CN113768849B - Orchid bud embryo composite microcrystal capsule composition, preparation method thereof and application thereof in aging resistance - Google Patents

Abstract

The orchid germ composite microcrystalline capsule composition of the present invention includes: a carrier formed into a shell-like structure; and an orchid germ extract embedded in or covered with the shell-like structure, and a method for preparing the orchid germ extract The method includes: extracting the growth point germ tissue from the terminal bud or lateral bud of the orchid; removing the wall membrane of the growth point germ tissue; grinding the growth point germ tissue into powder at low temperature; covering and soaking the powder with water or ethanol to obtain a mixed solution; Ultrasonic oscillation extraction equipment is used at low temperature to oscillate the mixed liquid with a total energy of 300 to 600W; the mixed liquid is centrifuged to obtain the supernatant; and the solvent of the supernatant is removed. The composition of the present invention can prevent orchid germ extract from deterioration and provide stable efficacy. In addition, the composition of the present invention can promote cell detoxification, energy activation, anti-glycation and anti-aging.

Description

Orchid germ composite microcrystalline capsule composition, its preparation method and anti-aging method use

Technical field

The present invention relates to a microcrystalline capsule composition, and in particular to an orchid germ composite microcrystalline capsule composition. In addition, the present invention also relates to the preparation method of the above composition and its use for promoting cell detoxification, energy activation and anti-aging.

Background technique

Taiwan, China, has mature orchid planting and tissue culture technology, including a wide variety of varieties, sophisticated tissue culture skills, virus detection capabilities, and engineering technology for large-scale mass production. Moreover, Taiwan, China currently has the advantageous conditions to promote the orchid industry, including unique natural geographical conditions, a diversified variety that ranks first in the world, a group of top-notch breeding experts, a super-level agricultural science and technology foundation, and agricultural experimental improvement and academic research all over the island. and other various agricultural technical assistance institutions, as well as the world-famous information industry and modern logistics channels, etc.

In Taiwan, China, the annual output of Phalaenopsis is about 132.94 million plants, and the total annual export sales is about 40 million plants, accounting for 20% of the global supply. The sales areas are all over Europe, the United States and Japan, so it is necessary to use advanced biotechnology to propagate in large quantities to meet the demand. Supply requirements. There are five common methods of cultivating and propagating orchids: sowing propagation, pedicel germination propagation, heart-cut germination propagation, stem cutting propagation, and tissue culture. Appropriate propagation methods can be selected according to the characteristics and needs of the variety. The "tissue culture method" mostly selects the apical bud part with meristematic ability for culture; while the production of the stem apex growing point part mostly uses bud proliferation (bud growth) or the method of inducing pseudosphere meristematic seedlings, bud proliferation It can reduce the dominance of terminal buds, making it easier for lateral buds to grow, and in this way, the buds can be multiplied.

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 created a greater demand for orchids. Orchids must be extracted into extracts before they are made into cosmetics or skin care products. Due to the different extraction parts of orchids, appropriate extraction techniques must be used. If the extraction process changes, the extracted ingredients will also be different, and the achievable whitening, antioxidant, or anti-aging effects will also be affected.

Since human skin is often affected by external environments such as ultraviolet rays, air, or temperature, there is a risk of accelerated skin aging or skin cancer. Taking skin aging as an example, exogenous factors account for 60%, of which ultraviolet rays account for up to 60%. Research on skin aging has found that ultraviolet rays not only cause melanocytes to become active, causing spots on the skin, but also degenerate dermal tissue, accelerate skin oxidation, and produce free radicals. These free radicals can destroy fibroblasts and cause collagen damage. Protein denaturation or reduction of content may cause the degeneration of elastic fiber tissue and produce aging wrinkles. It will also accelerate DNA damage in cells, reducing the renewal capacity 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. The active ingredients of the additives are such as anthocyanins, flavonoids, or polyphenols, etc., which are good for skin whitening and Anti-oxidant, anti-aging, or anti-wrinkle effects are quite significant, so many cosmetics or skin care products on the market often contain active ingredients extracted from various plants. Based on different plant species, different parts, or different requirements and effects of extracted ingredients, different extraction techniques must be used. Take orchid as an example. As an additive to 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 an orchid extract and its preparation method and application. The orchid is a genus Phalaenopsis. The orchid extract is prepared by the following steps: (1) Extraction: extract the orchid Mix and crush or break the wall with a solvent in a weight ratio of 0.5:1 to 10:1 to obtain orchid slurry. The solvent is water, alcohol, or alcohol aqueous solution, and the alcohol is ethanol, propanol, or butanol; (2) Solid-liquid separation: the orchid slurry is subjected to solid-liquid separation, leaving a liquid orchid filtrate; (3) Activity division: the separated orchid filtrate is purified with molecular sieves to obtain orchid extraction sieved liquid; (4) Concentration: The purified orchid extract sieved liquid is concentrated by molecular sieve treatment, or concentrated by vacuum or cooking methods to obtain active precipitate or active active extract.

