A multifunctional composition derived from bamboo leaves and its preparation method and application

Abstract

The present invention provides a multifunctional composition derived from bamboo leaves, belonging to the technical field of bamboo leaf resource development. The composition includes exosomes and nanofibers, both of which are derived from bamboo leaves, and the mass ratio of the exosomes to the nanofibers is 1: (1-100). All components of the composition are extracted from bamboo leaves, and have the functions of synergistically promoting cell proliferation, anti-oxidation and inhibiting bacterial growth. The composition is rich in ingredients, high in stability, and free of organic solvent residues. It also has the characteristics of high retention targeting and long half-life, and has great application transformation prospects. The present invention also provides a preparation method and application of a multifunctional composition derived from bamboo leaves.

Description

Technical Field

[0001] The invention belongs to the technical field of bamboo leaf resource development, and particularly relates to a multifunctional composition derived from bamboo leaves, a preparation method and application thereof. Background Art

[0002] Bamboo is one of the cash crops in agriculture and forestry, and the bamboo industry has become a globally recognized green industry with huge economic, ecological, cultural and medicinal value. However, in the comprehensive utilization of bamboo resources, the development of bamboo leaves has lagged behind and is basically in its primitive stage. According to data provided by growers, bamboo leaves are usually regarded as agricultural waste. Most fallen leaves become high-quality organic fertilizer after rotting, and some are used as green fodder for livestock in winter and spring (Journal of Natural Fibers, 2023, 20(1): 2178581.). In recent years, studies have reported that bamboo leaves contain a variety of active substances, including flavonoids, phenolic acids, polysaccharides, bamboo quinones, coenzyme Q10, etc., which have antibacterial, anti-inflammatory, antioxidant, cardiovascular disease and metabolic disease prevention, anti-diabetes and other pharmacological effects (J Ethnopharmacol. 2023 Apr 24;306:116166; Antioxidants (Basel). 2023 Jun 8;12(6):1239.; Nutrients. 2022 Feb 14;14(4):793.), which has attracted attention to the development and utilization of bamboo leaf resources. However, traditional plant extracts are mostly extracted by organic solvent extraction, which is easy to cause organic solvent residues, irritating or harmful to the human body; and the components of the obtained chemical extracts are easily destroyed, generally containing only a single or several active substances; the active substances are directly in contact with the air and are easily oxidized and deteriorated; chemical extracts usually cannot directly enter the cells to exert their effects.

[0003] Studies have shown that compared with chemical extracts, plant exosome extraction technology does not require other media and no impurities are introduced, so the safety of raw materials will be higher and the production cost will be lower. Plant exosomes are membrane-structured vesicles secreted by living cells. They are natural nanoparticles that are enriched with a large number of plant secondary metabolites, as well as a variety of active substances such as RNA, proteins, and lipids. Therefore, they can retain pharmacological activities similar to those of the original plants. Compared with single plant extracts, they have the characteristics of multi-component, multi-cross-scale, and multi-effect. They have significant advantages in cell uptake and functional synergy, and can be used as a new type of pharmacological substance (Pharmaceutics. 2022 Sep 8;14(9):1905.; J ExtracellVesicles. 2021 Feb;10(4):e12069.; Cell Host Microbe. 2018 Nov 14;24(5):637-652.e8.). In addition, the surface of plant exosomes is protected by a phospholipid bilayer, which is more stable. However, existing plant exosomes still have problems such as poor retention and targeting, short in vivo half-life, and difficulty in controlled release, which limits the practical application of plant exosomes. Summary of the invention

[0004] In order to solve the problems of single ingredients, poor stability and organic solvent residues in traditional bamboo leaf extracts, the present invention provides a multifunctional composition derived from bamboo leaves. All ingredients of the composition are extracted from bamboo leaves, and have the functions of synergistically promoting cell proliferation, anti-oxidation and inhibiting bacterial growth. The composition is rich in ingredients, high in stability and free of organic solvent residues. It also has the characteristics of high retention targeting and long half-life, and has great application transformation prospects.

[0005] The invention also provides a preparation method and application of a multifunctional composition derived from bamboo leaves.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a multifunctional composition derived from bamboo leaves. The composition comprises exosomes and nanofibers. Both the exosomes and the nanofibers are derived from bamboo leaves. The mass ratio of the exosomes to the nanofibers is 1: (1-100).

[0008] Furthermore, the molecular weight of the exosomes is 3 kD to 100 kD, and the particle size is 30 to 500 nm.

[0009] Furthermore, the nanofibers include at least one of cellulose nanofibers, cationic cellulose nanofibers, anionic cellulose nanofibers and cellulose nanocrystals.

[0010] Furthermore, the diameter of the cellulose nanofibers is 5 to 60 nm and the length is 200-1000 nm, and the diameter of the cellulose nanocrystals is 5 to 60 nm and the length is less than 100 nm;

[0011] The cationic cellulose nanofibers and the anionic cellulose nanofibers are both obtained by modifying the cellulose nanofibers.

[0012] Based on the same inventive concept, the present invention provides a multifunctional composition derived from bamboo leaves, comprising an exosome extract and nanofibers, wherein both the exosome extract and the nanofibers are derived from bamboo leaves, and the volume ratio of the exosome extract to the mass ratio of the nanofibers is (1-1000) mL:1 g.

