A plant nanovesicle composition and its preparation method and application

Abstract

The present invention provides a plant nanovesicle composition and a preparation method and application thereof, and relates to the fields of cell biology, molecular biology and drug development technology. The plant nanovesicle composition of the present invention includes Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles. The plant nanovesicle composition of the present invention can significantly improve the biological effects of Torreya grandis fruit, walnut and Platycladus orientalis leaf, promote the proliferation of dermal papilla cells of hair follicles, reduce the expression of inflammatory factor IL-6, scavenge DPPH free radicals, inhibit the proliferation of Propionibacterium acnes CCSM0331, inhibit the proliferation of Malassezia restricta CCSM1060, prolong the growth period of hair follicles, and prevent hair loss.

Description

Technical Field

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

[0002] Epidemiological surveys show that about 2% of the world's population suffers from hair loss diseases. Due to the obvious disfiguring characteristics of hair loss, patients are adversely affected both physically and mentally. According to data previously released by the National Health Commission, the number of people with hair loss in my country has exceeded 250 million, with one in every six people suffering from hair loss. According to a report released by the Chinese Academy of Social Sciences, 52% of people born after 2000 have begun to worry about hair loss.

[0003] Hair loss can be divided into two types, physiological hair loss and pathological hair loss. Hair follicles have their own unique life cycle: growth phase, regression phase, and resting phase. There are about 100,000 to 150,000 hair follicles on the scalp, and 10 to 15% are in the resting phase. The so-called physiological hair loss is the resting phase hair loss, and no more than 100 hairs are lost per day. There are many reasons for severe hair loss, including hormones, age, stress, and nutritional deficiencies.

[0004] Alopecia areata (AA) and androgenetic alopecia (AGA) are the most common types of hair loss in clinical practice. Although both of them have hair loss as the main clinical manifestation, their pathological mechanisms are different. Modern studies have shown that the onset of AA is mainly caused by the collapse of the immune immunity of hair follicles, which causes an inflammatory response, while AGA is caused by the miniaturization of hair follicles mediated by the increased sensitivity of hair follicles to androgens. AGA is considered to be a chronic disease caused by the combined action of genetic and environmental factors, and the scalp microecology is one of the factors. Studies have shown that the load of Malassezia is positively correlated with the incidence of AGA, especially Malassezia restricta and Malassezia globosa. Propionibacterium acnes can cause inflammation of hair follicles by releasing porphyrins to produce reactive oxygen species, which directly leads to hair loss. In addition to the direct hair loss effect, Propionibacterium acnes can also cause hyperkeratosis of the epidermis and excessive sebum secretion.

[0005] There are many treatments for hair loss, including drug therapy, low-energy laser irradiation, autologous platelet-rich plasma injection, hair transplantation, etc., but drug therapy has strong side effects, a long treatment process and unsatisfactory results. Therefore, there is a need for improved compositions or methods for treating hair loss.

[0006] Compendium of Materia Medica records that if you take three Torreya nuts, two walnuts, and one or two taels of Platycladus orientalis leaves, mash them and comb your hair, your hair will never fall out. Torreya grandis belongs to the Taxaceae family and is an evergreen, unique economic forest tree species. It is a tertiary relict plant, a gymnosperm, with a 1,300-year cultivation history. Torreya grandis is a natural longevity tree that can survive for more than a thousand years and continue to bear fruit. Torreya grandis is an important component of many classic prescriptions, with anti-inflammatory, antioxidant, microbial regulation, neurotrophic and other activities, and is used to prevent hair loss. Walnut plants contain a variety of bioactive ingredients such as xanthoquinones, ketones, phenolic acids and volatile oils, which have multiple activities such as antibacterial, antioxidant and anti-cancer. Platycladus orientalis leaves are the branches and leaves of Platycladus orientalis, a plant of the Cupressaceae family. They have been often used to treat hair loss since ancient times. Studies have shown that Platycladus orientalis leaves and their compound preparations have significant effects in preventing hair loss and promoting hair growth. Platycladus orientalis leaf extract significantly upregulated the transcription level of Wnt10bmRNA (P<0.001); downregulated the expression of TGF-β2mRNA in the back skin of mice, reducing it by 35.10%; it increased the levels of VEGF and IGF-1 in the back skin of mice, and reduced the content of TGF-β1 in the back skin of mice; it downregulated the transcription level of BAX mRNA. The Wnt / β-catenin signaling pathway is one of the important pathways for regulating the growth and development of hair follicles and inducing hair follicle regeneration. CN114306432B mentioned that the Platycladus orientalis leaf exosome composition can effectively promote the proliferation of hair follicle cells, balance the scalp biological flora, stimulate new hair growth, and thus improve the anti-hair loss and hair growth effect of the composition. However, the composition of plant extracts is complex and difficult to produce industrially, and the biologically active ingredients in plant extracts are difficult to be absorbed by human skin, so it is very necessary to purify the active ingredients in plant extracts.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a plant nanovesicle composition and a preparation method and application thereof. The plant nanovesicle composition of the present invention can promote hair growth, prevent hair loss, and improve the overall health of the scalp.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides a plant nanovesicle composition, comprising Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles.

[0011] Preferably, the particle number ratio of the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles is (1-3):(2-4):(4-10).

[0012] Preferably, the content of the Torreya grandis fruit nanovesicles is 1×10 9 ~1×10 11 Pieces / mL;

[0013] Preferably, the content of the walnut nanovesicles is 1×10 9 ~1×10 11 Pieces / mL;

[0014] Preferably, the content of the Platycladus orientalis nanovesicles is 1×10 9 ~1×10 11 Pieces / mL.