Chinese Invention Patent Publication No. CN105878122A discloses a natural extract cosmetic with freckle-removing and whitening effects, which includes the functional ingredients adenophora extract and Chlorophytum extract. The weight ratio of the Edenophyllum extract and Chlorophytum extract is 2 to 4:1. The preparation method of the extract is: extract the dried adenophora or Chlorophytum with ethanol hot reflux, and combine the filtrate and concentrate until there is no alcohol smell to obtain an ethanol extraction concentrate; after diluting with water, use petroleum ether, ethyl acetate, and Extract with water-saturated n-butanol; dissolve the n-butanol extract with water and filter, use macroporous resin to gather the active ingredients in the filtrate, and spray-dry it. The anti-freckle and whitening cosmetic mentioned in this patent uses plant extracts as functional ingredients, and controls the content ratio of adenophora extract and Chlorophytum extract to maximize the anti-freckle and whitening effect.

Taiwan Invention Patent Application No. 102140137 discloses an extract of Phalaenopsis alba petals and its preparation method and use. It is prepared by the following steps: extracting Phalaenopsis alba petals with supercritical carbon dioxide, thereby obtaining Phalaenopsis alba. The fat-soluble extract and residue of the petals; and the residue is extracted with water to obtain the water-soluble extract of Phalaenopsis albicans petals, which can be used to promote skin whitening, improve skin moisturizing ability, and prevent or delay skin aging.

In the first case mentioned above, the whole orchid is used for extraction, solid-liquid separation, activity classification, and concentration. In the second case, the whole adenophora or Chlorophytum is used for drying, hot reflux extraction, concentration, dilution, dissolution, and filtration. and other steps; the third case uses the petals of Phalaenopsis albicans for supercritical carbon dioxide extraction and water extraction. However, the parts of the orchid used in the above-mentioned cases are different. The first and second cases were carried out on the whole plant, while the third case only used petals. Due to the different extraction methods, processes, equipment used, or environmental parameters, the results obtained The extraction ingredients and content will also be different.

Contents of the invention

Regardless of whether it is extracted from the whole orchid or petals, 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 that high-temperature processing steps may destroy the chemical structure of active ingredients or cause other potentially active substances to volatilize, resulting in the whitening and whitening of subsequent cosmetics or skin care products. The antioxidant 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 they enhance or inhibit activity. Therefore, the overall active quality of the extract obtained would be limited.

Plant stem nodes usually have growing points to provide excellent growth functions. Cells at the growth points can divide and differentiate rapidly to produce new buds, and can continuously elongate the bud axis, just like the regeneration function of animal stem cells. For example, if plant stem nodes can be harvested By extracting the growth point cells, the content and effectiveness of the active ingredients obtained may also be quite high. However, the well-known extraction technology related to orchids does not target the extraction of stem nodes; therefore, the inventor of this case provides an orchid growth point extraction technology to obtain an orchid germ extract with high content of active ingredients and good biological effects.

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

Another object of the present invention is to provide an extraction product of orchid germ tissue. The extraction process is operated at a non-high temperature to reduce heat loss caused by temperature, avoid low boiling points and volatilization of effective substances, and preserve growth point tissue. growth active ingredients.

Another object of the present invention is to provide a composite microcrystalline capsule containing an extracted product of orchid germ tissue, which can protect the extracted product of orchid germ tissue, so that the extracted product of orchid germ tissue does not deteriorate and can be preserved for a long time. Stability and thus maintain biological activity.

Another object of the present invention is to provide a composite microcrystalline capsule containing an extraction product of orchid germ tissue, which has high percutaneous penetration ability to effectively transport the extraction product of orchid germ tissue into the skin.