[0013] Based on the same inventive concept, the present invention provides a method for preparing a multifunctional composition derived from bamboo leaves, the preparation method comprising:

[0014] The fresh bamboo leaf fragments are soaked in an osmotic buffer solution, and then the juice is extracted and centrifuged to obtain the supernatant and collect the sediment at the bottom layer;

[0015] The supernatant is first centrifuged at a centrifugal force of 2000-6000 xg for 40-80 min, and then centrifuged at a centrifugal force of 8000-12000 xg for 40-80 min, the supernatant is taken, and components with a molecular weight greater than 100 kD and less than 3 kD are removed to obtain an exosome extract;

[0016] The lower precipitate is sequentially subjected to alkali treatment and bleaching treatment, and then at least one of the following operations is performed to obtain nanofibers:

[0017] a. After the bleaching treatment is completed, mechanical treatment is performed to obtain cellulose nanofibers;

[0018] Wherein, the mechanical treatment includes any one of grinding, high-speed shearing, high-emulsification shearing, high-pressure homogenization and ultrasonic treatment;

[0019] b. After the bleaching treatment is completed, the cellulose is mechanically treated, and then chemically treated or biodegraded to obtain cationic cellulose nanofibers or anionic cellulose nanofibers;

[0020] Wherein, the chemical treatment includes any one of ultra-low acid hydrolysis, organic acid hydrolysis, quaternary ammonium salt cationization modification, TEMPO oxidation degradation, periodate oxidation and low eutectic solvent treatment, and the biodegradation treatment includes enzymatic hydrolysis or microbial fermentation;

[0021] c. After the bleaching treatment is completed, cellulose nanocrystals are prepared by any one of strong acid hydrolysis, strong oxidant oxidative degradation, ionic liquid treatment, organic acid hydrolysis, solid acid hydrolysis, subcritical hydrolysis and AVAP method; the nanofibers are evenly mixed with the exosome extract to obtain a multifunctional composition.

[0022] Based on the same inventive concept, the present invention provides an application of a multifunctional composition derived from bamboo leaves in the preparation of at least one of a drug for preventing and treating microbial infectious diseases, a drug for preventing and treating diseases related to oxidative damage caused by active oxygen and skin care and beauty products, wherein the drug for preventing and treating microbial infectious diseases comprises at least one of a viral infectious disease, a bacterial infectious disease and a chlamydia infectious disease;

[0023] In the drug for preventing and treating active oxygen and oxidative damage-related diseases caused by it, the active oxygen and oxidative damage-related diseases caused by it include at least one of neurodegenerative diseases, cancer, diabetes, eye diseases, mental illnesses, respiratory tract infections and rheumatoid arthritis.

[0024] Based on the same inventive concept, the present invention provides a drug with antibacterial and antioxidant functions, wherein the active ingredient of the drug comprises the above-mentioned multifunctional composition derived from bamboo leaves.

[0025] Furthermore, the dosage form of the drug includes any one of lyophilized powder, granules, microspheres, capsules, films, gels and sprays.

[0026] Based on the same inventive concept, the present invention also provides a beauty and skin care product with antibacterial and antioxidant functions, wherein the effective ingredients of the beauty and skin care product include the above-mentioned multifunctional composition derived from bamboo leaves.

[0027] Based on the same inventive concept, the present invention also provides an application of a multifunctional composition derived from bamboo leaves in the manufacture of medical devices with antibacterial and antioxidant functions.

[0028] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0029] 1. The present invention discloses a multifunctional composition derived from bamboo leaves, comprising exosomes and nanofibers, both of which are extracted from bamboo leaves. The composition has natural ingredients, good biocompatibility, is rich in active substances, has high stability, and is free of organic solvent residues. The bamboo leaf exosomes mainly play an antioxidant role, and the bamboo leaf nanofibers mainly play an antibacterial role. The combination of the two can not only solve the problems of poor retention targeting and short half-life of bamboo leaf exosomes, but also achieve synergistic effects in terms of anti-oxidation, inhibition of bacterial growth, and promotion of cell proliferation, and has great application transformation prospects.

[0030] 2. The present invention discloses a multifunctional composition derived from bamboo leaves, comprising exosomes and nanofibers derived from bamboo leaves. The extraction process is simple and environmentally friendly, the ingredients are natural, the cell compatibility is high, the biological activity is high, and the composition is beneficial to the adhesion, proliferation, growth and differentiation of cells and tissues. The composition can be used to prepare coatings for cell culture and tissue repair biomaterials. The present invention shows through experiments that the composition can significantly inhibit the growth of bacteria and has a strong DPPH free radical scavenging ability. Therefore, the composition can be prepared into preparations such as films, gels, sprays, etc., which can be used in the fields of medicines or functional cosmetics, medical devices, etc. The present invention is expected to become a new strategy for the development and utilization of bamboo leaf resources, which is of great significance to the development of bamboo leaf value-added products and the application of bamboo leaf exosome functional products in various fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 Suspensions of bamboo leaf exosomes, bamboo leaf nanofibers and their combination: (A) Particle size of BL-EVNs detected by NTA; (B) Morphology of different bamboo leaf fibers, scale bar = 100 nm; (C) Mixed suspension of BL-EVNs and different types of bamboo leaf nanocellulose.