[0015] Preferably, the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles are all nanovesicles with a double-layer membrane vesicle structure;

[0016] Preferably, the particle sizes of the Torreya grandis berry nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles are independently 100 to 900 nm.

[0017] In a second aspect, the present invention provides a method for preparing the plant nanovesicle composition, the preparation method comprising the following steps:

[0018] (1) respectively breaking the wall of Torreya grandis fruit, walnut and Platycladus orientalis leaves to obtain Torreya grandis fruit juice, walnut juice and Platycladus orientalis leaf juice;

[0019] (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, walnut juice, and Platycladus orientalis leaf juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, walnut nanovesicles, and Platycladus orientalis leaf nanovesicles;

[0020] (3) The Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed to obtain the plant nanovesicle composition.

[0021] Preferably, in step (1), the Torreya grandis fruit includes Torreya grandis fruit aril and / or Torreya grandis fruit kernel.

[0022] Preferably, in step (1), the temperature of the cell wall breaking treatment is 2-6°C.

[0023] Preferably, in step (2), the specific steps of centrifugation are:

[0024] The Torreya grandis fruit juice, walnut juice and Platycladus orientalis leaf juice obtained in step (1) are centrifuged respectively, and the supernatants are collected to obtain Torreya grandis fruit supernatant, walnut juice supernatant and Platycladus orientalis leaf supernatant.

[0025] Preferably, the centrifugal force of the centrifugation is 2000-3000×g.

[0026] Preferably, the centrifugation temperature is 2-6°C.

[0027] Preferably, the centrifugation time is 20 to 40 minutes.

[0028] Preferably, in step (2), the filtration is performed using a Nan filter.

[0029] Preferably, the capsule filter is a Bricap C01 capsule filter.

[0030] Preferably, the pore size of the Nas filter is 0.2-0.45 μm, preferably 0.45 μm.

[0031] Preferably, the membrane area of ​​the Nas filter is 160 to 200 cm 2 , preferably 180cm 2 .

[0032] Preferably, in step (2), the chromatography is performed using a Capto Core 700 chromatography column.

[0033] Preferably, the diameter-to-height ratio of the chromatography column is 1:(8-12).

[0034] Preferably, the column volume of the chromatography column is 100-200 mL.

[0035] Preferably, during the chromatography process, the eluent used is sterile physiological saline.

[0036] Preferably, during the chromatography, the flow rate of the eluent is 180-360 cm / h.

[0037] Preferably, during the chromatography, the sample loading volume is 100-500% of the column volume.

[0038] Preferably, in step (2), the ultrafiltration concentration is performed using a Cobetter hollow fiber column.

[0039] Preferably, the molecular weight cut-off range of the ultrafiltration concentration is 100-300 KD.

[0040] Preferably, the flow rate of the raw water end of the ultrafiltration concentration is 100 to 300 mL / min.

[0041] Preferably, the transmembrane pressure difference at the reflux end of the ultrafiltration concentration is 1 to 5 psi.

[0042] 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:

[0043] (1) Products that extend the growth phase of hair follicles;

[0044] (2) Products that improve dandruff effects;

[0045] (3) Products with anti-inflammatory effects;

[0046] (4) Products that regulate the balance of dandruff scalp flora;

[0047] (5) Products that inhibit the growth of Malassezia restricta;

[0048] (6) Products that inhibit the growth of Propionibacterium acnes.

[0049] In a fourth aspect, the present invention provides a plant nanovesicle-based preparation, wherein the plant nanovesicle-based preparation comprises any one of the following forms:

[0050] (A) Nanovesicle suspension: The nanovesicle suspension comprises a solvent and the plant nanovesicle composition;

[0051] (B) Sustained-release nanovesicle complex: the sustained-release nanovesicle complex comprises a carrier and the plant nanovesicle composition;

[0052] (C) Preparations with nanovesicles as additives: The preparations with nanovesicles as additives include the plant nanovesicle composition.

[0053] Preferably, the solvent is selected from physiological saline and / or phosphate buffer.

[0054] Preferably, the carrier is selected from any one or a combination of at least two of silica nanoparticles, chitosan and its derivatives, polylactic acid-co-glycolic acid nanoparticles or hyaluronic acid microspheres.

[0055] Preferably, the dosage form of the preparation with nanovesicles as additives includes any one of drops, ointments, emulsions, films, spreads, gels, pastes, sprays, aerosols or patches, or a combination of at least two of them.

[0056] In a fifth aspect, the present invention provides a preparation containing an anti-hair loss active ingredient, wherein the preparation containing an anti-hair loss active ingredient comprises the anti-hair loss active ingredient and a carrier; wherein the carrier comprises the plant nanovesicle composition.

[0057] Preferably, the anti-hair loss active ingredient includes any one or a combination of at least two of minoxidil, biotin, caffeine, peptides, vitamin E, zinc, keratin or growth factors.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) Improving bioavailability and promoting the proliferation of hair follicle dermal papilla cells: The plant nanovesicle composition of the present invention can effectively encapsulate and protect the active ingredients, preventing them from being decomposed or inactivated prematurely on the skin surface. Due to their smaller size, the nanovesicles can more easily penetrate the stratum corneum and enter deep into the hair follicles, thereby improving the absorption rate of the active ingredients and promoting the proliferation of hair follicle dermal papilla cells.

[0060] (2) Naturalness and safety: Compared with synthetic materials, the plant nanovesicle compositions of the present invention as a source of nanocarriers are generally considered to be more gentle and human-friendly; and they often have good biocompatibility and low irritation, and are suitable for use on sensitive skin.

