TW202143948A - Microspherulite composition composed of orchid germ extract, and its manufacturing method and use for cell detoxification, energy activation, and aging resistance - Google Patents

Abstract

A microspherulite composition composed of orchid germ extract includes a carrier formed in a shell, and an orchid germ extract embedded or enclosed in the shell. The orchid germ extract is manufactured by the method comprising the steps of: taking germ tissues from growth point of an orchid’s apical bud or an orchid’s lateral bud; removing an outer membrane of the germ tissues; grinding the germ tissues to powders in a low temperature condition; soaking the powders in water or ethanol to obtain a mixture; shaking the mixture in an ultrasonic shaker with total power of 300-600 W in a low temperature condition; centrifuging the mixture to obtain a supernatant; and removing a solvent of the supernatant. The present composition can avoid the extract spoiling so that stable effects can be provided. Additionally, the composition has the properties of cell detoxification, energy activation, glycation resistance, and aging resistance.

Description

Orchid bud embryo composite microcapsule composition, method for making the same, and use for promoting cell detoxification, energy activation and anti-aging

The present invention relates to a microcapsule composition, and particularly relates to an orchid bud embryo composite microcapsule composition. In addition, the present invention also relates to the preparation method of the above-mentioned composition and its use for promoting cell detoxification, energy activation and anti-aging.

Known as the "Orchid Kingdom", my country has mature planting and tissue culture technologies, including a wide variety of varieties, sophisticated tissue culture skills, virus testing capabilities, and mass-production engineering technologies. Moreover, my country's agriculture currently has the advantageous conditions to promote the orchid industry, including the unique natural geographical conditions, the diversified varieties that are proud of the world, the top-notch breeding expert group, the super-standard agricultural science and technology foundation, and the agricultural experiment improvement and academic research all over the country. and other agricultural technology auxiliary institutions, as well as the world-famous information industry and modern logistics channels.

In my country, the annual output of Phalaenopsis is about 132.94 million, and the total annual export volume is about 40 million, accounting for 20% of the global supply. cargo demand. There are five common methods of orchid cultivation and reproduction: seeding propagation method, pedicel budding propagation method, broken heart budding propagation method, stem cutting propagation method, and tissue culture method. Appropriate propagation methods can be selected according to the characteristics and needs of the variety. In the "tissue culture method", apical buds with meristematic ability are often harvested for culture; while the production of the growth point at the top of the stem mostly adopts bud proliferation (bud growth) or induction of pseudo-protosphere meristematic seedlings, bud proliferation The dominance of terminal buds can be reduced, the lateral buds can be easily grown, and buds can be proliferated in this way.

In addition to being used for ornamental purposes, orchids have also been widely used as raw materials for cosmetics or skin care products in recent years, which has greatly increased the economic value of orchids and increased demand for orchids. Orchids must be extracted into extracts, and then made into cosmetics or skin care products. Due to the different extraction parts of orchids, appropriate extraction techniques are required. If the extraction process is changed, the extracted ingredients will also be different, which will also affect the whitening effect, anti-oxidation, or anti-aging effect that can be achieved.

Because human skin is often affected by external environmental influences such as ultraviolet rays, air, or temperature, there is a risk of accelerated skin aging or skin cancer. Taking skin aging as an example, external factors account for 60%, and ultraviolet rays account for up to 60%. Researches on skin aging have found that ultraviolet rays not only activate melanocytes and cause skin spots, but also degenerate dermal tissue, accelerate skin oxidation, and generate free radicals. These free radicals can damage fibroblasts and cause collagen. Protein denaturation or reducing its content, or causing the degeneration of elastic fibrous tissue and resulting in aging wrinkles, will also accelerate DNA damage in cells, thereby reducing the renewal ability of skin cells and slowing down the rate at which new cells replace old cells.

At present, different cosmetic raw materials are developed for the above-mentioned skin problems, and other additives with special effects are added together. The active ingredients of the additives are: anthocyanins, flavonoids, or polyphenols, etc. The effects of anti-oxidation, anti-aging, or anti-wrinkle are quite significant, so many cosmetic or maintenance products on the market often have various plant extract active ingredients added to them. Based on different plant species, different parts, or the requirements and efficacy of the extracted ingredients, the extraction techniques to be used are different. Taking orchid as an example, as an additive for cosmetics or skin care products, there are many different extraction techniques, and the extraction steps, processes and environmental parameters are also different.

Chinese Invention Patent Application No. 201510295194.7 discloses "orchid extract and its preparation method and application", characterized in that: the orchid is Phalaenopsis, and the orchid extract is prepared by the following steps: (1) Extraction: the The orchid and the solvent are mixed and pulverized or broken to obtain orchid pulp in a weight ratio of 0.5:1 to 10:1, the solvent is water, alcohol, or an aqueous alcohol solution, and the alcohol is ethanol, propanol, or butanol; (2) Solid-liquid separation: the orchid pulp is subjected to solid-liquid separation, leaving a liquid orchid filtrate; (3) Activity division: the separated orchid filtrate is purified by molecular sieves to obtain orchid extraction sieve liquid; (4) Concentration: The purified orchid extract sieve solution is concentrated by molecular sieve treatment, or concentrated by vacuum or cooking to obtain active precipitate or active active extract.