[0033] Figure 2 Evaluation of the cytocompatibility of bamboo leaf exosomes: (A) Cytotoxicity of BL-EVNs; (B) Cytotoxicity of BL-EVNs (10 8 The proliferation rate of cells after incubation with 4% paraformaldehyde (Particles / mL) for 72 h.

[0034] Figure 3 Evaluation of the cell compatibility and cell proliferation of bamboo leaf nanocellulose film layers: (A) Image of the nanocellulose coating at the bottom of the well plate; cell adhesion (B) and proliferation (C) on CNF, a-CNF, b-CNF, and CNC nanofiber coatings.

[0035] Figure 4 To evaluate the ability of bamboo leaf compositions to promote cell proliferation.

[0036] Figure 5 Evaluation of the antioxidant activity of bamboo leaf compositions: DPPH radical scavenging rate of BL-EVNs (A), nanocellulose and its composition with BL-EVNs (B).

[0037] Figure 6Evaluation of the antibacterial activity of bamboo leaf compositions: Bacterial growth rate of Staphylococcus aureus in culture medium containing BL-EVNs (A), nanocellulose and its composition with BL-EVNs (B).

[0038] Figure 7 It is a preparation type of bamboo leaf composition. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0040] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0042] The overall idea of ​​the present invention is as follows:

[0043] A large number of studies have shown that when exosomes are fixed on scaffold materials, the advantages of exosomes can be maximized compared to using exosomes alone (Theranostics. 2023;13(12):4266-4286; Acta Biomater. 2023;171:68-84.; J Control Release. 2023;361:280-296.; J Control Release. 2024;368:355-371.). Therefore, in recent years, some biomaterials such as hydrogels, nanofibers, collagen scaffolds, keratin sponges, polymer-based scaffolds and inorganic materials have been used to load exosomes in a controlled release manner to prolong their retention time in the body and endow exosomes with retention and targeting ability in surface tissues (Int. J. Mol. Sci. 22 (24) (2021)13347.; Front. Bioeng. Biotechnol. 11 (2023) 1077825.). Among them, plant-derived nanofibers have attracted widespread attention due to their attractive properties, including low density (1.6 g / cm 3 ), high strength (2-3 GPa), high specific surface area (up to 800 m 2 / g), rich hydroxyl groups, strong water absorption, sustainability, biocompatibility and biodegradability. In addition, nanofibers can now be mass-produced in industrial operations. Since nanofibers can be used to prepare structure-controllable nanomaterials, they are suitable for various surface modification strategies for functionalization and can be easily prepared into various preparation types such as gels, films, capsules, sprays, etc., nanofibers have great application potential in biomaterials, food packaging and preservation technology, wastewater treatment, and new energy. For example, studies have shown that plant-derived nanocellulose, as a low-cost, naturally derived biomaterial, is a renewable alternative to traditional non-biodegradable petroleum-based polymers. It can not only maintain exosome function and sustained release, but also deliver exosomes directly to the disease site and increase the localization and persistence of exosomes in specific tissues or organs. It also has certain tissue repair properties, including promoting cell adhesion, proliferation, growth and differentiation, as well as angiogenesis and skin re-epithelialization (J Control Release. 2023;361:280-296.; Acta Biomater. 2023;171:68-84.). In addition, the exosome-loaded nanofibers can maintain their fiber structure for up to 12 weeks regardless of temperature, and the sterilization of nanofiber biomaterials can be achieved by simple, routine laboratory sterilization methods such as autoclaving and ultraviolet irradiation sterilization. Therefore, plant-derived nanofibers are expected to become an effective exosome formulation strategy and be widely used in various fields.

[0044] Based on this, the present invention uses bamboo leaf nanocellulose to load bamboo leaf exosomes. While extracting bamboo leaf exosomes, the generated bamboo leaf residues are recycled, and the bamboo leaf nanofibers extracted therefrom can be used to load bamboo leaf exosomes, thereby maximizing the utilization of bamboo leaf resources.

[0045] The multifunctional composition derived from bamboo leaves of the present invention includes bamboo leaf nanofibers and bamboo leaf exosomes (or bamboo leaf exosome extract). The development and utilization of bamboo leaf nanofibers of the present invention can turn bamboo leaves into "waste and treasure", and provide a new direction for further developing bamboo leaves into value-added products. Compared with the extraction of bamboo fiber, bamboo leaf fiber production has obvious advantages in energy consumption, time and economic cost. The nanofibers include at least one of cellulose nanofibers, cationic cellulose nanofibers, anionic cellulose nanofibers and cellulose nanocrystals. The cellulose nanofibers are slender fibers with a diameter of 5 to 60 nm and a length of 200-1000 nm, which are intertwined to form a network structure, and the cellulose nanocrystals are short rod-shaped fibers with a diameter of 5 to 60 nm and a length of <100 nm. The obtained bamboo leaf nanofibers can be applied to the fields of biomedicine, food packaging, preservation technology, materials and beauty, and specific uses include cell culture, nano drug carriers, wound dressings, degradable tableware, shopping bags, cling film, facial masks, mechanical reinforcement materials and 3D printing materials. Among them, surface-modified anionic or cationic cellulose nanofibers can be used in wastewater treatment and to capture or recover metal particles from solutions.