Therefore, the present invention proposes an orchid germ composite microcrystalline capsule composition, including: a carrier, which is formed into a shell-like structure; and an orchid germ embryo extract, which is embedded in or covered by the shell-like structure. , and the preparation method of orchid germ embryo extract includes the following steps: extracting the growth point germ tissue from the terminal bud or lateral bud of the orchid; removing the wall membrane of the growth point germ tissue; grinding the growth point germ tissue into powder at low temperature; Cover the powder with water or ethanol and soak it to obtain a mixed liquid; use ultrasonic oscillation extraction equipment at low temperature to oscillate the mixed liquid with a total energy of 300 to 600W; centrifuge the mixed liquid to obtain the supernatant; and remove the solvent of the supernatant to obtain Orchid germ extract.

The orchid germ embryo extract in the microcrystalline capsule composition of the present invention is prepared under a non-high temperature process, which can avoid the volatilization of low boiling points and effective substances and maintain the growth activity of the germ embryo tissue, and the prepared extract It has high content of total polyphenols and total flavonoids. When added to cosmetics or skin care products, it can promote cell detoxification, energy activation, anti-glycation, anti-aging, anti-oxidation, whitening, promote ATP production, maintain telomere function, and protect and repair. Effect of light damage caused by UVB. However, the present invention protects the orchid germ extract through the shell-like structure formed by the carrier, and can provide the orchid germ extract with long-term stability without deterioration, thus without damaging the promotion of cell detoxification, energy activation, anti-glycation, anti-aging, Antioxidant, whitening, promoting ATP production, maintaining telomere function, and protecting and repairing light damage caused by UVB. In this way, the microcrystalline capsule composition of the present invention can be made to promote skin cell detoxification and skin cell energy activation, resist glycation, anti-aging, antioxidant, whitening, promote ATP production, maintain telomere function, and protect and repair damaged cells. A composition that causes photodamage caused by UVB, and the composition may be in the form of pharmaceuticals, cosmetics, skin care products, fragrances or human body cleaning products, but is not limited thereto.

Description of the drawings

Figure 1 is a photograph to illustrate the appearance changes of orchid embryo extract after being stored at different ambient temperatures for different days.

Figure 2 is a statistical graph of free radical scavenging experiments to illustrate the relative free radical scavenging activity of orchid germ 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 microcrystalline capsule composition.

Figure 4 is a high performance liquid chromatography statistical chart to illustrate the stability of the orchid germ extract and the orchid germ composite microcrystalline capsule composition in water.

Figure 5 is a diagram of the results of an autophagy experiment to illustrate the autophagy effect of the orchid germ composite microcrystalline capsule composition on skin cells.

Figure 6 is a diagram of ROS reactive oxide experiment results to illustrate the inhibition of ROS reactive oxide induction in skin cells by the orchid germ composite microcrystalline capsule composition.

Figure 7 is a diagram of ATP content experimental results to illustrate the promotion of the ATP content of skin cells by the orchid germ composite microcrystalline capsule composition.

Figure 8 is a graph showing the results of the SA-β-gal experiment to illustrate the delay of aging of skin cells by the orchid germ composite microcrystalline capsule composition.

Figure 9 is a PCR electrophoresis photograph to illustrate the effect of the orchid embryo composite microcrystalline capsule composition on the expression of SIRT-1 gene.

Figure 10 is a qPCR experiment result diagram to illustrate the effect of the orchid germ embryo composite microcrystalline capsule composition on the expression of genes such as mTOR, TOP1, and TPP1.

Detailed ways

In order to make the above and/or other objects, effects, and features of the present invention more obvious and understandable, preferred embodiments are listed below and described in detail as follows:

One embodiment of the present invention provides an orchid germ composite microcrystalline capsule composition, which includes: a carrier and an orchid germ extract.

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

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

First, the growth point bud tissue is extracted from the terminal bud or lateral bud of the orchid. An example of the orchid is Phalaenopsis, but it is not limited to this.

Secondly, remove the wall membrane of the germ tissue at the growing point. In a preferred example, the growth point germ tissue is quickly frozen in liquid nitrogen to remove the wall membrane.