Chinese Invention Patent Publication No. CN105878122A discloses "Natural Extract Cosmetics with Freckle Removal and Whitening Effects", which comprises functional ingredients Zelan extract and Chlorophytum extract, and the weight ratio of the Zelan extract and Chlorophytum extract is: 2 to 4:1. And the preparation method of the extract is as follows: extracting the dried zephyr orchid with ethanol under heat reflux, and concentrating the combined filtrate until there is no alcohol smell to obtain the ethanol extraction concentrate; after diluting with water, using petroleum ether, ethyl acetate, Extract with water-saturated n-butanol; dissolve the n-butanol extract with water and filter, use macroporous resin to aggregate the active ingredients in the filtrate, and spray dry to obtain it. The freckle-removing and whitening cosmetic proposed in this patent uses plant extracts as functional ingredients, and maximizes the freckle-removing and whitening effect by controlling the content ratio of Zelan extract and Chlorophytum extract.

Taiwan Invention Patent Application No. 102140137 discloses "Extract of Phalaenopsis phalaenopsis and its preparation method and use", which is prepared by the following steps: extracting Phalaenopsis phalaenopsis petals with supercritical carbon dioxide, thereby obtaining Phalaenopsis phalaenopsis petals Fat-soluble extracts and residues of , and extracting the residues with water to obtain water-soluble extracts of Phalaenopsis petals, which can be used to promote skin whitening, enhance skin moisturizing ability, and prevent or delay skin aging.

The above-mentioned first case uses the whole orchid for extraction, solid-liquid separation, activity division, and concentration processes; the second case uses the whole orchid orchids for drying, heat reflux extraction, concentration, dilution, dissolution, and filtration. and other steps; the third case is to use the petals of Phalaenopsis phalaenopsis to perform supercritical carbon dioxide extraction and water extraction. However, the parts of the orchid used in the above-mentioned previous cases were different. The first and second cases were carried out with the whole plant, and the third case only used the petals. Due to different extraction methods, procedures, equipment used, or environmental parameters, the obtained Extraction composition and content will also vary.

Whether the whole orchid or the petals are extracted, the content and effect of the extracted active ingredients are still limited. Chinese Invention Patent Publication No. CN105878122A and Taiwan Invention Patent Application No. 102140137 disclose high temperature steps, which may destroy the chemical structure of active ingredients or cause other potentially active substances to volatilize, resulting in the subsequent whitening and anti-aging of cosmetics or skin care products. Oxidation, or anti-aging effects are affected. Furthermore, in the past, the whole orchid was used for extraction, so that all parts of the orchid could be extracted regardless of whether or not the components that enhance the activity or inhibit the activity, so the overall active quality of the extracted extract will be limited.

Plant stem nodes usually have growth points to provide excellent growth function. Growth point cells can rapidly divide and differentiate to produce new buds, and can continuously elongate the bud axis, just like the regeneration function of animal stem cells. The content and benefits of active ingredients obtained by spotting cells for extraction may also be quite high. However, the known extraction technologies related to orchids do not extract the stem nodes; therefore, the present inventor provides an orchid growing point extraction technology to obtain orchid embryo extracts with high content of active ingredients and good biological effects.

Therefore, an object of the present invention is to provide an extraction product of orchid bud tissue, which mainly extracts and collects orchid bud embryo regeneration cells with high active components for extraction operation, and the prepared extract has a high content of active components, and It has the functions of promoting cell detoxification, energy activation, anti-glycation, anti-aging, anti-oxidation, whitening, promoting ATP production, maintaining telomere function, and protecting and repairing light damage caused by UVB. It can be added to cosmetics or skin care products.

Another object of the present invention is to provide an extraction product of orchid bud embryo tissue, the extraction process of which is operated at non-high temperature, so as to reduce heat loss caused by temperature, and also to avoid low boiling point and volatilization of effective substances and maintain growth point tissue. Growth active ingredients.

Another object of the present invention is to provide a composite microcapsule containing the extract product of orchid bud embryo tissue, which can protect the extract product of orchid bud embryo tissue, so that the extract product of orchid bud embryo tissue does not deteriorate and maintains stability for a long time to maintain biological activity.

Another object of the present invention is to provide a composite microcapsule containing the extract of orchid bud embryo tissue, which has high transdermal penetration ability to effectively transport the extract product of orchid bud embryo tissue into the skin.

Therefore, the present invention provides an orchid bud embryo composite microcapsule composition, which comprises: a carrier, formed into a shell-like structure; and an orchid bud embryo extract, embedded in the shell-like structure or in a shell-like structure The structure is coated, and the preparation method of the orchid bud embryo extract comprises the following steps: extracting a growth point embryo tissue from the terminal bud or lateral bud of an orchid; removing the wall membrane of the growth point embryo tissue; cryogenic grinding of the growth point embryo Organize into powder; cover and soak the powder with water or ethanol to obtain a mixed solution; use an ultrasonic vibration extraction device at low temperature to shake the mixed solution with a total energy of 300 to 600 W; centrifuge the mixed solution to obtain a supernatant; and removal of the solvent from the supernatant to obtain orchid bud embryo extract.