[0046] The present invention is a multifunctional composition derived from bamboo leaves. The molecular weight of bamboo leaf exosomes is 3 kD to 100 kD, the particle size is about 30 to 500 nm, and it has a lipid membrane structure. It is rich in nucleic acids, proteins and lipids. It has pharmacological activities similar to those of the original plants, including antibacterial, antioxidant, anti-inflammatory, anti-tumor, etc., which is better than a single chemical extract. It can be applied to the field of beauty and skin care, such as preparing moisturizing, antioxidant, antibacterial and anti-inflammatory masks, sprays, nanoemulsions and other products. The bamboo leaf exosome preparation process of the present invention can avoid the pollution and toxic side effects caused by organic reagents. In the composition prepared by the present invention, the combination strategy of bamboo leaf exosomes and bamboo leaf nanofibers can achieve synergistic synergy, and has significant effects in promoting cell proliferation, anti-oxidation, antibacterial and other aspects.

[0047] The bamboo leaf composition provided by the present invention also has good biodegradability, and its application in the food field can include degradable tableware, straws, food packaging materials, fresh-keeping sprays, fresh-keeping films, etc.

[0048] The multifunctional composition derived from bamboo leaves of the present application, its preparation method and application will be described in detail below in combination with embodiments and experimental data.

[0049] Example 1

[0050] The present embodiment is a suspension of a multifunctional composition derived from bamboo leaves, which is prepared by the following method:

[0051] 1. Extraction of bamboo leaf exosomes: Weigh 200 g of fresh bamboo leaves, cut them at the junction of the leaf blade and petiole with a scalpel, gently rinse the leaves in deionized water for 3 times, wipe the surface water with absorbent paper, cut them into 1-2 cm fragments and immediately immerse them in a glass bottle containing infiltration buffer (50 mM Tris-HCl pH 7.5, 0.6% NaC, 0.1% 2-mercaptoacetic acid in ethanol), and vacuum and soak for 30 min until the buffer fills the entire fragment tissue. Use a filter to remove the leaves, wipe the surface water with absorbent paper, squeeze the juice, collect the juice, centrifuge the juice, collect the supernatant, and centrifuge at 4000 xg for 60 min at 0-4°C, and then centrifuge at 10000 xg for 60 min. The supernatant was taken and first centrifuged at 4°C, 5000 x g for 30 min using a 100 kD ultrafiltration tube to remove components with a molecular weight greater than 100 kD in the supernatant. It was then transferred to a 3 kD ultrafiltration tube and centrifuged at 4°C, 5000 x g for 30 min to remove components with a molecular weight less than 3 kD. Finally, an extract containing components with a molecular weight of 3 kD-100 kD was obtained, which was the bamboo leaf exosome extract.

[0052] 2. Pretreatment of bamboo leaf residue: The bamboo leaf residue after collecting the supernatant in step 1 was subjected to wall breaking treatment, that is, the leaves were broken using a wall breaking machine, and the broken leaf tissue was dispersed in a beaker containing 1000 mL of distilled water at a ratio of 2 wt % and placed on a magnetic stirrer. The leaf tissue dispersion was washed at 80°C for 2 h with gentle stirring, the speed was set to 300 rpm, and then vacuum filtered using double-layer quantitative filter paper and dried in a 50°C oven to constant weight.

[0053] Then the bamboo leaf fibers were extracted by alkali treatment + bleaching:

[0054] 1) Prepare acetate buffer by adding 27 g NaOH and 75 mL acetic acid to 925 mL distilled water;

[0055] 2) Dissolve 21.25 g NaClO2 (80%) in 1000 mL water to prepare sodium chlorite solution;

[0056] 3) Place a 1:1:1 volume mixture of acetate buffer, sodium chlorite solution and distilled water into a 1000 mL glass beaker. Then add the crushed bamboo leaf tissue and disperse it in the treatment solution. Keep the reaction temperature at 90°C and stir gently for 2 h. Then vacuum filter, wash thoroughly with distilled water until neutral, and dry in a 50°C oven for later use.

[0057] 3. Mechanical preparation of bamboo leaf cellulose nanofibers (CNF): 5 g of bamboo leaf fibrils were dispersed in 500 mL of deionized water. First, they were fully sheared using a high-speed shear mixer at 25,000 rpm for 15 min, and then ultrasonicated at 100 W for 15 min to obtain nanocellulose (CNF) prepared by a simple mechanical method. The nanocellulose was dried at 60°C for later use.

[0058] 4. Preparation of bamboo leaf composition: 1 g of bamboo leaf cellulose nanofibers and 10 mL of bamboo leaf exosome extract were thoroughly mixed.

[0059] Example 2

[0060] The present embodiment is a suspension of a multifunctional composition derived from bamboo leaves, which is prepared by the following method:

[0061] 1. Preparation of bamboo leaf exosome extract: same as Example 1.

[0062] 2. Pretreatment of bamboo leaf residue: same as in Example 1.