[0061] (3) Multifunctional properties: The three components of the plant nanovesicle composition of the present invention, Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles, will produce a synergistic effect, further enhancing the overall efficacy of the product, which can promote hair growth in multiple ways, prevent hair loss, and improve the overall health of the scalp.

[0062] (4) Environmentally friendly: Green nanotechnology produced using renewable resources is in line with the concept of sustainable development and helps reduce the impact on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0064] Figure 1 Transmission electron micrographs of Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles provided in Example 1.

[0065] Figure 2 The figure is a comparison chart of the results of each sample on prolonging the growth phase of isolated hair follicles.

[0066] Figure 3 The figure is a comparison chart of the test results of promoting the proliferation of human hair follicle dermal papilla cells of each sample.

[0067] Figure 4 The figure is a comparison chart of the anti-inflammatory effect test results of each sample.

[0068] Figure 5 This is a comparison chart of the results of the DPPH free radical scavenging test for each sample.

[0069] Figure 6This is a comparison chart of the effects of the plant nanovesicle composition provided in Example 1 on the proliferation of Propionibacterium acnes CCSM0331.

[0070] Figure 7 This is a comparison chart of the effects of the plant nanovesicle composition provided in Example 1 on the proliferation of Malassezia restricta CCSM1060. DETAILED DESCRIPTION

[0071] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0072] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0073] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0074] In a first aspect, the present invention provides a plant nanovesicle composition, comprising Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles.

[0075] It should be noted that plant nanovesicles are different from plant extracts. They are nano-scale lipid bilayer vesicles, including exosomes and microvesicles. As a mechanism of intercellular communication, exosomes / microvesicles carry a variety of bioactive substances including DNA, RNA, proteins, etc. into effector cells, effectively regulating the body's cell signal transduction, and exerting anti-inflammatory, anti-oxidation, and promoting the proliferation of effector cells.

[0076] In the present invention, the plant nanovesicle composition contains three types of vesicles, Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles, which carry multiple functional properties. The three cooperate with each other to produce a synergistic effect, further enhancing the overall efficacy of the product, and can preferably reduce the expression of inflammatory factor IL-6, scavenge DPPH free radicals, inhibit the proliferation of Propionibacterium acnes CCSM0331, inhibit the proliferation of Malassezia restricta CCSM1060, and prolong the growth period of hair follicles, thereby regulating the scalp microecology, regulating oil secretion, protecting the scalp, and preventing hair loss.

[0077] In the present invention, Torreya grandis and Platycladus orientalis leaves both have strong antioxidant and anti-inflammatory effects, and can jointly reduce scalp inflammation and protect the scalp from oxidative stress damage. At the same time, Platycladus orientalis leaves and walnuts can improve scalp blood circulation and increase blood supply to hair follicles, thereby providing more nutrition for hair. At the same time, the nutrients rich in walnuts can nourish the scalp, while the antioxidant and anti-inflammatory effects of Torreya grandis and Platycladus orientalis leaves can further maintain the health of the scalp. Therefore, when the three are used in combination, they will produce a synergistic effect, which can promote hair growth in all directions, prevent hair loss, and improve the overall health of the scalp.

[0078] As an optional embodiment, the particle number ratio of the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles is (1-3):(2-4):(4-10);

[0079] Here, “1-3” can be, for example, 1, 1.5, 2, 2.5, 3, etc.;

[0080] Here, “2-4” can be, for example, 2, 2.5, 3, 3.5, 4, etc.;

[0081] Here, “4-10” can be, for example, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc.

[0082] 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 / mL, 5×10 9 / mL, 6×10 9 / mL, 7×10 9 / mL, 8×10 9 / mL, 9×10 9 / mL, 1×1010 / 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.

[0083] As an optional embodiment, the content of the walnut 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.

[0084] As an optional embodiment, the content of the Platycladus orientalis leaf 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, 9×109 / 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.

[0085] As an optional embodiment, the Torreya grandis berry nanovesicle is a nanovesicle with a double-layer membrane vesicle structure.

[0086] As an optional embodiment, the walnut nanovesicle is a nanovesicle with a double-layer membrane vesicle structure.

[0087] As an optional embodiment, the Platycladus orientalis leaf nanovesicles are all nanovesicles with a double-layer membrane vesicle structure.

[0088] In the present invention, the properties of the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles are identified by electron microscopy, particle size analysis and other methods, and the results are consistent with the characteristics of nanovesicles, showing a double-layer membrane vesicle structure under a transmission electron microscope.

[0089] 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.

[0090] As an optional embodiment, the particle size of the walnut 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.

[0091] As an optional embodiment, the particle size of the Platycladus orientalis leaf nanovesicles 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.

[0092] In a second aspect, the present invention provides a method for preparing the plant nanovesicle composition, the preparation method comprising the following steps:

[0093] (1) respectively breaking the wall of Torreya grandis fruit, walnut and Platycladus orientalis leaves to obtain Torreya grandis fruit juice, walnut juice and Platycladus orientalis leaf juice;

[0094] (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, walnut juice, and Platycladus orientalis leaf juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, walnut nanovesicles, and Platycladus orientalis leaf nanovesicles;

[0095] (3) The Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed to obtain the plant nanovesicle composition.

[0096] As an optional embodiment, in step (1), the Torreya grandis fruit includes Torreya grandis fruit aril and / or Torreya grandis fruit kernel.

[0097] 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.

[0098] 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.

[0099] As an optional embodiment, in step (2), the specific steps of the centrifugation are:

[0100] The Torreya grandis fruit juice, walnut juice and Platycladus orientalis leaf juice are centrifuged respectively, and the supernatants are collected to obtain Torreya grandis fruit supernatant, walnut juice supernatant and Platycladus orientalis leaf supernatant.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] As an optional embodiment, in step (2), the filtration is performed using a Nascher filter.