Then, the growing point embryo tissue is cryogenically ground into powder. In a preferred example, the growth point germ tissue with the wall membrane 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 prevent the temperature generated by the grinding tools or the grinding process from affecting the subsequently obtained active ingredients, the grinding tools (such as tissue grinding bowls and tissue grinders) can be pre-cooled at -80°C.

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

Then, use ultrasonic oscillation extraction equipment at low temperature to oscillate the mixed liquid with a total energy of 300 to 600W. In this step, water or ethanol is used to extract active ingredients from the germ tissue of the growth point into the solvent through shaking. In a preferred example, the shaking time is 60 minutes. In another preferred example, shake for 3 minutes and rest for 2 minutes until the total shaking time reaches 60 minutes (20 shaking times in total). In another preferred example, the oscillation temperature is 50 to 60°C.

Finally, the mixture is centrifuged to obtain the supernatant, and the solvent of the supernatant is removed to obtain the orchid germ extract. In a preferred example, the mixture is centrifuged at 15,000 rpm for 15 minutes at 4°C to obtain the supernatant. In another preferred example, after the supernatant is vacuum-filtered to prepare an extract, the solvent of the extract is removed using a vacuum concentrator and a freeze dryer to obtain the orchid germ extract.

In addition, another embodiment of the present invention proposes a method for preparing an orchid germ composite microcrystalline capsule composition, as detailed below:

After the orchid germ extract is obtained according to the above, the orchid germ extract is mixed with the carrier material, so that the carrier material is dispersed to form a colloidal complex. In a preferred example, the carrier material is dissolved in a solvent, the solvent is removed using a rotary vacuum concentrator to form a lipid film, and then orchid germ extract is added to the lipid film to disperse the lipids into a colloidal complex.

Next, a high-power ultrasonic oscillator is used to vibrate the colloidal composite to obtain an orchid germ composite microcrystalline capsule composition. In a preferred example, the shaking time is 30 minutes and the shaking temperature is 45°C.

The above embodiments are illustrated by the following examples:

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

From the terminal bud or lateral bud of the orchid, extract the embryo tissue of the growing point. Secondly, the "rapid liquid nitrogen freezing ultrasonic oscillation method" is used. The specific operation is as follows: the germ tissue is rapidly frozen in liquid nitrogen to remove the wall membrane, and the tissue grinding bowl and tissue grinder are pre-placed at -80°C to cool; After that, use a knife to cut the tissue with the wall membrane removed into pieces and put them into a grinding bowl, add a little liquid nitrogen to solidify the tissue pieces, and then grind them into powder; then, put the tissue powder into the ultrasonic oscillation extraction equipment, and Cover the tissue powder with pure water or ethanol and soak it; then, shake the mixture of solvent and powder at low temperature with a total energy of 300 to 600W for 60 minutes (shake for 3 minutes, rest for 2 minutes, until the shaking time reaches 60 minutes); After centrifugation at 15,000 rpm for 15 minutes at 4°C, the supernatant was collected and filtered under vacuum to prepare an extract. Finally, use a vacuum concentrator (Rotary evaporator R-2000) and a freeze dryer (Eco nomic freeze dryer CT5020D) to remove the solvent from the extract to obtain the orchid germ extract (hereinafter used interchangeably with the symbol "OG") .

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

As shown in Figure 1 and Table 1, the appearance and color Lab value of OG did not change significantly after being placed at 25°C for 7 days; on the other hand, the appearance became darker after being placed at 50°C for 7 days, and the L value decreased from 22.3 to 20.1, and the a value It increased from 3.4 to 4.7, and the b value increased from 3.4 to 4.2.

Table 1. Appearance changes of OG after being placed at different ambient temperatures for different days.

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

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

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

<Example 2: Preparation of orchid germ composite microcrystalline capsule composition and related tests>

d-alpha tocopheryl polyethyleneglycol 1000succinate (TPGS) is a vitamin E derivative that is safe and has excellent oral biocompatibility with effective active ingredients. TPGS is currently used in liposome drug delivery systems to increase the stability of liposomes. Previous studies have used TPGS to form microcells as anti-cancer drug carriers. Previous studies have also added TPGS to polymer carriers as carriers for the controlled release of anticancer drugs (Cao & Feng, 2008). Therefore, TPGS is an excellent functional auxiliary for the microcrystalline capsule composition of this embodiment, and it also has anti-aging and antioxidant activities.