[0063] 3. Mechanical method combined with cationic etherification agent to prepare bamboo leaf cationic cellulose nanofibers (a-CNF): CNF was prepared in the same manner as in Example 1. 5 g of ultrasonically treated CNF was mixed with 225 mL of isopropanol solution. After stirring at room temperature for 1 h, 2.5 mL of 10 mol / L NaOH solution was added dropwise. After stirring for 30 min, 17.8 mL of EPTMAC was added dropwise. Deionized water was added to make the total volume of the solvent 250 mL. After stirring and reacting at 60°C for 3 h, glacial acetic acid was added to neutralize to neutrality. The mixture was washed with anhydrous ethanol, filtered, and cationic nanofibers (a-CNF) were obtained. Dry at 60°C for use.

[0064] 4. Preparation of bamboo leaf composition: 1 g of bamboo leaf cationic cellulose nanofiber (a-CNF) was thoroughly mixed with 10 mL of bamboo leaf exosome extract.

[0065] Example 3

[0066] The present embodiment is a suspension of a multifunctional composition derived from bamboo leaves, which is prepared by the following method:

[0067] 1. Preparation of bamboo leaf exosome extract: same as Example 1.

[0068] 2. Pretreatment of bamboo leaf residue: same as in Example 1.

[0069] 3. Mechanical method combined with TEMPO oxidation method to prepare bamboo leaf anionic cellulose nanofibers (b-CNF): CNF was prepared as in Example 1, 50 mL of 1 wt% CNF suspension treated with ultrasound was taken; TEMPO (50 mg, 0.32 mmol) and sodium bromide (500 mg, 4.86 mmol) were dissolved in 50 mL of distilled water, and 0.5 mol / L NaOH solution was used to control the pH of the system to about 10, and then slowly added to the dispersed suspension of CNF under stirring at room temperature; 15 mL of 12wt% NaClO solution was slowly added to the mixed solution for oxidation reaction, and 0.5 mol / L NaOH solution was added at the same time to keep the pH of the reaction solution at 10.8 (pH meter detection). When the pH no longer decreased, 1 mL of anhydrous ethanol was added to terminate the reaction, and the pH was adjusted to 7 with 0.5 mol / L HCl. Finally, the oxidized nanocellulose (b-CNF) was thoroughly washed with distilled water for more than five times and dried at 60°C for use.

[0070] 4. Preparation of bamboo leaf composition: 1 g of bamboo leaf anionic cellulose nanofiber (b-CNF) and 10 mL of bamboo leaf exosome extract were thoroughly mixed.

[0071] Example 4

[0072] The present embodiment is a suspension of a multifunctional composition derived from bamboo leaves, which is prepared by the following method:

[0073] 1. Preparation of bamboo leaf exosome extract: same as Example 1.

[0074] 2. Pretreatment of bamboo leaf residue: same as in Example 1.

[0075] Preparation of bamboo cellulose nanocrystals CNC by 3.64% sulfuric acid hydrolysis method: 20 g of bamboo leaf fibrils were placed in a 500 mL glass beaker, and 64% H2SO4 was slowly poured into the beaker while stirring. The reaction was stirred at 45°C for 45 min. After the reaction was completed, it was transferred to a 5L glass beaker, and a large amount of deionized water was added and stirred evenly. The mixture was allowed to stand overnight, the supernatant was removed, and then deionized water was added for washing, 4400 rpm, 20 min, and centrifuged 3 times; finally, the CNC was transferred to a dialysis bag with a molecular weight of 3500 Da, and then dialyzed in deionized water for 3-7 days, ultrasonicated for 5 min, and dried at 60°C for use.

[0076] 4. Preparation of bamboo leaf composition: 1 g of bamboo leaf cellulose nanocrystals CNC was thoroughly mixed with 10 mL of bamboo leaf exosome extract.

[0077] Example 5

[0078] A composite suspension of this embodiment is prepared by the following method:

[0079] 1. Preparation of bamboo leaf exosome extract: same as Example 1.

[0080] 2. Mix 1 g of carboxymethyl cellulose (CMC) and 10 mL of bamboo leaf exosome extract. The raw material of carboxymethyl cellulose is wood.

[0081] Example 6

[0082] A composite suspension of this embodiment is prepared by the following method:

[0083] 1. Preparation of bamboo leaf exosome extract: same as Example 1

[0084] 2. Mix 1 g of microcrystalline cellulose (MCC) and 10 mL of bamboo leaf exosome extract. The raw material of microcrystalline cellulose is rice husk.

[0085] Example 7

[0086] In this example, bamboo leaf exosomes and bamboo leaf nanofibers are characterized.

[0087] 1. Characterization of bamboo leaf exosomes: The bamboo leaf exosome extract was diluted 10 times with 1× PBS solution, and the particle size and concentration of bamboo leaf exosomes were determined using nanoparticle tracking analyzer (NTA), and the Zeta potential of bamboo leaf exosomes was determined using dynamic laser light scattering (DLS).

[0088] The results are as follows Figure 1 As shown in A, the average particle size of BL-EVNs (bamboo leaf exosomes) was 132.2 ± 7.5 nm, and the particle concentration was 2.6×10 10 particles / mL.

[0089] 2. Characterization of bamboo leaf nanocellulose: Take 0.1 g of nanocellulose (CNF, a-CNF, b-CNF, CNC) in the above examples respectively, disperse it in 10 mL of deionized water to prepare a 1 wt% nanocellulose suspension, pipette 20 μL and drop it on a glass slide, cover it with a cover slip, observe under an optical microscope and take pictures.