[0105] As an optional embodiment, the capsule filter is a Bricap C01 capsule filter.

[0106] 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.

[0107] 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 .

[0108] 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.

[0109] As an optional embodiment, in step (2), the chromatography is performed using a Capto Core 700 chromatography column.

[0110] 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.

[0111] 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.

[0112] 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).

[0113] As an optional embodiment, during the chromatography process, the eluent used is sterile physiological saline.

[0114] 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.

[0115] As an optional embodiment, during the chromatography process, the loading volume is 100-500% of the column volume, for example, it can be 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, etc.

[0116] As an optional embodiment, in step (2), the ultrafiltration concentration is performed using a Cobetter hollow fiber column.

[0117] 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.

[0118] As an optional embodiment, in step (2), the ultrafiltration concentration uses a Cobetter hollow fiber column (model: HFELA01001530P, 100KD, 236cm 2 )conduct.

[0119] 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.

[0120] 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.

[0121] As an optional embodiment, after obtaining Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles, any one or a combination of at least two of the following post-processing steps are also included: filtration, sterilization, sample packaging, inspection, or long-term storage at -80°C.

[0122] 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:

[0123] (1) Products that extend the growth phase of hair follicles;

[0124] (2) Products that improve dandruff effects;

[0125] (3) Products with anti-inflammatory effects;

[0126] (4) Products that regulate the balance of dandruff scalp flora;

[0127] (5) Products that inhibit the growth of Malassezia restricta;

[0128] (6) Products that inhibit the growth of Propionibacterium acnes.

[0129] As an optional embodiment, the present invention provides a use of the plant nanovesicle composition as described in the first aspect in the preparation of cosmetics for preventing and alleviating hair loss.

[0130] In the present invention, a plant nanovesicle composition containing Torreya grandis fruit (aril or kernel) nanovesicles, walnut nanovesicles, and Platycladus orientalis leaf nanovesicles is used to alleviate hair loss. The mixed suspension of the three plant nanovesicles is introduced into the scalp by smearing or microneedle injection, and the alleviating effects of the three plant nanovesicles on hair loss are observed.

[0131] In a fourth aspect, the present invention provides a plant nanovesicle-based preparation, wherein the plant nanovesicle-based preparation comprises any one of the following forms:

[0132] (A) Nanovesicle suspension: The nanovesicle suspension comprises a solvent and the plant nanovesicle composition;

[0133] (B) Sustained-release nanovesicle complex: the sustained-release nanovesicle complex comprises a carrier and the plant nanovesicle composition;

[0134] (C) Preparations with nanovesicles as additives: The preparations with nanovesicles as additives include the plant nanovesicle composition.

[0135] It should be noted that in the above (A) nanovesicle suspension: the plant nanovesicle composition is dissolved in a solvent so that the plant nanovesicle composition exists in the solvent in the form of a suspending agent to obtain the nanovesicle suspension.

[0136] It should be noted that in the above (B) sustained-release nanovesicle complex: the plant nanovesicle composition is combined with a carrier to form a sustained-release nanovesicle complex.

[0137] It should be noted that in the above-mentioned (C) preparation with nanovesicles as additives: plant nanovesicles are additives with effective ingredients.

[0138] As an optional embodiment, in the above (A) nanovesicle suspension: the solvent is selected from physiological saline and / or phosphate buffer.

[0139] As an optional embodiment, in the above (A) nanovesicle suspension: the nanovesicle suspension can be used by directly applying, injecting or spraying on the tissue site.

[0140] As an optional embodiment, in the above (B) sustained-release nanovesicle complex: the carrier is selected from any one or a combination of at least two of silica nanoparticles, chitosan and its derivatives, polylactic acid-glycolic acid copolymer nanoparticles or hyaluronic acid microspheres.

[0141] As an optional embodiment, in the above (B) sustained-release nanovesicle complex: the sustained-release nanovesicle complex can be used in the form of implantation into a tissue injury site.

[0142] As an optional embodiment, in the above-mentioned (C) preparation with nanovesicles as additives: the dosage form of the preparation with nanovesicles as additives includes any one of drops, ointments, emulsions, films, smears, gels, pastes, sprays, aerosols or patches, or a combination of at least two of them.

[0143] As an optional embodiment, in the above-mentioned (C) preparation with nanovesicles as an additive: the preparation C can be a cosmetic in various forms, such as shampoo, conditioner, scalp stimulating foam or a combination thereof, which is topically applied to the scalp.

[0144] In a fifth aspect, the present invention provides a preparation containing an anti-hair loss active ingredient, wherein the preparation containing an anti-hair loss active ingredient comprises the anti-hair loss active ingredient and a carrier; wherein the carrier comprises the plant nanovesicle composition.

[0145] As an optional embodiment, the anti-hair loss active ingredient includes any one or a combination of at least two of minoxidil, biotin, caffeine, peptides (such as copper tripeptide-1), vitamin E, zinc, keratin or growth factors.

[0146] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0147] Example 1

[0148] This embodiment provides a plant nanovesicle composition, which is prepared by the following steps:

[0149] (1) The aril of Torreya grandis, the leaf of Platycladus orientalis, and the walnut are respectively squeezed out of juice by a homogenizer to obtain Torreya grandis fruit juice, walnut juice, and Platycladus orientalis leaf juice.

[0150] (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, walnut juice, and Platycladus orientalis leaf juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, walnut nanovesicles, and Platycladus orientalis leaf nanovesicles;

[0151] 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.