The orchid bud embryo extract in the microcapsule composition of the present invention is prepared in a non-high temperature process, which can avoid low boiling point and volatilization of effective substances and maintain the growth activity of bud embryo tissue, and the prepared extract The content of total polyphenols and total flavonoids is high. When added to cosmetics or skin care products, it can promote cell detoxification, energy activation, anti-glycation, anti-aging, anti-oxidation, whitening, promotion of ATP generation, maintenance of telomeres, and protection and repair. The effect of light damage caused by UVB. However, the present invention protects the orchid bud embryo extract through the shell-like structure formed by the carrier, and can provide the orchid bud embryo extract with long-term stability and no deterioration, and thus does not impair the promotion of cell detoxification, energy activation, anti-glycation, anti-aging, anti- Oxidizes, whitens, promotes ATP production, maintains telomere function, and protects and repairs light damage caused by UVB. In this way, the microcapsule composition of the present invention can be made to promote skin cell detoxification and skin cell energy activation, anti-glycation, anti-aging, anti-oxidation, whitening, promoting ATP production, maintaining telomere function, and protecting and repairing receptors. The composition for photodamage caused by UVB, and the form of the composition can be pharmaceuticals, cosmetics, skin care products, fragrances or body cleaning products, but not limited thereto.

In order to make the above-mentioned and/or other purposes, effects and features of the present invention more obvious and easy to understand, preferred embodiments are given below, and are described in detail as follows:

One embodiment of the present invention provides an orchid bud embryo composite microcapsule composition, which comprises: a carrier and an orchid bud embryo extract.

The carrier is formed into a shell-like structure, and an example thereof may be hydrogenated soybean lecithin or d-α-tocopheryl polyethylene glycol 1000 succinate, but not limited thereto. In a preferred embodiment, the carrier comprises hydrogenated soybean lecithin and d-α-tocopheryl polyethylene glycol 1000 succinate, and based on the total weight of the carrier, the weight percentage of hydrogenated soybean lecithin is 50 to 99%, And the weight percentage of d-α-tocopheryl polyethylene glycol 1000 succinate is 1 to 50%.

The orchid bud embryo extract is embedded in the shell-like structure or covered by the shell-like structure. In a preferred embodiment, based on the total weight of the composite microcapsule composition, the weight percentage of the orchid bud embryo extract is 2% to 10%. In another preferred embodiment, based on the total volume of the composite microcapsule composition, the total volume molar concentration of the orchid bud embryo extract and the carrier is 0.1 to 20 mM. The preparation method of orchid bud embryo extract is detailed as follows:

First, a growing point bud embryo tissue is extracted from the terminal bud or lateral bud of an orchid, and an example of the orchid is Phalaenopsis, but not limited thereto.

Next, the parietal membrane of the growth point bud embryo tissue is removed. In a preferred embodiment, the growth point embryo tissue is snap-frozen in liquid nitrogen to remove the parietal membrane.

Then, the growth point embryos were cryogenically ground into powder. In a preferred example, the growth point embryo tissue from which the parietal membrane has been removed is first cut into pieces, then liquid nitrogen is added to solidify the tissue pieces, and finally the tissue pieces are ground into powder. In order to avoid the temperature of the grinding tools or grinding process affecting the subsequent active ingredients, grinding tools (such as tissue grinding bowls and tissue grinders) can be pre-cooled at -80°C.

Next, the powder is covered and soaked with water or ethanol to obtain a mixed solution. In a preferred embodiment, the weight ratio of the powder to water or ethanol is 1:1 to 1:10, preferably 1:5.

Then, the mixed solution is shaken with a total energy of 300 to 600 W by using an ultrasonic vibration extraction device at low temperature. In this step, the active ingredients are extracted into the solvent by water or ethanol from the growth point embryo tissue by shaking. In a preferred example, the shaking time is 60 minutes. In another preferred example, every 3 minutes of shaking, there is a 2-minute rest until the total shaking time reaches 60 minutes (a total of 20 shakings). In another preferred embodiment, the shaking temperature is 50 to 60°C.

Finally, the mixture was centrifuged to obtain a supernatant, and the solvent of the supernatant was removed to obtain orchid bud embryo extract. In a preferred example, the mixture was centrifuged at 15,000 rpm for 15 minutes at 4°C to obtain the supernatant. In another preferred embodiment, after vacuum filtration of the supernatant to prepare an extract, the solvent of the extract is removed by a vacuum concentrator and a freeze dryer to obtain the orchid bud embryo extract.

In addition, another embodiment of the present invention provides a method for preparing an orchid bud embryo composite microcapsule composition, as detailed below:

After obtaining the orchid bud embryo extract according to the above, the orchid bud embryo extract is mixed with the carrier material to disperse the carrier material to form a colloidal complex. In a preferred embodiment, the carrier material is dissolved in a solvent, the solvent is removed by a rotary decompression concentrator to form a lipid film, and then the orchid germ embryo extract is added to the lipid film to disperse the lipid into a colloidal complex.

Next, the colloidal compound is shaken by a high-power ultrasonic oscillator to obtain an orchid bud embryo compound microcapsule composition. In a preferred example, the shaking time is 30 minutes, and the shaking temperature is 45°C.