[0090] The results are as follows Figure 1 As shown in Figure B, cellulose nanofibers CNF, a-CNF, and b-CNF are all intertwined slender fibers with a diameter of 10-20 nm and a length of 200-1000 nm; cellulose nanocrystals CNC are short rods with a diameter of 5-10 nm and a length of 10-60 nm.

[0091] Preparation of bamboo leaf composite suspension: Accurately weigh 0.2 g of the four nanocelluloses and mix them thoroughly with 2 mL of bamboo leaf exosome extract. Figure 1 As shown in C.

[0092] Example 8

[0093] In this example, various properties of bamboo leaf exosomes, bamboo leaf nanofibers and a combination of the two were tested.

[0094] 1. Cell experiments:

[0095] Test the cytocompatibility of bamboo leaf exosomes: L929 cells in logarithmic growth phase were taken and 5×10 4 The cells were inoculated in a 96-well plate and cultured at 37°C for 12 h. The culture supernatant was carefully removed and the wells were washed twice with PBS. 9 , 10 8 , 10 7 , 10 6 200 μL of 1640 medium containing 500 μg / mL (100 μg / mL) was added to each well and incubated at 37 °C for 24 h. The supernatant of the culture medium was carefully removed, the wells were washed twice with PBS, and 100 μL of serum-free medium containing CCK8 (5 mg / mL) was added to each well. The wells were incubated at 37 °C for another 4 h, and then the absorbance was measured by enzyme labeling at a wavelength of 450 nm.

[0096] The results are as follows Figure 2 As shown in A and B, when the concentration of BL-EVNs is equal to or less than 10 8 particles / mL, the cell survival rate was about 100%, and there was no cytotoxicity ( Figure 2 A), and compared with TC plates, it can significantly promote cell proliferation within 72 h ( Figure 2 B).

[0097] 2. Cell adhesion and proliferation test on bamboo leaf nanocellulose coating:

[0098] 5×10 4L929 cells were seeded in 96-well plates at a cell density of 100 / well. The experimental groups were as follows: 1) Control group: untreated blank well plate; 2) CNF group: CNF film layer with simple mechanical treatment; 3) a-CNF group: cationized CNF film layer; 4) b-CNF group: anionized CNF film layer; 5) CNC group: cellulose nanocrystal film layer; the blank film layer was used as the negative control for each film layer, n = 3. After 12 h of co-culture, the medium in the well plate was carefully aspirated and washed with 100 μL 1× PBS, and repeated twice. 100 μL serum-free medium containing CCK8 (5 mg / mL) was added to each well, and incubated at 37 °C for 4 h, and then the absorbance was measured by enzyme labeling at a wavelength of 450 nm. For cell proliferation, the medium in the well plate was carefully aspirated and washed twice with 1× PBS at 24, 48, and 72 h, and the cells were detected by CCK8 method as above.

[0099] The results are as follows Figure 3 As shown, nanocellulose forms a dense thin coating on the bottom of the well plate ( Figure 3 A); 12 h after cell seeding, the adhesion rates on CNF, a-CNF, b-CNF and CNC coating were 43.22% ± 4.25%, 84.19% ± 2.09%, 81.24% ± 5.59%, 62.78% ± 5.99%, respectively ( Figure 3 B); 72 h later, the cell proliferation rates were 55.64% ± 0.77%, 56.71% ± 0.71%, 67.98% ± 0.14%, and 43.11% ± 2.23%, respectively, which were significantly higher than those in the control group (39.06% ± 0.45%) ( Figure 3 C). These results show that among the four nanofiber coatings, a-CNF and b-CNF are more conducive to cell adhesion, and b-CNF has the strongest ability to promote cell proliferation.

[0100] 3. Cell adhesion and proliferation test on the bamboo leaf composite film layer:

[0101] Cells were dispersed in a medium containing bamboo leaf exosomes (10 8 After adding 5×10 4 L929 cells were seeded in a 96-well plate at a cell density of cells / well. The experimental grouping and subsequent experimental operations were the same as step 2, and compared with carboxymethyl cellulose and microcrystalline cellulose on the market, that is, 6) CMC group: carboxymethyl cellulose film layer; 7) MCC group: microcrystalline cellulose film layer.

[0102] The results are as follows Figure 4As shown, the bamboo leaf composition has a significantly better effect on promoting cell proliferation than a single bamboo leaf exosome or bamboo leaf nanocellulose component, and among them, the combination of bamboo leaf exosomes and anionized cellulose nanofibers (b-CNF) has the best effect.

[0103] 4. Antioxidant test:

[0104] DPPH free radical scavenging experiment: The antioxidant activity of bamboo leaf composites and CMC, MCC + bamboo leaf exosome composites was evaluated by microplate method. Weigh 0.05 g of 6 cellulose samples, add 0.8 mL nitrogen free radical extract, soak in 40℃ water bath for 60 min, centrifuge at 10000 rpm for 10 min, take the supernatant, and store at 4℃ for later use. BL-EVNs (10 10 particles / mL) was diluted to 10 9 , 10 8 , 10 7 After adding 100 μL of BL-EVNs to 900 μL of nitrogen radical extract, mix them in a ratio of 1:9, that is, add 100 μL of BL-EVNs to 900 μL of nitrogen radical extract, shake and mix, centrifuge at 10000 rpm for 10 min, take the supernatant, and store at 4°C for later use. For bamboo leaf composites, mix the nitrogen radical extract of nanocellulose with the nitrogen radical extract of BL-EVNs in a ratio of 1:1. Prepare vitamin C solution: fully dissolve a 10 mg vitamin C tube in 1 ml of nitrogen radical extract to form a 10 mg / ml vitamin C solution; it can be divided into 0.1 ml portions and stored at -20°C. Prepare Vc standard working solution: dilute 10 mg / ml vitamin C solution to 50, 40, 30, 20, 10, 5 μg / ml Vc standard working solution with nitrogen free radical extract to prepare standard curve; preheat the microplate reader for more than 30 minutes, detect wavelength at 517nm, and adjust to zero with anhydrous ethanol. DPPH addition: set blank tube, sample test tube, sample control tube, and positive control tube according to the table below. The solutions should be added in order, mixed, and left to stand at room temperature and away from light for 30 minutes.

[0105] Table 1 DPPH nitrogen free radical scavenging experiment

[0106] Added component (mL) Blank tube Sample test tube Sample control tube Positive control tube (standard tube) Nitrogen free radical extract 0.05 - - - Supernatant - 0.05 0.05 - Vc standard working solution - - - 0.05 Anhydrous ethanol 0.225 0.225 0.45 0.225 DPPH solution 0.225 0.225 - 0.225

[0107] OD value determination: Take a 96-well plate, pipette 300ul of the solution from each tube into the 96-well plate in turn, and use an ELISA reader to detect the absorbance value of each tube, which are recorded as A0, A1, A2, and A3 in turn.

[0108] Positive control·DPPH clearance rate (%) = (A0- A3) / A0×100%

[0109] Test sample·DPPH removal rate (%) = [A0-(A1-A2)] / A0×100%

[0110] Note: A0 = absorbance value of blank tube

[0111] A1 = absorbance value of the sample measuring tube

[0112] A2 = absorbance value of sample control tube

[0113] A3 = absorbance value of the positive control tube.

[0114] The results are as follows Figure 5 As shown, BL-EVNs (10 10 particles / mL) had strong antioxidant capacity, and the DPPH nitrogen free radical scavenging ability of BL-EVNs was concentration-dependent. The nitrogen free radical scavenging abilities of CNF, a-CNF, b-CNF, and CNC were 10.51% ± 0.87%, 15.88% ± 1.85%, 12.47% ± 0.21%, and 24.35% ± 0.46%, respectively. BL-EVNs were the main antioxidants in the bamboo leaf combination, and the addition of nanocellulose a-CNF, b-CNF, and cellulose nanocrystals CNC could significantly enhance the DPPH nitrogen free radical scavenging ability of BL-EVNs.

[0115] 5. Antibacterial test:

[0116] Preparation of Staphylococcus aureus solution: Preserve Staphylococcus aureus ( S. aureus ) Take out the strain and melt it quickly in a 37℃ water bath, then use an inoculation loop to dip an appropriate amount of bacterial solution and streak it. After inverting and culturing in a 37℃ constant temperature incubator for 24 hours, pick a single colony and put it in 10 mL of liquid culture medium. After fully mixing, place it in a constant temperature shaking incubator with the parameters set to 37℃, 100 rpm, and culture for 16 hours. Then use a UV-visible spectrophotometer to detect OD 600 = 2.5, and the concentration of Staphylococcus aureus in the culture medium solution was calculated to be 5×10 9 CFU / mL, diluted to 1×10 7 CFU / mL is reserved.

[0117] The experimental groups were 1) 1× PBS solution; 2) penicillin-streptomycin solution as positive control group; 3) bamboo leaf exosome solution (10 10particles / mL); 4) four 1 wt% nanocellulose suspensions (CNF, a-CNF, b-CNF, CNC); 5) bamboo leaf exosome solution (10 10 particles / mL) and four 1 wt% nanocellulose suspensions were mixed in a 1:1 volume ratio; 6) bamboo leaf exosome solution (10 10 The solution of bamboo leaf exosome solution (10 10 A solution of 1000 wt% microcrystalline cellulose (MCC) suspension mixed with 1 wt% microcrystalline cellulose (MCC) suspension in a volume ratio of 1:1 was used as control group 2. 1 mL of the above solution was pipetted and added into 2 mL of 100 wt% S. aureus concentration. 7 CFU / mL liquid culture medium, mix thoroughly and place in a constant temperature shaking incubator, set the parameters to 37°C, 100 rpm, and culture overnight. Use a pipette to draw 300 uL into a 96-well plate and use an enzyme reader to detect the OD of each sample. 600 The absorbance value at 100 μm was used to quantitatively evaluate the antibacterial activity of different groups. The bacterial growth rate was calculated as follows:

[0118] Bacterial growth rate (%) = (A2-A1) / A2×100%

[0119] A1 is the initial bacterial solution at OD 600 The absorbance value at

[0120] A2 is the OD of the bacterial solution to be tested after overnight culture. 600 The absorbance value at .

[0121] The results are as follows Figure 6 As shown, the antibacterial effect is mainly exerted by bamboo leaf cellulose nanofibers. When bamboo leaf exosomes are combined with bamboo leaf nanocellulose, the antibacterial ability is significantly enhanced and is significantly better than that of the CMC group and the MCC group.

