Biomembrane microparticles derived from plant petals

Abstract

The economic loss of discarded flowering plants (waste flowers) is a problem, and there is a need for effective utilization of petals. The purpose of this disclosure is to provide a method for producing biomembrane microparticles from plant petals, biomembrane microparticles obtained by this method, and raw materials for cosmetic compositions and cosmetic compositions containing said biomembrane microparticles. [Solution] A method for producing biomembrane microparticles derived from plant petals, comprising the steps of: (a) obtaining a petal extract from petals, and (b) purifying the petal extract to obtain biomembrane microparticles.

Description

Technical Field

[0001] The present disclosure relates to a method for producing biomembrane microparticles from plant petals, the biomembrane microparticles obtained by the method, a raw material for a cosmetic composition containing the biomembrane microparticles, and a cosmetic composition.

Background Art

[0002] Flowering plants have long been mainly produced for ornamental and decorative purposes. Alternatively, extracts of the petals of flowering plants are industrially used as dyes having unique colors or fragrances having unique scents, for example. However, the industrial use of plant petals is limited, and petals after being used for ornamental or decorative purposes and petals excluded as off-specification during the distribution process are also discarded without being effectively utilized.

[0003] It is known that various types of cells including animals and plants secrete various forms of vesicles (biomembrane microparticles) mainly composed of cell membrane components. It has been found that these vesicles contain bioactive substances such as cytokines and RNAs inside and can contribute to intercellular signal transduction in vivo, and research on their components and functions has been actively conducted (Non-Patent Documents 1 and 2).

[0004] In recent years, the application of such biomembrane microparticles in the medical or cosmetic field has been studied. For the production of biomembrane microparticles suitable for application to humans, there is a need in this field for a simpler production of biomembrane microparticles using inexpensive raw materials.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] The economic loss of discarded flowering plants (waste flowers) is a problem, and there is a need for effective utilization of petals. The purpose of this disclosure is to provide a method for producing biomembrane microparticles from plant petals, biomembrane microparticles obtained by this method, and raw materials for cosmetic compositions and cosmetic compositions containing said biomembrane microparticles. [Means for solving the problem]

[0007] This disclosure provides the following: [Section 1] The following steps: (a) A step of obtaining a petal extract from the petals, and (b) A step of purifying the petal extract to obtain biomembrane microparticles. A method for producing biomembrane microparticles derived from plant petals, including [the specified ingredient]. [Section 2] The method according to item 1, wherein step (a) is not the crushing of petals. [Section 3] The method according to claim 1, wherein step (a) includes cutting the stalk portion of the petal and collecting the exuded biological fluid. [Section 4] The method according to claim 1, wherein step (a) includes the step of vacuum impregnating the petals with a buffer solution. [Section 5] The method according to item 1, wherein in step (a), the petals are dried petals. [Section 6] The method according to item 1, wherein the purification in step (b) is performed by chromatography. [Section 7] The method according to item 1, wherein the petals are rose or bitter orange petals. [Section 8] The method according to claim 1, wherein the total amount of triglycerides, diglycerides, and ceramides in the biomembrane microparticles is at least 30% of the total amount of substance, or the total amount of ceramides in the biomembrane particles is at least 5% of the total amount of substance. [Section 9] A raw material for cosmetic compositions, including biomembrane microparticles manufactured by the method described in item 1. [Section 10] A cosmetic composition comprising biomembrane microparticles manufactured by the method described in item 1. [Effects of the Invention]

[0008] The method for producing biomembrane microparticles from plant petals in the present disclosure does not include the step of crushing tissues or cells, and thus provides a simpler and cheaper method for producing biomembrane microparticles that does not require multi-step purification and isolation operations or ultracentrifugation. In the method of the present disclosure, even wilted and dried petals that are discarded can be used, which is also industrially advantageous.

Brief Description of Drawings

[0009] [Figure 1] A graph summarizing the results of nanoparticle tracking analysis (NTA) for a sample containing biomembrane microparticles obtained from fresh rose flowers is shown. [Figure 2] A transmission electron microscope (TEM) image of biomembrane microparticles obtained from fresh rose flowers is shown. [Figure 3] For each fraction of a sample containing biomembrane microparticles obtained from fresh rose flowers, the number of microparticles by NTA analysis and the quantification results of the contained phospholipids are shown. [Figure 4] A pie chart showing the main components constituting the biomembrane microparticles obtained from fresh rose flowers by HPLC-LC / MS is shown. [Figure 5] A graph summarizing the results of NTA for a sample containing biomembrane microparticles obtained from dried rose petals is shown. [Figure 6] A graph summarizing the results of NTA for a sample containing biomembrane microparticles obtained from fresh bitter orange flowers is shown. [Figure 7] A TEM image of biomembrane microparticles obtained from fresh bitter orange flowers is shown.