The above embodiments are illustrated with the following examples:

<Example 1: Preparation of orchid bud embryo extract and stability test>

From the terminal bud or lateral bud of the orchid, the growth point bud embryo tissue was harvested. Secondly, the "rapid liquid nitrogen freezing ultrasonic oscillation method" is used, and the specific operation is as follows: the embryonic tissue is rapidly frozen in liquid nitrogen to remove the parietal membrane, and the tissue grinding bowl and the tissue grinder are pre-cooled at -80°C. ; After that, the parietal membrane-removed tissue was cut into pieces by a knife and placed in a grinding bowl, and a little liquid nitrogen was added to solidify the tissue pieces, and then ground into powder; then, the tissue powder was placed in the ultrasonic vibration extraction equipment , and cover and soak the tissue powder with pure water or ethanol; then, shake the mixture composed of solvent and powder at low temperature with a total energy of 300 to 600W for 60 minutes (shaking for 3 minutes, rest for 2 minutes, until the shaking time reaches 60 minutes ); after centrifugation at 15,000rpm for 15 minutes at 4°C, the supernatant was collected and vacuum filtered to prepare an extract. Finally, the solvent of the extract is removed by a vacuum concentrator (Rotary evaporator R-2000) and a freeze dryer (Economic freeze dryer CT5020D) to obtain orchid germ embryo extract (the symbol "OG" can be used interchangeably below) .

In order to understand the stability of the orchid bud embryo extract, it was placed at 25 °C or 50 °C for 7 days to observe the appearance and measure the color Lab value with a color difference meter (MET-CM6).

As shown in Figure 1 and Table 1, the appearance and color Lab value of OG had no obvious change after being placed at 25 °C for 7 days; on the contrary, the appearance became darker after being placed at 50 °C for 7 days, and the L value decreased from 22.3 to 20.1, The a value increased from 3.4 to 4.7, and the b value changed from 3.4 to 4.2. Table 1. Appearance changes of OG after being placed in different ambient temperatures for different days Color Lab value days Ambient temperature 25℃ Ambient temperature 50℃ L value 0 22.2 22.3 7 22.0 20.1 a value 0 3.2 3.4 7 4.0 4.7 b value 0 3.5 3.4 7 3.7 4.2

DPPH·(2,2-diphenyl-1-picrylhydrazyl) is a stable free radical, and a purple DPPH ethanol solution is used to generate a specific absorbance value under irradiation at a wavelength of 517 nm. If the DPPH·radical reacts with the sample, the absorbance value will decrease; therefore, the ability of the sample to scavenge DPPH·radical can be known from the change of the absorbance value. Specifically, the lower the absorbance value, the stronger the ability of the sample to scavenge DPPH·radicals.

Here, OG was prepared at different concentrations before and after 7 days at 50°C, and 90 μL of freshly prepared 100 μM DPPH·ethanol solution was added to react in a 96-well plate. Next, the absorbance at 517 nm of the reaction solution was detected by an enzyme immunoassay analyzer.

As shown in Figure 2, the free radical scavenging capacity of OG before being placed at 50 °C was 82.3%; after being placed at 50 °C for 7 days, the free radical scavenging capacity was 73.7%.

<Example 2: Preparation of orchid bud embryo composite microcapsule composition and related tests>

d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) is a vitamin E derivative, which is safe and has excellent oral biocompatibility with active active ingredients sex. Currently, TPGS is used in liposome drug delivery systems to increase liposome stability. Previous studies have used TPGS to form micelles as anticancer drug carriers. Previous studies have also added TPGS to polymer carriers as a carrier for controlled release of anticancer drugs (Cao & Feng, 2008). Therefore, TPGS is an excellent functional adjuvant for the microcapsule composition of this embodiment, and also has anti-aging and antioxidant activities.

Specifically, in this example, the orchid embryo composite microcapsule composition is prepared by using hydrogenated soybean lecithin (HL) and d-α-tocopheryl polyethylene glycol 1000 succinate. The detailed process is as follows : Add HL and TPGS to the solvent according to the experimental design ratio to dissolve (based on the total of HL and TPGS, the weight percentage of HL is 50 to 99%, and that of TPGS is 1 to 50%), and the solvent is removed by a rotary decompression concentrator, In order to form a lipid film on the wall of the flask; then, add OG to interact with the film to disperse the lipid to form a colloidal complex; finally, use a high-power ultrasonic oscillator at 45 ° C to vibrate the colloidal complex for 30 minutes to obtain The orchid embryo composite microcapsule composition (hereinafter can be used interchangeably with the symbol "OG-L") and its volume molar concentration is 0.1 to 20 mM.

The particle structure of OG-L was observed by transmission electron microscope (TEM), and OG-L was attached to the polyvinyl formal support film (formvar film) by liposome dispersion droplets before observation. The carbon-coated copper mesh was left for 2 minutes to embed the sample particles on the copper mesh, and then wiped off the excess sample with a wiping paper; then 0.5wt% uranyl acetate was added dropwise to the copper mesh to stain the sample. After standing for 2 minutes, excess dye was removed by wiping, and after being stored in a drying box for 12 hours, the morphological structure of OG-L was observed by transmission electron microscope.