[0152] (3) The Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed at a particle number ratio of 3:2:5 to obtain the plant nanovesicle composition; the content of the Torreya grandis aril nanovesicles in the composition is 6.00×10 9 / mL, the content of the walnut nanovesicles is 4.00×10 9 / mL, the content of the Platycladus orientalis nanovesicles is 1.00×10 10 Pieces / mL.

[0153] Test Example 1

[0154] Extraction and identification of plant nanovesicles

[0155] Test samples: Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles provided in Example 1.

[0156] Test method: The vesicle structure was observed by transmission electron microscopy, and the particle sizes of Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles were statistically analyzed by particle size analysis.

[0157] The specific test results are as follows:

[0158] Figure 1 The transmission electron micrographs of the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles provided in Example 1 are shown. Figure 1 As shown in the figure, the three plant nanovesicles are double-layer membrane vesicle structures under a transmission electron microscope. The particle sizes of the three plant nanovesicles are in the range of 100 to 900 nm.

[0159] Example 2

[0160] This embodiment provides a plant nanovesicle composition, which is different from the embodiment 1 only in that, in step (3), the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed at a particle number ratio of 2:2:10 to obtain the plant nanovesicle composition; the content of the Torreya grandis fruit nanovesicles in the composition is 2.90×10 9 / mL, the content of the walnut nanovesicles is 2.90×10 9 / mL, the content of the Platycladus orientalis nanovesicles is 1.45×10 10 Pieces / mL.

[0161] Example 3

[0162] This embodiment provides a plant nanovesicle composition, which is different from the embodiment 1 only in that, in step (3), the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed in a particle number ratio of 1:2:4 to obtain the plant nanovesicle composition; the content of the Torreya grandis fruit nanovesicles in the composition is 2.86×10 9 / mL, the content of the walnut nanovesicles is 5.72×10 9 / mL, the content of the Platycladus orientalis nanovesicles was 1.14×10 10 Pieces / mL.

[0163] Example 4

[0164] This embodiment provides a plant nanovesicle composition, which is different from the embodiment 1 only in that, in step (3), the Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed in a ratio of 3:4:6 in terms of the number of particles to obtain the plant nanovesicle composition; the content of the Torreya grandis fruit nanovesicles in the composition is 4.62×10 9 / mL, the content of the walnut nanovesicles is 6.16×10 9 / mL, the content of the Platycladus orientalis nanovesicles is 9.24×10 9 Pieces / mL.

[0165] Comparative Example 1

[0166] This comparative example provides a plant composition, which comprises Torreya grandis fruit extract, walnut extract and Platycladus orientalis leaf extract in a mass ratio of 3:2:5;

[0167] The Torreya grandis fruit extract is prepared by the following steps: extracting juice from Torreya grandis aril through a homogenizer, centrifuging and filtering;

[0168] The walnut extract is prepared by the following steps: squeezing the walnuts through a homogenizer to extract juice, centrifuging and filtering;

[0169] The Platycladus orientalis leaf extract is prepared by the following steps: squeezing juice from the Platycladus orientalis leaves through a homogenizer, and centrifuging and filtering.

[0170] Comparative Example 2

[0171] This comparative example provides a plant composition, which comprises Torreya grandis fruit extract, walnut extract and Platycladus orientalis leaf extract in a mass ratio of 2:2:10;

[0172] The Torreya grandis fruit extract is prepared by the following steps: extracting juice from Torreya grandis aril through a homogenizer, centrifuging and filtering;

[0173] The walnut extract is prepared by the following steps: squeezing the walnuts through a homogenizer to extract juice, centrifuging and filtering;

[0174] The Platycladus orientalis leaf extract is prepared by the following steps: squeezing juice from the Platycladus orientalis leaves through a homogenizer, and centrifuging and filtering.

[0175] Comparative Example 3

[0176] This embodiment provides a plant nanovesicle composition, which is different from the embodiment 1 only in that the Torreya grandis fruit nanovesicle is no longer added, but the content of the walnut nanovesicle is increased to 5.80×10 9 / mL, the content of the Platycladus orientalis nanovesicles increased to 1.45×10 10 Pieces / mL.

[0177] Comparative Example 4

[0178] This embodiment provides a plant nanovesicle composition, which is different from the embodiment 1 only in that walnut nanovesicles are no longer added, but the content of Torreya grandis fruit nanovesicles is increased to 7.80×10 9 / mL, the content of the Platycladus orientalis nanovesicles increased to 1.30×10 10 Pieces / mL.

[0179] Comparative Example 5

[0180] This embodiment provides a plant nanovesicle composition, which is different from the embodiment 1 only in that the Platycladus orientalis leaf nanovesicle is no longer added, but the content of the Torreya grandis fruit nanovesicle is increased to 1.20×10 10 / mL, the content of the walnut nanovesicles increased to 8.00×10 9 Pieces / mL.

[0181] Test Example 1

[0182] Prolong the growth phase of isolated hair follicles

[0183] Test samples: the plant nanovesicle compositions provided in Examples 1 to 4, the plant compositions provided in Comparative Examples 1 to 2, and the plant nanovesicle compositions provided in Comparative Examples 3 to 5.

[0184] Test method: 10 μg / mL of each of the above test samples were cultured in hair follicle ex vivo organ culture for 5 days. Human hair follicles were taken from 3 donors, a total of 30 follicles, all of which were subjected to 3 independent experiments. Hair growth was evaluated by microscopic hair cycle staging.

[0185] The specific test results are shown in Table 1 and Figure 2 As shown:

[0186] Table 1

[0187]

[0188]

[0189] Figure 2 Note: *Compared with the blank control group, P<0.01; **Compared with the blank control group, P<0.0001; #Compared with Example 1, P<0.05; ##Compared with Example 1, P<0.001.