Modes for Carrying Out the Invention

[0010] The present disclosure provides a method for producing biomembrane microparticles from plant petals, biomembrane microparticles derived from plant petals produced by the method, a raw material for a cosmetic composition containing the biomembrane microparticles, or a cosmetic composition.

[0011] In one embodiment, the present disclosure provides a method for producing biomembrane microparticles from petals, comprising the steps of (a) obtaining a petal extract from the petals, and (b) purifying the petal extract to obtain biomembrane microparticles.

[0012] In some embodiments, the method of the present disclosure does not involve the step of crushing the petals. In some embodiments, the step (a) of obtaining a petal extract from the petals is carried out by cutting at least the stalk portion of the petal and collecting the exuded biosap.

[0013] In some embodiments, the method described herein includes, in step (a), a step of vacuum impregnating the petals with water or a buffer solution. Vacuum impregnation (vitrification) is known in the art for improving the efficiency of extracting extracellular components of plants. By immersing a plant sample in any solution under reduced pressure, the solution can be widely penetrated into parts of the plant sample that are exposed to the outside air, such as inside tubes, allowing for efficient extraction of components outside the plant cell tissue. In some embodiments, the buffer solution includes, but is not limited to, PBS, phosphate buffer, acetate buffer, and citrate buffer.

[0014] For the collection of biological fluids, the petals may be gently centrifuged with the receptacle facing the direction of the load. Centrifugation can be performed, for example, at 500-3000g for about 1-30 minutes.

[0015] In some embodiments, the petals may be further cut one or more times in a direction perpendicular to the vein cross-section or at an angle of ±45° from the perpendicular.

[0016] The methods described herein do not include a step of crushing the petals. A step of crushing the petals means destroying most of the cells of the petals so that impurities, including cell walls and components within the nucleus, seep into the petal extract. Examples of steps of crushing the petals include grinding, blending, and pressing the petals. In some embodiments, the methods of the herein do not include a step of further cutting the petal fragment, which has been cut at the stalk portion, one or more times in a direction parallel to the vein cross-section or at an angle of within ±45° from the parallel direction.

[0017] By minimizing damage to the petal tissue, the inclusion of impurities in the petal extract is minimized. Because the extract contains virtually no impurities, the method described herein is advantageous in that it allows for the isolation of biomembrane microparticles more simply and inexpensively in the subsequent purification step, without the need for ultracentrifugation, which is commonly used in the art for the isolation of biomembrane microparticles.

[0018] In some embodiments, the step may include filtering the petal extract obtained prior to step (b).

[0019] In step (b), the biomembrane microparticles can be isolated and purified by any method known in the art. In some embodiments, step (b) includes size exclusion chromatography. In some embodiments, step (b) includes ultrafiltration. In some embodiments, step (b) includes size exclusion chromatography and ultrafiltration. In some embodiments, step (b) does not include ultracentrifugation or density gradient centrifugation.

[0020] In some embodiments, size exclusion chromatography is aqueous chromatography. In the methods of this disclosure, size exclusion chromatography is performed according to conventional methods in the art. The exclusion limits for size exclusion chromatography to obtain biomembrane nanoparticles may be approximately 5000 nm or less, for example, approximately 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 220, 200, 100, or 70 nm or less.

[0021] In some embodiments, for the purpose of separating biomembrane microparticles according to their size, step (b) may further include one or more size exclusion chromatography steps.

[0022] In some embodiments, the biomembrane microparticles obtained in step (b) may be subjected to further appropriate treatments, including but not limited to filtration using a pore-size filter, dialysis, dilution, concentration, solvent replacement, desalting, freezing, and drying, provided that the biomembrane microparticles are not destroyed.

[0023] In some embodiments, the petals are fresh, treated, or more commonly, dried. In some embodiments, the petals are dried. The drying process is carried out by any method including air drying and heat treatment. Dried, wilted petals are often discarded during distribution, but are advantageous in terms of weight, volume, and storability, and are therefore suitable as starting material in the methods of this disclosure.

[0024] In some embodiments, the petals are microwave-treated petals. Microwave treatment may be carried out by any method known in the art.