As shown in Figure 3, OG-L formed colloidal particle complexes with particle sizes ranging from 50 to 400 nm.

OG-L particle size and interface potential were measured using a dynamic light scattering particle size and interface potential element analyzer (Dynamic Light Scattering, DLS, model Brookhaven 90 Plus Particle Size Analyzer, purchased from Brookhaven Inc., U.S.A). The analysis results are: the average particle size (APS) of OG-L is 286.13±10.17 nm, the distribution coefficient (polydispersity index, Pd.I) is 0.217±0.01, and the average zeta potential (AZP) is -7.13±4.51mV, the OG combined percentage (CP) was 94.31±3.76% (the OG concentration ratio in OG-L).

High performance liquid chromatography (HPLC) was used for quantitative analysis of OG. The pump model used was L-7100, the automatic injector model was L-7200, and the UV-vis model was used. were L-4200, both purchased from Hitachi (Japan), and the column model used was Microsorb-MV 100-5 C18 (250 mm x 4.6 mm id, 5 μm particle size).

As shown in Figure 4, it shows that the concentration of OG active ingredients in OG aqueous solution and OG-L aqueous solution changes with time; it can be seen that OG in OG-L is more stable in water and easier to store than pure OG. In other words, OG-L can improve the problem of unstable activity of OG in water.

<Example 3: Skin cell autophagy test>

Autophagy is the process of encapsulating aging organelles or protein aggregates and sending them to lysates for decomposition into small molecules. Cells age when they are unable to combat the damage caused by oxidative stress.

Human skin keratinocyte HaCaT was ^{cultured in a 96-well plate at a density of 1.5x10 5} cells/mL and grown at 37°C in a 5% carbon dioxide incubator for more than 24 hours. Afterwards, the OG-L reaction was added, and after the reaction time, the supernatant was removed and washed with PBS. Next, after UVB irradiation at 20 mJ/cm ² , a fresh culture solution containing 0.1 mM hydrogen peroxide was added for 1 hour. Then, add the autophagosome detection reagent working solution and place it in the incubator for 15 minutes to 1 hour, then wash the cells 3 to 4 times with wash buffer. Finally, quantitative PBS was added and absorbance was measured at excitation 360 nm and emission 520 nm (BioTek, Synergy ^™ 2, USA).

As shown in Figure 5, only under the action of oxidation inducers (UVB and hydrogen peroxide), the autophagy of HaCaT in human skin keratinocytes can be induced, but under the action of oxidation inducers combined with OG-L, the autophagy can be enhanced. Phagocytosis ability to increase the ability of cells to resist oxidative stress and enhance the ability of cells to detoxify. Taken together, OG-L promotes autophagy in skin cells to combat oxidative stress.

<Example 4: Cell Active Oxide Content Test>

In order to evaluate whether OG-L reduces the production of ROS reactive oxides in skin cells induced by hydrogen peroxide, fluorescently labeled DCFH2DA (2',7'-dichlorofluorescin diacetate) was used to quantitatively analyze the content of reactive oxides. The detailed process is as follows:

Human skin keratinocyte HaCaT was ^{cultured in a 96-well dish at a density of 1.5x10 5} cells/mL and grown at 37°C in a 5% carbon dioxide incubator for more than 24 hours. Afterwards, the OG-L reaction was added, and after the reaction time, the supernatant was removed and washed with PBS. Next, a fresh culture medium containing 0.1 mM hydrogen peroxide was added for 1 hour, and then the culture medium was removed, and then a fresh culture medium containing 10 μM DCFH2DA was added, and the culture medium was removed after being protected from light for one hour. Finally, quantitative PBS was added and absorbance was measured at excitation 502 nm and emission 524 nm (BioTek, Synergy ^™ 2, USA).

As shown in Figure 6, without the action of hydrogen peroxide, the cellular ROS active oxide content was set as the benchmark 100.0 ± 3.1%; under the action of hydrogen peroxide only, the relative content of the cellular ROS active oxide was 121.0 ± 6.1%; and Under the action of hydrogen peroxide and OG-L, the relative content of ROS reactive oxides in cells was 113.0±4.2%. This means that OG-L protects cells from 66.3% of the effects of oxidative damage. In conclusion, OG-L can reduce the oxidative damage induced by oxidative hydrogen oxidation inducers, thereby protecting cells from damage caused by oxidative stress.

<Example 5: ATP activation test>

ATP is a kind of nucleotide. As a "molecular currency" for cellular energy transfer, it stores and transmits chemical energy. Therefore, ATP is the direct source of energy for life activities. The energy required by the human body almost comes from ATP, such as heart beating and muscle movement. , and different cells perform different biological functions. On the contrary, lack of ATP will cause various organs and tissues of the human body to strike one after another, resulting in physiological phenomena such as heart failure, muscle soreness, or easy fatigue.