[0190] From table 1 above and below Figure 2 As shown, the nanovesicle composition and extraction composition treatments of Examples 1 to 4 and Comparative Examples 1, 2, and 5 all prolonged the growth period of hair follicles after 5 days of culture; in contrast, the treatment of the plant nanovesicle composition of the present invention can further significantly prolong the growth period of hair follicles.

[0191] Test Example 2

[0192] Promotes proliferation of human hair follicle dermal papilla cells

[0193] Test samples: the plant nanovesicle compositions provided in Examples 1 to 4, the plant compositions provided in Comparative Examples 1 to 2, and the plant nanovesicle compositions provided in Comparative Examples 3 to 5.

[0194] Test method: Human hair follicle dermal papilla cells (HFDPC, passage 5) were cultured using hair follicle dermal papilla cell growth medium (Sigma-Aldrich) and incubated at 37°C and 5% CO2. The proliferation experiment was divided into a baseline experiment and a stress experiment, and each experiment was divided into a blank control group and each test sample group with 10 μg / mL added. 300 nM cortisol was added to the stress experiment. HFDPCs were seeded in a 96-well plate at a density of 4000 cells / well, and the culture medium was replaced after 24 hours, and the culture medium was replaced with or without extracts or nanovesicles. The cells were cultured for a further 72 hours, and the cell viability was detected at 450 nm using a CCK-8 kit (Dojindo Laboratories, CK04).

[0195] The specific test results are shown in Table 2 and Figure 3 As shown:

[0196] Table 2

[0197]

[0198]

[0199] From table 2 above and below Figure 3 As shown, at a concentration of 10 μg / mL, the extract and the nanovesicles can promote the increase in HFDPC growth both at baseline and in the presence of the growth inhibitor cortisol, and the nanovesicles of the present invention promote the proliferation of HFDPC more significantly; among them, the 10 μg / mL group of the three nanovesicle compositions (3:2:5) of Torreya grandis aril, walnut, and Platycladus orientalis leaves of Example 1 promoted the proliferation rate of HFDPC by 133% at baseline, and promoted the proliferation of HFDPC more significantly.

[0200] Test Example 3

[0201] Anti-inflammatory effects

[0202] Test samples: the plant nanovesicle compositions provided in Examples 1 to 4, the plant compositions provided in Comparative Examples 1 to 2, and the plant nanovesicle compositions provided in Comparative Examples 3 to 5.

[0203] Test method:

[0204] (1) RAW264.7 cell viability assay:

[0205] RAW264.7 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum under the conditions of 5% CO2 and 37°C. When the cells grew to 80-90% confluence, they were digested with trypsin and plated at 8×10 5cells / mL were inoculated in 96-well plates. After the cells adhered to the wall for 24 hours, different concentrations of the samples to be tested were added to treat the cells for 24 hours respectively. At the same time, a high-glucose DMEM medium containing 10% fetal bovine serum was set up as a solvent control group. Three parallel wells were set up for each experimental group. After the cells in the wells were washed once with DPBS, the cell viability was detected at 450nm using a CCK-8 kit (Dojindo Laboratories, CK04). Relative cell activity % = (OD value of the sample group / OD value of the solvent control group) × 100%.

[0206] (2) LPS-induced IL-6 content in RAW264.7 cells:

[0207] The cell lines were cultured normally in high-glucose DMEM medium containing 10% fetal bovine serum under the conditions of 5% CO2 and 37°C. When the cells grew to 80-90% confluence, they were digested with trypsin and plated at 8×10 5 The cells were inoculated in a 96-well plate at a density of 10 cells / mL. After the cells adhered to the wall for 24 hours, the cells were treated with samples, and 3 parallel wells were set up for each group. After 24 hours of treatment, the supernatant was taken and the IL-6 inflammatory factor was detected using an IL-6 ELISA kit. IL-6 relative production % = (IL-6 content in the sample group / IL-6 content in the solvent control group) × 100%.

[0208] The specific test results are shown in Table 3 and Figure 4 As shown:

[0209] Table 3

[0210] sample IL-6 relative production% Blank control group 2 LPS 100 Example 1 29 Example 2 37 Example 3 53 Example 4 40 Comparative Example 1 81 Comparative Example 2 77 Comparative Example 3 75 Comparative Example 4 42 Comparative Example 5 72

[0211] According to the results of cell viability, compared with the control group, the extract composition and the nanovesicle composition did not show cytotoxicity when the addition amount was 1 to 40 μg / mL. And according to the results of cell viability, the concentration of 40 μg / mL of the extract composition and the nanovesicle composition was selected, and the LPS (1 μg / mL) induced cell inflammation experiment.

[0212] From Table 3 above and below Figure 4 As shown, normal cells produce a small amount of IL-6 cytokines, while after LPS treatment, cells secrete a large amount of inflammatory factor IL-6. After adding the extract composition and the nanovesicle composition, the expression of inflammatory factor IL-6 can be significantly reduced (P < 0.01). The inhibition rate of the extract composition of comparative example 2 on inflammatory factor IL-6 is 23%, and the inhibition rate of the nanovesicle composition of example 1 on inflammatory factor IL-6 is 71%. It can be seen that the nanovesicle composition can significantly reduce the expression of inflammatory cytokine IL-6 caused by LPS, showing a good anti-inflammatory effect.

[0213] Test Example 4

[0214] Antioxidant Effect

[0215] Test samples: the plant nanovesicle compositions provided in Examples 1 to 4, the plant compositions provided in Comparative Examples 1 to 2, and the plant nanovesicle compositions provided in Comparative Examples 3 to 5.