[0025] Any plant petals may be used in the methods of this disclosure. In some embodiments, the petals used in the methods of this disclosure include petals of plants belonging to the Rosaceae, Liliaceae, Asteraceae, or Rutaceae families. In some embodiments, the petals used in the methods of this disclosure include petals of roses, tulips, cherries, plums, peaches, apricots, apples, loquats, chrysanthemums, or oranges. In some embodiments, the petals used in the methods of this disclosure are rose petals or bitter orange petals.

[0026] In one embodiment, the present disclosure relates to biomembrane microparticles derived from plant petals produced by the method described herein. In the present disclosure, “biomembrane microparticles” means cell-derived microparticles composed of components of the same kind as those that make up cell membranes. The biomembrane microparticles of the present disclosure include, but are not limited to, various subtypes classified on the basis of size, origin or structure, such as exosomes, ectosomes, microvesicles, shedding vesicles, oncosomes, prostagosomes, lipid nanoparticles, solid lipid nanoparticles and nanostructured lipid carriers (NLCs). In some embodiments, the biomembrane microparticles of the present disclosure may be extracellular vesicles (EVs) secreted from cells.

[0027] The biomembrane microparticles of this disclosure may include particles of various sizes. The particle size of the biomembrane microparticles of this disclosure is approximately 30 to 5000 nm, for example, approximately 30 to approximately 1000 nm, approximately 70 to 500 nm, or approximately 70 to 200 nm.

[0028] The biomembrane microparticles of this disclosure are microparticles whose outer surface is covered by a single or double lipid membrane, and which may contain an aqueous solution and / or lipid components internally. In addition to the lipid components and membrane proteins that constitute the lipid membrane, the biomembrane microparticles of this disclosure may contain various physiologically active substances. These physiologically active substances may include, for example, various components in the apoplast or symplast, such as proteins such as cytokines, nucleic acid molecules such as DNA and RNA, aroma components, and antioxidant components.

[0029] The lipid membranes constituting the biomembrane microparticles of this disclosure are mainly composed of the same lipid components as those constituting the cell membranes of plant petal cells. Therefore, the types and proportions of lipid components constituting the biomembrane microparticles of this disclosure vary depending on the plant used. Examples of lipid components contained in the biomembrane microparticles of this disclosure include, but are not limited to, triglycerides, diglycerides, ceramides, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, triacylglycerol, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), lysophosphatidylserine, lysophosphatidylethanolamine, and lysophosphatidylcholine.

[0030] In some embodiments, the biomembrane microparticles of this disclosure have a high content of triglycerides, diglycerides, or ceramides. In some embodiments, the amount of triglycerides, diglycerides, or ceramides in the biomembrane particles is at least 1% of the total amount of substance, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% or more. In some embodiments, the amount of triglycerides, diglycerides, or ceramides in the biomembrane particles is at least 5% of the total amount of substance.

[0031] In some embodiments, the biomembrane microparticles of this disclosure have a high content of triglycerides, diglycerides, and ceramides. In some embodiments, the total amount of triglycerides, diglycerides, and ceramides in the biomembrane particles is at least 15% of the total amount of substance, for example, 20, 25, 30, 35, 40, 45, 50, 55, or 60% or more. In some embodiments, the total amount of triglycerides, diglycerides, and ceramides in the biomembrane particles is at least 30% of the total amount of substance.

[0032] In some embodiments, the biomembrane microparticles of this disclosure may contain antioxidants as bioactive substances. In this disclosure, the antioxidant may be any substance having antioxidant activity measured by the method described in the examples of this disclosure. The type and amount of antioxidants contained in the biomembrane microparticles of this disclosure may vary depending on the type of plant used. Antioxidants known in the art include polyphenols, including flavonoids and catechins; carotenoids, including carotenes and xanthophylls; organic compounds such as butylhydroxyanisole (BHA); and inorganic compounds such as sodium sulfite.

[0033] In some embodiments, the biomembrane microparticles of this disclosure exhibit antioxidant activity.

[0034] In some embodiments, the biomembrane microparticles of this disclosure exhibit membrane permeability.

[0035] In some embodiments, the biomembrane microparticles described herein may be derived from the petals of plants belonging to the Rosaceae, Liliaceae, Asteraceae, or Rutaceae families. In some embodiments, the biomembrane microparticles described herein may be derived from the petals of roses, tulips, cherries, plums, peaches, apricots, apples, loquats, chrysanthemums, or oranges. In some embodiments, the biomembrane microparticles described herein may be derived from rose petals or bitter orange petals.

[0036] In one embodiment, the present disclosure relates to a raw material for a cosmetic composition or a cosmetic composition comprising biomembrane microparticles of the present disclosure. In some embodiments, the compositions of the present disclosure are raw materials for a cosmetic composition or a cosmetic composition for anti-aging and skincare purposes, for example, for moisturizing, whitening and wrinkle improvement.