Human skin keratinocyte HaCaT was ^{cultured in a 24-well dish at a density of 1.5x10 5} cells/mL and grown at 37°C in a 5% carbon dioxide incubator for more than 24 hours. After that, OG-L was added for 24 hours, and then placed in an incubator for another 24 hours. After the reaction time, the culture medium and samples were first removed, then PBS was added to wash, and then a new culture medium was replaced. Next, after adding lysis buffer for 30 minutes, centrifuge at 1,500 rpm for 2 minutes, then take 50 μL of supernatant to a white 96-well plate, and use ATP determination kit (Molecular Probes, Eugene, OR, USA) to measure the absorbance value (BioTek, Synergy ^T M2, USA) to assess intracellular ATP content (μmol/g-cell).

As shown in Figure 7, after the action of OG-L, the ATP content of cells was higher than that of cells without OG-L (control group).

<Example 6: Anti-glycation test>

Aging is an irreversible phenomenon in the human body. When cells divide for many generations, they may be excessively stimulated by the outside world, resulting in growth arrest and aging. Senescence-associated β-galactosidase (SA-β-gal) is overexpressed in senescent cells and can be used as one of the indicators of cellular senescence.

Human skin keratinocyte HaCaT was ^{cultured in a 24-well dish at a density of 1.5×10 5} cells/mL, and after 24 hours of cell attachment, the culture medium was removed. After pretreatment with OG-L for 24 hours, ^{cells were irradiated with 50mJ/cm 2} UVB, and then cultured with serum-free medium for 24 hours. After the reaction time, the cells were stained with senescence β-galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA), and then the blue senescent cells were observed and counted under a microscope.

As shown in Figure 8, after UVB irradiation, the β-galactosidase of cells was greatly increased; and after UVB irradiation and OG-L, the cells could reduce the activation of β-galactosidase caused by UVB. In addition, 25μg/mL vitamin C in the positive control group could effectively inhibit the activity of β-galactosidase, especially the activation of β-galactosidase caused by UVB.

<Example 7: Longevity gene SIRT-1 expression test>

Although aging is an irreversible phenomenon in the human body, the activation of some genes can maintain cell function and promote individual health. These genes are called "longevity genes", and SIRT-1 is one of the typical examples. Previous studies have confirmed that up-regulating the expression of SIRT-1 can effectively protect cell repair and maintain normal function, preventing cells from aging and causing diseases.

Using reverse transcription-polymerase chain reaction (reverse transcription-PCR, RT-PCR) experiments to analyze the expression of OG-L regulation of longevity gene SIRT-1.

Human skin keratinocyte HaCaT was ^{cultured in a 24-well dish at a density of 1.5×10 5} cells/mL, and after 24 hours of cell attachment, the culture medium was removed. Next, OG-L was added for pretreatment for 24 hours, and the ^{cells were irradiated with 50 mJ/cm 2 of} UVB, and then cultured without serum was added for another 24 hours. Afterwards, after removing the culture medium, the cells were removed with Trypsin, and the cells were collected with PBS and centrifuged to remove the supernatant. Then, 1 mL of REzol ^™ C&T was added and the cells were lysed with uniform mixing for 5 minutes at room temperature. After adding 200 μL of chloroform and mixing for 15 seconds, the RNA was extracted by reacting at 4°C for 5 minutes. Then, after centrifugation at 12,000 rpm for 10 minutes at 4° C., the upper transparent layer was taken into a microcentrifuge tube soaked in DEPC water and sterilized. After that, an equal volume of isopropanol was added, mixed well and left at 4°C for 5 minutes to precipitate the RNA. Centrifuge again at 12,000 rpm for 10 minutes at 4°C and remove the supernatant. After adding 200 μL of 75% ice alcohol to wash the RNA, centrifuge at 12,000 rpm for 10 minutes at 4°C, and remove the supernatant. Finally, invert it to dry in the shade on a sterile operating table to completely evaporate the remaining water vapor in the centrifuge tube, add 100 μL of 0.1% DEPC water to disperse and dissolve the RNA, and store at -80°C. In addition, 2 μL of RNA was taken into 198 μL of 0.1% DEPC water, mixed evenly, and 100 μL was taken into a micro-quartz tube, and the absorbance at OD260nm and OD280nm was measured by a spectrophotometer (BioTek, Synergy ^TM 2, USA).

After mixing 3 μg of RNA with an appropriate amount of DEPC water, 1 μL of Oligo(dT) ₁₈ primer was added, placed at 70°C for 2 minutes, and then quickly moved to ice. Next, 4 μL of 10x MMLV RT buffer solution, 1 μL of dNTP mixture (10 mM each dNTP), 0.5 μL of recombinant RNase inhibitor (1 unit/ml), and 1 μL of MMLV reverse transcriptase (5 units) were added to make the total volume 20 μL. After mixing the mixture evenly, it was incubated at 42°C for 1 hour, and then heated at 94°C for 5 minutes to remove the MMLV reverse transcriptase activity to terminate the reaction and complete the cDNA template preparation. Finally, 80 μL of DEPC water was added and stored at -20°C for later use.