[0216] Test method:

[0217] DPPH free radical scavenging test: control group: take 1mL of 2×10 -4 mol / L DPPH solution, add water to make the final volume 4mL, after 30min in dark at room temperature, measure the absorbance at 517nm, record as a0; sample solution: take 1mL of DPPH solution and mix with 1mL of 20, 40, 60, 80 and 100μg / mL samples, add water to make the final volume 4mL, after 30min in dark at room temperature, measure the absorbance of nanovesicle composition or extract composition of different concentrations, record as a. The experiment uses Vc of the same mass concentration as the positive control, and each sample is repeated 3 times. DPPH free radical scavenging rate (%) = (a0-a) / a0×100%.

[0218] The specific test results are shown in Table 4 and below Figure 5 As shown:

[0219] Table 4

[0220]

[0221]

[0222] From table 4 above and below Figure 5 As shown, the scavenging ability of the nanovesicle composition and the extract composition on DPPH free radicals increases with the increase of mass concentration, especially the scavenging rate of the nanovesicle composition in Example 1 can reach up to 83±1.9% (P<0.001). The results show that the nanovesicle composition has a good antioxidant free radical scavenging ability, and the free radical scavenging effect is better than that of the extract composition.

[0223] Test Example 5

[0224] Effect of regulating scalp microecology on the proliferation of Propionibacterium acnes CCSM0331

[0225] Test samples: plant nanovesicle compositions provided in Example 1 at different concentrations.

[0226] Test method:

[0227] (1) Preparation of bacterial suspension of Propionibacterium acnes CCSM0331:

[0228] Take out the preserved Propionibacterium acnes strain milk powder freeze-dried tube from the -80℃ refrigerator, pick the milk powder with a disposable inoculation loop and put it on the TSA plate, streak it with the three-zone streak method, place it in a 37℃ constant temperature incubator for anaerobic culture for 48 to 60 hours to make a first-generation activation plate, pick a single colony from the colonies grown on the first-generation plate, streak it on the second-generation plate, place it in a 37℃ constant temperature incubator for anaerobic culture for 48 to 60 hours. Pick a single colony of Propionibacterium acnes that has been activated for two generations and inoculate it in a test tube containing 5mL of TSB liquid culture medium, place it in a 37℃ incubator, culture it for 48 to 60 hours, adjust the bacterial OD600 to about 0.3, and set it aside.

[0229] (2) The growth of the strain was determined by the half-dilution method:

[0230] Use a sterile 96-well plate to perform 5 half-dilution gradients, and set up 4 duplicate wells for each group; first, take 100 μL of the above samples with a concentration of 40 μg / mL and add them to the first well to mix evenly, then take 50 μL of the solution in the first well to add to the second well, add 50 μL of sterile TSB medium to mix thoroughly, and repeat the above operation to the fifth well, add 50 μL of sterile TSB medium to the fifth well, mix evenly, and then take 50 μL of liquid and discard it, then add 50 μL of Propionibacterium acnes suspension and 100 μL of sterile TSB medium to the first to sixth wells of the 96-well plate. Experimental group: 100 μL sterile TSB medium + 50 μL of each half-dilution sample + 50 μL 10 6 cfu / mL of Propionibacterium acnes suspension; blank group: 50 μL of each half-dilution sample + 150 μL of sterile TSB medium; growth control group: 50 μL 10 6 cfu / mL of Propionibacterium acnes suspension + 150μL sterile TSB medium; sterile control group: 200μL sterile TSB medium.

[0231] The specific addition amount of each group is shown in Table 5 below:

[0232] Table 5

[0233]

[0234] The 96-well plate was incubated anaerobically at 37°C for 48 h, and the OD600 value was measured using a microplate reader to calculate the inhibition rate. Inhibition rate (%) = 1-(OD value of the experimental group-OD value of the experimental blank group) / (OD value of the growth control well-OD value of the sterile control well) × 100%.

[0235] From the bottom Figure 6As shown, the three nanovesicle compositions (3:2:5) of Torreya grandis aril, walnut, and Platycladus orientalis leaves have inhibitory effects on Propionibacterium acnes CCSM0331 at multiple concentrations. At a concentration of 10 μg / mL, the growth inhibition rate of Propionibacterium acnes is about 50%, and at a maximum concentration of 40 μg / mL, the growth inhibition rate of Propionibacterium acnes can reach more than 90%, indicating that the nanovesicle composition can effectively inhibit the growth of Propionibacterium acnes.

[0236] Test Example 6

[0237] Effect of regulating scalp microecology on the proliferation of Malassezia restricta CCSM1060

[0238] Test samples: plant nanovesicle compositions provided in Example 1 at different concentrations.

[0239] Test method:

[0240] (1) Preparation of Malassezia bacterial suspension:

[0241] Take out the preserved restricted Malassezia CCSM1060 strain milk powder freeze-dried tube from the -80℃ refrigerator, pick the milk powder with a disposable inoculation loop and put it on the mDixon agar plate, streak it with the three-zone streak method, place it in a 32℃ constant temperature incubator and culture it for 72h as the first generation activation plate, pick a single colony from the colonies grown on the first generation plate, streak it on the second generation plate and place it in a 32℃ constant temperature incubator and culture it for 72h. Pick a single colony of the Malassezia that has been activated for two generations and inoculate it in a conical flask filled with 100mL of mDixon liquid culture medium, place it in a 32℃ shaker, shake and culture it at a speed of 150r / min for 72h, adjust the bacterial OD600 to the range of 0.7-0.8, and continue to dilute it 100 times for use.

[0242] (2) Use a sterile 96-well plate to perform 5 half-dilution gradients, with 4 replicate wells in each group; first, take 100 μL of each test sample with a concentration of 40 μg / mL and mix it with sterile double-concentration mDixon medium in a 1:1 ratio to obtain a composition with concentrations of 40, 20, 10, 5, and 2.5 μg / mL.

