In step (1), the Torreya grandis fruit includes Torreya grandis fruit aril and / or Torreya grandis fruit kernel; Preferably, in step (1), the temperature of the cell wall breaking treatment is 2-6°C.

7. The method for preparing the plant extract combination according to claim 5 or 6, characterized in that: In step (2), the specific steps of centrifugation are: Centrifuging the Torreya grandis fruit juice, Scutellaria baicalensis root juice and Momordica grosvenori juice obtained in step (1) respectively, collecting the supernatants to obtain Torreya grandis fruit supernatant, Scutellaria baicalensis root juice supernatant and Momordica grosvenori supernatant; Preferably, the centrifugal force of the centrifugation is 2000-3000×g; the centrifugal temperature is 2-6°C; the centrifugal time is 20-40 min; Preferably, in step (2), the filtration is performed using a Nassau filter; Preferably, the capsule filter is a Bricap C01 capsule filter; Preferably, the pore size of the Nas filter is 0.2-0.45 μm; the membrane area of ​​the Nas filter is 160-200 cm 2 ; Preferably, in step (2), the chromatography is performed using a Capto Core 700 chromatography column; Preferably, the diameter-to-height ratio of the chromatography column is 1:(8-12); the column volume of the chromatography column is 100-200 mL; Preferably, during the chromatography, the eluent used is sterile physiological saline; the flow rate of the eluent is 180-360 cm / h; the loading volume is 100-500% of the column volume; Preferably, in step (2), the ultrafiltration concentration is performed using a Cobetter hollow fiber column; Preferably, the molecular weight cut-off range of the ultrafiltration concentration is 80 to 120 KD; Preferably, the flow rate of the raw water end of the ultrafiltration concentration is 100-300 mL / min; the transmembrane pressure difference of the reflux end of the ultrafiltration concentration is 1-5 psi.

8. Use of the plant extract combination according to any one of claims 1 to 4 in the preparation of any one of the following products: (1) Products with whitening effect; (2) Products that promote the decomposition of melanin; (3) Products that delay the production of melanin.

9. The use according to claim 8, characterized in that The product is an external product or an internal product, wherein the external product is selected from skin care products, cosmetics, and hair care products; the internal product is selected from solid beverages, liquid beverages, dairy products, seasoning powders, nutritional products, and internal health care products; Preferably, the topical product is selected from emulsions, gels, creams, microneedle patches, and facial masks.

10. The use according to claim 8 or 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 ingredients: the preservative ingredients are selected from one or more of methylparaben, ethylparaben, propylparaben, formaldehyde releasers, methylchloroisothiazolinone, methylisothiazolinone, phenoxyethanol, imidazolidinyl urea, isothiazolinone, phenol, benzoic acid, benzyl alcohol, potassium sorbate, bromopol, p-hydroxyacetophenone, chlorphenesin, triclosan, quaternary ammonium salt-15, dehydroacetic acid and its salts; 2) containing emulsifying ingredients: the emulsifying ingredients are selected from one or more of polyethylene glycols, glycerides, sugar derivatives, anionic emulsifiers, cationic emulsifiers, and phospholipids; 3) Containing modifying auxiliary materials: the modifying ingredients are selected from flavors and / or pigments; The oral product further contains a food-friendly excipient, wherein the excipient has any one or more of the following characteristics: 1) containing flavoring agents, wherein the flavoring agents are selected from one or more of oligofructose, lactose, stevioside, xylitol, honey, syrup, mint oil, instant tea powder, fruit juice powder, edible flavors, and citric acid; 2) Containing dosage form excipients, wherein the dosage form excipients are selected from one or more of maltodextrin, calcium stearate, magnesium stearate, hydroxypropyl methylcellulose, microcrystalline cellulose, and silicon dioxide.

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