[0037] Generally, extracellular vesicles derived from plant cells are known to have lower immunogenicity and be less likely to induce inflammatory responses when applied to humans compared to those derived from animal cells. Furthermore, the biomembrane microparticles of the present invention exhibit antioxidant activity and membrane permeability, making them particularly advantageous for cosmetic applications applied to the skin.

[0038] The raw materials or compositions of this disclosure may include any carriers, additives, or excipients known in the art that are acceptable as cosmetic raw materials or cosmetics, provided that they do not destroy the biomembrane microparticles of this disclosure. In some embodiments, the biomembrane microparticles of this disclosure may include any carriers, additives, or excipients known in the art that are acceptable as cosmetic raw materials or cosmetics, which are suitable for enhancing their preservation or activity. [Examples]

[0039] Example 1: Biomembrane microparticles from rose petals • Isolation Approximately 100g of fresh rose petals were carefully removed from the receptacle to avoid damaging the tissue. The lower part of the petal was cut perpendicular to the vein to expose the main vein, and the petal fragments were immersed in phosphate buffer (pH = 7.2) and vacuum-impregnated. The sufficiently vitrified petal fragments were cut perpendicular to the vein cross-section and placed in a centrifuge tube with the receptacle side facing the load direction. The apoplast solution discharged from the vein and accumulated at the bottom of the centrifuge tube was collected by subsequent centrifugation (700xg, 5 min). The obtained apoplast solution was filtered using a 0.22 μm filter, and further, microparticles were eluted with phosphate buffer (pH = 7.2) using a pore size exclusion column with a pore size of 70 nm to obtain multiple fractions containing purified microparticles.

[0040] • Particle number analysis using particle tracking analysis (NTA), transmission electron microscopy (TEM) observation, and phospholipid quantification. The obtained fractional components were subjected to microparticle tracking analysis (NTA) using NanoSight NS300 (Malvern Panalytical) (Figure 1). Combining all fractions, the microparticles with a particle size of 100-200 nm were found to be up to 5.0 x 10⁻¹⁶. 9It was confirmed that approximately [number] particles were present. The obtained microparticles were stained with phosphomolybdic acid and observed using a transmission electron microscope (TEM, JEOL Ltd.), confirming the presence of spherical particles with a diameter of 100-200 nm (Figure 2). Lipids were extracted from the obtained fractional components using the Lipid Extraction Kit (Choroform Free) (Cell Biolabs), and the contained phospholipids were quantified by the choline oxidase (DAOS) method using a phospholipid measurement kit (Fujifilm Wako Pure Chemical Industries, Ltd.). The lipid content in each fraction and the increasing / decreasing trend of the number of microparticles obtained from NTA analysis were consistent, suggesting that the obtained microparticles are derived from lipid bilayers (Figure 3).

[0041] • Evaluation of antioxidant activity The antioxidant activity of the obtained microparticles was measured based on the reducing action of DPPH radicals using the DPPH Antioxidant Assay Kit (Dojin Chemical Laboratory) (Figure 4). Curcumin, TBHA, microparticles, microparticles + curcumin, and microparticles + TBHA were used as samples. Here, curcumin and TBHA are positive controls known to exhibit antioxidant activity. The DPPH radical scavenging rate for each sample is shown in Table 1 below, and the relative values ​​of the scavenging rates are shown in Table 2. Microparticles alone showed superior antioxidant activity compared to curcumin or TBHA (7.6 × 10⁻⁶ compared to curcumin). 4 Twice, TBHA 1.3 × 10 7 (Twice as much). Furthermore, combinations of microparticles with curcumin or TBHA showed superior antioxidant activity compared to each sample alone. [Table 1] [Table 2]

[0042] Lipidomics Lipid components in the obtained microparticles were measured by HPLC-LC / MS (Figure 4). HPLC was performed using an Imtakt Unison UK-18MF (UK022F) column (φ2mm × 50mm, particle size 3mm), with mobile phase (A) being 1:1:3 (v / v / v)ACN:MeOH:water, ammonium acetate (5mM), and EDTA (10nM), and mobile phase (B) being 100% IPA, ammonium acetate (5mM), and EDTA (10nM). Mass spectrometry was performed under the following conditions: CID: collision energy 30V, OAD: H2O flow rate 0.3 sccm / H2 flow rate 0.2 sccm.