Take 1 μL of cDNA into a microcentrifuge tube and add 5 μL of 10x reaction buffer, 0.8 μL of 10 mM dNTPs (200 mM each dNTP) and 1 μL of each 50 mM forward primer (5'-tcgcaactatacccagaacatagaca-3') and reverse primer (5' -ctgttgcaaaggaaccatgaca-3'), Taq DNA polymerase (5 unit/μL), and finally deionized water was added to bring the total volume to 50 μL. After mixing evenly, the PCR reaction was carried out in an automatic temperature cycler (Techne Progene). The reaction conditions were as follows: denaturation, 98°C, 3 minutes; then, denaturation reaction, 94°C, adhesion reaction, 60°C, synthesis Reactions, 72, and 1 min of PCR each, for a total of 35 PCR cycles. After the reaction, an appropriate amount of the reaction product was taken and subjected to 2% agarose gel electrophoresis to analyze the reference gene (β-actin) and the target gene. In addition, quantification was performed using imaging software (Image J).

As shown in Figure 9, without UVB damage, the expression level of SIRT-1 was set at 1.0 (control group), while after UVB irradiation, its expression level was significantly reduced to 0.2. In addition, after UVB irradiation and OG-L, the expression of SIRT-1 can be significantly increased to 0.6, indicating that OG-L can effectively regulate and protect skin cells from UV damage to the longevity gene SIRT-1.

<Example 8: Expression test of cell rejuvenation gene mTOR and telomerase-regulated gene TERT>

mTOR is a serine-threonine protein kinase. It is an important factor in aging and canceration. It can promote cells to absorb heat, close the autophagy mechanism and prevent cell renewal. Therefore, in recent years, researches related to inhibiting the expression of mTOR have pointed out that by inhibiting Expression of mTOR in aged cells enables cell regeneration.

The role of telomeres is to maintain the integrity of eukaryotic chromosomes and control the cell division cycle. After cell division, telomeres stabilize the replicated DNA and ensure the integrity of the genetic sequence. However, after each cell division, telomeres are shortened; when telomeres are exhausted, cells initiate apoptosis, so telomeres are considered the best marker of aging. Telomerase replenishes telomeres, prolongs the number of cell divisions, and slows down the rate of aging. TOP1 and TPP1 are telomere protection proteins, and promoting their expression can improve the protection of telomeres.

Here, real-time quantitative PCR (qPCR) was used to analyze the expression of OG-L-regulated rejuvenation gene mTOR and telomerase-regulated gene TERT-related genes.

Human skin keratinocyte HaCaT was ^{cultured in a 6-well plate at a density of 1.5×10 5} cells/mL, and after culturing for 24 hours, OG-L was added to mix serum-free culture medium for 72 hours. Afterwards, after removing the culture medium, the cells were removed with Trypsin, and the cells were collected with PBS and centrifuged to remove the supernatant. Then, 1 mL of REzol ^™ C&T was added and the cells were lysed with uniform mixing for 5 minutes at room temperature. After adding 200 μL of chloroform and mixing for 15 seconds, the RNA was extracted by reacting at 4°C for 5 minutes. Then, after centrifugation at 12,000 rpm for 10 minutes at 4° C., the upper transparent layer was taken into a microcentrifuge tube soaked in DEPC water and sterilized. After that, an equal volume of isopropanol was added, mixed well and left at 4°C for 5 minutes to precipitate the RNA. Centrifuge at 12,000 rpm for 10 minutes at 4°C and remove the supernatant. After adding 200 μL of 75% ice alcohol to wash the RNA, centrifuge at 12,000 rpm for 10 minutes at 4°C, and remove the supernatant. Finally, invert it to dry in the shade on a sterile operating table to completely evaporate the remaining water vapor in the centrifuge tube, add 100 μL of 0.1% DEPC water to disperse and dissolve the RNA, and store at -80°C. In addition, 2 μL of RNA was taken into 198 μL of 0.1% DEPC water, mixed well and 100 μL was placed in a micro-quartz tube, and the absorbance at OD260nm and OD280nm was measured with a spectrophotometer (BioTek, Synergy ^TM 2, USA).

After mixing 3 μg of RNA with an appropriate amount of DEPC water, 1 μL of Oligo(dT) ₁₈ primer was added, placed at 70°C for 2 minutes, and then quickly moved to ice. Next, 4 μL of 10x MMLV RT buffer solution, 1 μL of dNTP mixture (10 mM each dNTP), 0.5 μL of recombinant RNase inhibitor (1 unit/mL), and 1 μL of MMLV reverse transcriptase (5 units) were added to make the total volume 20 μL. After mixing the mixture evenly, it was incubated at 42°C for 1 hour, and then heated at 94°C for 5 minutes to remove the MMLV reverse transcriptase activity to terminate the reaction and complete the cDNA template preparation. Finally, 80 μL of DEPC water was added and stored at -20°C for later use.

Take 10 μL of cDNA into a microcentrifuge tube, and add OmicsGreen 5x qPCR masterMix (ROX) (Omics Bio) and 5 μL each of 50mM forward primer and reverse primer to mix evenly for qPCR analysis. Placed in a qPCR machine (MyGo Pro) for real-time quantitative PCR reaction, the reaction conditions are as follows: denaturation, 98 °C, 3 minutes; followed by denaturation reaction at 94 °C, annealing reaction (annealing), 60 °C, synthesis reaction (extension), polymerase chain reaction at 72°C for 1 minute each, and the polymerase chain reaction was carried out for a total of 25 to 35 cycles. Data were calculated using 2- ^ΔΔCt expression to calculate relative expression magnitude values for genes.