[0243] The specific addition amount of each group is shown in Table 6 below:

[0244] Table 6

[0245]

[0246] The 96-well plate was incubated at 32°C for 72 h, the OD600 value was measured using a microplate reader, and the inhibition rate was calculated.

[0247] Inhibition rate = 1-(OD value of the experimental group-OD value of the experimental blank group) / (OD value of the growth control well-OD value of the sterile control well) × 100%.

[0248] From top to bottom Figure 7 As shown, the nanovesicle composition exhibited an inhibitory effect on Malassezia restricta CCSM1060, and also had an inhibitory effect on the growth of Malassezia restricta CCSM1060 at a lower concentration (2.5 μg / mL). At a concentration of 40 μg / mL, it significantly inhibited the growth of Malassezia restricta CCSM1060, with an inhibition rate of 62.05%.

[0249] In summary, the plant nanovesicle composition of the present invention can well regulate the balance of scalp bacteria, reduce the abundance of Propionibacterium acnes and Malassezia restricta, thereby reducing follicular inflammation, epidermal hyperkeratosis, and excessive sebum secretion caused by the production of reactive oxygen species by porphyrin, and prevent hair loss.

[0250] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant nanovesicle composition, characterized in that: The plant nanovesicle composition comprises Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles.

2. The plant nanovesicle composition according to claim 1, characterized in that The particle number ratio of the Torreya grandis fruit nanovesicle, the walnut nanovesicle and the Platycladus orientalis leaf nanovesicle is (1-3):(2-4):(4-10); Preferably, the content of the Torreya grandis fruit nanovesicles is 1×10 9 ~1×10 11 Pieces / mL; Preferably, the content of the walnut nanovesicles is 1×10 9 ~1×10 11 Pieces / mL; Preferably, the content of the Platycladus orientalis nanovesicles is 1×10 9 ~1×10 11 Pieces / mL.

3. The plant nanovesicle composition according to claim 1, characterized in that The Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles are all nanovesicles with a double-layer membrane vesicle structure; Preferably, the particle sizes of the Torreya grandis berry nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles are independently 100 to 900 nm.

4. A method for preparing a plant nanovesicle composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) respectively breaking the wall of Torreya grandis fruit, walnut and Platycladus orientalis leaves to obtain Torreya grandis fruit juice, walnut juice and Platycladus orientalis leaf juice; (2) respectively centrifuging, filtering, chromatography, and ultrafiltration concentration the Torreya grandis fruit juice, walnut juice, and Platycladus orientalis leaf juice obtained in step (1) to obtain Torreya grandis fruit nanovesicles, walnut nanovesicles, and Platycladus orientalis leaf nanovesicles; (3) The Torreya grandis fruit nanovesicles, walnut nanovesicles and Platycladus orientalis leaf nanovesicles obtained in step (2) are mixed to obtain the plant nanovesicle composition.

5. The method for preparing the plant nanovesicle composition according to claim 4, 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.

6. The method for preparing the plant nanovesicle composition according to claim 4, characterized in that: In step (2), the specific steps of the centrifugation are: Centrifuging the Torreya grandis fruit juice, walnut juice and Platycladus orientalis leaf juice obtained in step (1) respectively, collecting the supernatants to obtain Torreya grandis fruit supernatant, walnut juice supernatant and Platycladus orientalis leaf 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 100 to 300 KD; Preferably, the flow rate at the inlet end of the ultrafiltration concentration is 100-300 mL / min; the transmembrane pressure difference at the reflux end of the ultrafiltration concentration is 1-5 psi.

7. Use of the plant nanovesicle composition according to any one of claims 1 to 3 in the preparation of any of the following products: (1) Products that extend the growth phase of hair follicles; (2) Products that improve dandruff effects; (3) Products with anti-inflammatory effects; (4) Products that regulate the balance of dandruff scalp flora; (5) Products that inhibit the growth of Malassezia restricta; (6) Products that inhibit the growth of Propionibacterium acnes.

8. A preparation based on plant nanovesicles, characterized in that: The plant nanovesicle-based preparation includes any one of the following forms: (A) A suspension of nanovesicles: The suspension of nanovesicles comprises a solvent and the plant nanovesicle composition according to any one of claims 1 to 3; (B) A complex of sustained-release nanovesicles: The complex of sustained-release nanovesicles comprises a carrier and the plant nanovesicle composition according to any one of claims 1 to 3; (C) Preparations with nanovesicles as additives: The preparations with nanovesicles as additives include the plant nanovesicle composition according to any one of claims 1 to 3.

9. The plant nanovesicle-based preparation according to claim 8, characterized in that The solvent is selected from physiological saline and / or phosphate buffer; Preferably, the carrier is selected from any one or a combination of at least two of silica nanoparticles, chitosan and its derivatives, polylactic acid-co-glycolic acid nanoparticles or hyaluronic acid microspheres; Preferably, the dosage form of the preparation with nanovesicles as additives includes any one of drops, ointments, emulsions, films, spreads, gels, pastes, sprays, aerosols or patches, or a combination of at least two of them.

10. A preparation containing an anti-hair loss active ingredient, characterized in that: The preparation loaded with anti-hair loss active ingredients comprises the anti-hair loss active ingredients and a carrier; wherein the carrier comprises the plant nanovesicle composition according to any one of claims 1 to 3; Preferably, the anti-hair loss active ingredient includes any one of minoxidil, biotin, caffeine, peptides, vitamin E, zinc, keratin or growth factors, or a combination of at least two thereof.

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