[0122] 6. Preparation of bamboo leaf composition preparation:

[0123] Preparation of composite film: Accurately weigh 2 g of dry a-CNF nanocellulose sample and prepare a 1 wt% suspension. Place the small dish filled with nanofiber suspension in a 60 ℃ constant temperature drying oven and dry overnight to obtain a film sample. At this time, pour 75% ethanol into the small dish and soak it for 30 min, so that the film can be carefully lifted from the bottom of the small dish with tweezers. After the ethanol evaporates, seal the dried film and store it at -20 ℃ for later use. Cut the dried film into 4 cm 2 After the size is soaked in bamboo leaf exosome solution (10 10particles / mL) for 12 h and then taken out to obtain the nanofiber film loaded with bamboo leaf exosomes. Figure 7 As shown in A.

[0124] Preparation of composite gel: First, the nanocellulose gel was prepared by repeated freeze-thaw method. Weigh 5 g of b-CNF nanocellulose and disperse it in 10 mL of deionized water, stir evenly to form a colloidal solution. The colloidal solution was frozen at -20 °C for 2 h, taken out, and melted at room temperature. After repeated freeze-thaw 5 times, the nanocellulose hydrogel was formed and freeze-dried. 1 g of nanocellulose hydrogel freeze-dried product can be used with 10 mL of bamboo leaf exosome solution (10 10 particles / mL) and stirred to prepare a composite hydrogel. Figure 7 As shown in B.

[0125] Preparation of the composite spray: bamboo leaf exosomes and bamboo leaf nanocellulose CNC are mixed at a ratio of 1000 mL:1 g, fully shaken and mixed, and then ultrasonicated at 100 W for 5 min. The spray can be evenly sprayed from the spray bottle to form a uniform water film on the skin surface. Figure 7 C. According to the test, each gram of nanocellulose can absorb about 10 9 Bamboo leaf exosome particles.

[0126] The bamboo leaf composition of the present invention can be directly prepared into various preparations, including films, gels and sprays, so that the bamboo leaf composition can be applied to different disease sites. In addition to the medical field, it can also be used in food packaging, preservation technology, skin care products and other fields.

[0127] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0128] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0129] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a multifunctional composition derived from bamboo leaves, characterized in that: The preparation method comprises: The fresh bamboo leaf fragments are soaked in an osmotic buffer solution, and then the juice is extracted and centrifuged to obtain the supernatant and collect the sediment at the bottom layer; The supernatant is first centrifuged at a centrifugal force of 2000-6000 x g for 40-80 min, and then centrifuged at a centrifugal force of 8000-12000 x g for 40-80 min, the supernatant is taken, and components with a molecular weight greater than 100 kD and less than 3 kD are removed to obtain an exosome extract; The lower precipitate is sequentially subjected to an alkali treatment and a bleaching treatment, and then subjected to at least one of the following operations to obtain nanofibers, wherein the nanofibers include at least one of cellulose nanofibers, cationic cellulose nanofibers, anionic cellulose nanofibers, and cellulose nanocrystals: a. After the bleaching treatment is completed, mechanical treatment is performed to obtain cellulose nanofibers; b. After the bleaching treatment is completed, the cellulose is mechanically treated, and then chemically treated or biodegraded to obtain cationic cellulose nanofibers or anionic cellulose nanofibers; c. After the bleaching treatment is completed, cellulose nanocrystals are prepared by any one of strong acid hydrolysis, strong oxidant oxidative degradation, ionic liquid treatment, organic acid hydrolysis, solid acid hydrolysis, subcritical hydrolysis and AVAP method; The nanofibers and the exosome extract are evenly mixed to obtain a multifunctional composition.

2. A multifunctional composition derived from bamboo leaves, characterized in that: The composition is prepared by the preparation method of a multifunctional composition derived from bamboo leaves according to claim 1, the volume ratio of the exosome extract and the mass ratio of the nanofibers is (1-1000) mL:1 g, the molecular weight of the exosomes is 3kD-100kD, and the particle size is 30-500nm.

3. The multifunctional composition derived from bamboo leaves according to claim 2, characterized in that: The diameter of the cellulose nanofiber is 5-60nm, and the length is 200-1000nm; the diameter of the cellulose nanocrystal is 5-60nm, and the length is less than 100nm.

4. Use of a multifunctional composition derived from bamboo leaves as claimed in claim 2 or 3 in the preparation of a drug for preventing and treating Staphylococcus aureus infectious diseases.

5. A drug having anti-Staphylococcus aureus and anti-oxidation functions, characterized in that: The active ingredient of the medicine comprises the multifunctional composition derived from bamboo leaves as claimed in claim 2 or 3.

6. The drug having anti-Staphylococcus aureus and anti-oxidation functions according to claim 5, characterized in that: The dosage form of the drug includes any one of lyophilized powder, granules, microspheres, capsules, films, gels and sprays.

7. A beauty and skin care product with anti-Staphylococcus aureus and anti-oxidation functions, characterized in that: The effective ingredients of the beauty and skin care product include the multifunctional composition derived from bamboo leaves as claimed in claim 2 or 3.

8. Use of a multifunctional composition derived from bamboo leaves as claimed in claim 2 or 3 in manufacturing medical devices with anti-Staphylococcus aureus and anti-oxidation functions.

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