As shown in Figure 10, OG-L down-regulated the expression of mTOR gene and up-regulated the gene expression of telomere protection proteins such as TOP1 and TPP1, indicating that OG-L has anti-aging potential.

However, the above are only preferred embodiments of the present invention, but cannot limit the scope of implementation of the present invention; therefore, any simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the contents of the description of the invention, All still fall within the scope of the patent of the present invention.

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Figure 1 is a photographic diagram to illustrate the change in appearance of orchid bud embryo extract after being placed at different ambient temperatures for different days. FIG. 2 is a statistical graph of a free radical scavenging experiment to illustrate the relative free radical scavenging activity of the orchid bud embryo extract after being placed at different ambient temperatures for different days. Figure 3 is a transmission electron microscope photograph illustrating the appearance of the orchid embryo composite microcapsule composition. FIG. 4 is a high performance liquid chromatography statistical chart to illustrate the stability of the orchid bud embryo extract in water of the orchid bud embryo extract and the orchid bud embryo composite microcapsule composition. FIG. 5 is a graph showing the results of an autophagy experiment to illustrate the autophagy effect of the orchid bud-embryo complex microcapsule composition on skin cells. FIG. 6 is a graph showing the results of a ROS reactive oxide experiment to illustrate the inhibition of ROS reactive oxide induction in skin cells by the orchid bud-embryo composite microcapsule composition. FIG. 7 is a graph showing the results of an ATP content experiment, to illustrate the promotion of the ATP content of skin cells by the orchid bud-embryo composite microcapsule composition. FIG. 8 is a graph showing the results of an SA-β-gal experiment to illustrate the delay of skin cell aging by the orchid bud-embryo complex microcapsule composition. Figure 9 is a picture of a PCR electrophoresis to illustrate the effect of the orchid bud-embryo complex microcapsule composition on the expression of SIRT-1 gene. Figure 10 is a graph of the results of a qPCR experiment to illustrate the effect of the orchid bud-embryo complex microcapsule composition on the expression of genes such as mTOR, TOP1, and TPP1.

Claims (10)

An orchid bud embryo composite microcapsule composition, comprising: a carrier formed into a shell-like structure; and an orchid bud embryo extract embedded in the shell-like structure or covering the shell-like structure; Wherein the preparation method of the orchid bud embryo extract comprises the following steps: Extracting a growing point bud embryo tissue from the terminal bud or lateral bud of an orchid; removing the parietal membrane of the germ embryo tissue of the growth point; Cryogenic grinding the growth point embryo tissue into powder; Cover and soak the powder with water or ethanol to obtain a mixed solution; Under low temperature, use an ultrasonic vibration extraction equipment to shake the mixture with a total energy of 300 to 600W; centrifuging the mixture to obtain a supernatant; and The solvent of the supernatant was removed to obtain the orchid embryo extract. The orchid embryo composite microcapsule composition according to claim 1, wherein the carrier comprises hydrogenated soybean lecithin and d-α-tocopheryl polyethylene glycol 1000 succinate. The orchid embryo composite microcapsule composition according to claim 2, wherein based on the total weight of the carrier, the weight percentage of the hydrogenated soybean lecithin is 50 to 99%, and the d-α-tocopherol-based poly The weight percent of ethylene glycol 1000 succinate is 1 to 50%. The orchid bud embryo composite microcapsule composition according to claim 1, wherein the orchid is Phalaenopsis. The orchid bud embryo composite microcapsule composition according to claim 1, wherein the shaking time in the shaking step of the mixed solution is 60 minutes. The orchid bud embryo composite microcapsule composition as claimed in claim 1, wherein the wall membrane removal step comprises: The growth point embryo tissue was snap frozen in liquid nitrogen. The orchid bud embryo composite microcapsule composition according to claim 1, wherein the step of growing point bud embryo tissue comprises: The growth point embryo tissue from which the parietal membrane has been removed is cut into pieces; adding liquid nitrogen to solidify the tissue mass; and The tissue mass is ground into the powder. A use of the composite microcapsule composition as claimed in claim 1 is used to prepare and promote skin cell detoxification, skin cell energy activation, anti-glycation, anti-aging, anti-oxidation, whitening, promotion of ATP generation, maintenance of A composition that acts as a particle and protects and repairs photodamage caused by UVB. A preparation method of an orchid bud embryo composite microcapsule composition, comprising: Extracting a growing point bud embryo tissue from the terminal bud or lateral bud of an orchid; removing the parietal membrane of the germ embryo tissue of the growth point; Grinding the growth point embryo tissue into powder; Cover and soak the powder with water or ethanol to obtain a mixed solution; Under low temperature, use an ultrasonic vibration extraction equipment to shake the mixture with a total energy of 300 to 600W; Centrifuge the mixture to obtain a supernatant; removing the solvent of the supernatant to obtain an orchid germ embryo extract; mixing the orchid embryo extract with a carrier material to disperse the carrier material to form a colloidal complex; and The colloidal complex was shaken by a high-power ultrasonic oscillator to obtain a microcapsule composition containing the orchid bud embryo extract. The preparation method according to claim 9, wherein the operating temperature of the colloid complex shaking step is 45°C, and the shaking time is 30 minutes.

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