[0043] • Evaluation of cell uptake efficiency Cells were cultured in a carbon dioxide atmosphere at 37°C until the number of cells per dish was approximately 1.0 x 10^4 to 1.0 x 10^6. Subsequently, microparticles stained with a suitable fluorescent dye were added to the cells. After a certain period of culture, the immobilized cells were observed using a confocal laser scanning fluorescence microscope. The results showed that the rose petal-derived biomembrane microparticles exhibited relatively high intracellular uptake behavior.

[0044] The method described herein allows for the isolation of microparticles derived from biological membranes, particularly exosomes, with a diameter of 100-200 nm, from fresh rose flowers. Advantageously, the method described herein does not involve crushing the petals, resulting in a simple purification process and eliminating the need for expensive experimental equipment such as ultracentrifuges. Furthermore, the plant-derived biological membrane microparticles described herein have shown excellent antioxidant properties and are readily taken up by cells, suggesting their usefulness as cosmetic ingredients, especially for skin whitening and anti-aging purposes.

[0045] Example 2: Isolation of biomembrane microparticles from dried petal samples and microwave-treated petal samples Furthermore, for the same weight of fresh flowers, microparticles were similarly isolated and purified from petal samples treated with microwave irradiation before vacuum impregnation, and from petal samples dried to 10-20% of their original weight. Nanoparticle tracking analysis (NTA) was performed on the obtained fractional components using NanoSight NS300 (Malvern Panalytical). For the dried petal samples, microparticles originating from biological membranes with a diameter of 100-200 nm were isolated, similar to the fresh flower samples (Figure 5). Relatively larger microparticles were recovered from the microwave-treated petal samples, which is thought to be due to the aggregation or fusion of microparticles.

[0046] Example 3: Biomembrane microparticles from bitter orange (neroli) • Isolation 224 g of fresh bitter orange petals were carefully removed from the receptacle to avoid damaging the tissue. The lower part of the petal was cut perpendicular to the vein to expose the main vein, and the petal fragments were immersed in phosphate buffer (pH = 7.2) and vacuum-impregnated. The sufficiently vitrified petal fragments were cut perpendicular to the vein cross-section and placed in a centrifuge tube with the receptacle side facing the load direction. The apoplast solution discharged from the vein and accumulated at the bottom of the centrifuge tube was collected by subsequent centrifugation (1400xg, 20 minutes). The obtained apoplast solution was filtered using 1.0 μm and 0.22 μm filters, and further, multiple fractions containing purified microparticles were obtained by eluting the microparticles with phosphate buffer (pH = 7.2) using a 70 nm pore size exclusion column. • Particle number analysis using particle tracking analysis (NTA), transmission electron microscopy (TEM) observation, and phospholipid quantification. Microparticle tracking analysis (NTA) was performed on the obtained fractional components. Combining all fractions, the maximum size of microparticles with a particle size of 100-200 nm was 5.0 x 10⁻¹⁶. 8 It was confirmed that approximately [number] particles were present (Figure 6). Furthermore, when the obtained microparticles were stained with phosphomolybdic acid and observed using a transmission electron microscope (TEM), the presence of spherical particles with a diameter of 100-200 nm was confirmed (Figure 7). [Industrial applicability]

[0047] This disclosure provides a method for producing biomembrane microparticles from plant petals, biomembrane microparticles obtained by this method, a raw material for a cosmetic composition containing biomembrane microparticles, and a cosmetic composition.

Claims

1. The following steps: (a) A step of obtaining a petal extract from the petals, and (b) A step of purifying the petal extract to obtain biomembrane microparticles. A method for producing biomembrane microparticles derived from plant petals, including [the specified ingredient].

2. The method according to claim 1, wherein step (a) does not involve crushing the petals.

3. The method according to claim 1, wherein step (a) includes cutting the stalk portion of the petal and collecting the exuded biological fluid.

4. The method according to claim 1, wherein step (a) includes a step of vacuum impregnating the petals with a buffer solution.

5. The method according to claim 1, wherein in step (a), the petals are dried petals.

6. The method according to claim 1, wherein the purification in step (b) is performed by chromatography.

7. The method according to claim 1, wherein the petals are rose or bitter orange petals.

8. The method according to claim 1, wherein the total amount of triglycerides, diglycerides, and ceramides in the biomembrane microparticles is at least 30% of the total amount of substance, or the total amount of ceramides in the biomembrane particles is at least 5% of the total amount of substance.

9. A raw material for a cosmetic composition, comprising biomembrane microparticles produced by the method described in claim 1.

10. A cosmetic composition comprising biomembrane microparticles produced by the method described in claim 1.

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