Specifically, this example uses hydrogenated soybean lecithin (HL) and d-α-tocopheryl polyethylene glycol 1000 succinate to prepare an orchid germ composite microcrystalline capsule composition. The detailed process is as follows : Add HL and TPGS to the solvent according to the experimental design ratio (based on the total of HL and TPGS, the weight percentage of HL is 50 to 99%, and TPGS is 1 to 50%), and use a rotary vacuum concentrator to remove the solvent. To form a lipid film on the wall of the flask; then, add OG to interact with the film to disperse the lipids and form a colloidal complex; finally, use a high-power ultrasonic oscillator to shake the colloidal complex for 30 minutes at 45°C, that is, An orchid germ composite microcrystalline capsule composition (hereinafter interchangeable with the symbol "OG-L") is obtained and its volume molar concentration is 0.1 to 20mM.

The particle structure of OG-L was observed using a transmission electron microscope (TEM). Before observation, the liposome dispersion liquid was used to attach OG-L to the polyvinyl formal support film (formvarfilm). Place it on a carbon-plated copper grid and let it sit for 2 minutes to allow the sample particles to embed on the copper grid. Then wipe it with clean paper to remove excess sample. Then add 0.5wt% uranyl acetate dropwise on the copper grid to dye the sample and let it stand for 2 minutes. After 12 minutes, the excess dye was removed by wiping, and then stored in a drying oven for 12 hours. The morphological structure of OG-L was then observed using a transmission electron microscope.

As shown in Figure 3, OG-L forms a colloidal particle complex with a particle size ranging from 50 to 400 nm.

The particle size and interface potential of OG-L were measured using a dynamic light scattering particle size and interface potential analyzer (Dynamic Light Scattering, DLS, model Brookhaven 90Plus Particle Size Analyzer, purchased from Brookhaven Inc., U.S.A.). The analysis results are: the average particle size (A.P.S.) of OG-L is 286.13±10.17nm, the distribution coefficient (polydispersity index, Pd.I) is 0.217±0.01, and the average interface potential (average zeta potential, A.Z.P.) is -7.13±4.51mV, OG combined percentage (C.P.) is 94.31±3.76% (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 ultraviolet light detector (UV-vis) model was used. It is L-4200, both purchased from Hitachi (Japan), and the column model used is Microsorb-MV 100-5C18 (250mmx 4.6mm i.d., 5μm 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 and easier to store in water 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 coating aged organelles or protein aggregates and sending them to the lysosome for breakdown into small molecules. When cells are unable to resist damage caused by oxidative stress, they will age.

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

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

<Example 4: Cell active oxide content test>

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

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

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

<Example 5: Adenosine triphosphate activation test>

ATP is a type of nucleotide. As the "molecular currency" of cellular energy transmission, it stores and transmits chemical energy. Therefore, ATP is the direct source of energy for life activities. Almost all the energy needed by the human body comes from ATP, such as heart beating and muscle. Movement, and different cells performing different biological functions. On the contrary, if there is a lack of ATP, various organs and tissues of the human body will fail one after another, leading to physiological phenomena such as heart failure, muscle soreness, or easy fatigue.

Human skin keratinocytes HaCaT were cultured in a 24-well plate at a density of 1.5x10 ⁵ cell/mL and grown in an incubator at 37°C and 5% carbon dioxide for more than 24 hours. After that, OG-L was added and incubated for 24 hours, then placed in an incubator and incubated for another 24 hours. After the reaction time is reached, remove the culture medium and samples, add PBS for cleaning, and then replace with new culture medium. Next, lysis buffer was added and incubated for 30 minutes, and then centrifuged at 1,500 rpm for 2 minutes. Then, 50 μL of the supernatant was transferred to a white 96-well plate, and the absorbance value was measured using an ATP determination kit (Molecular Probes, Eugene, OR, USA) (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 over-stimulated by the outside world, leading to growth stagnation 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 keratinocytes HaCaT were cultured in a 24-well plate at a density of 1.5x10 ⁵ cell/mL, and after 24 hours of cell attachment, the culture medium was removed. After adding OG-L for pretreatment for 24 hours, the cells were first irradiated with 50mJ/cm ² UVB, and then serum-free culture medium was added and cultured for 24 hours. After reaching the reaction time, stain with senescenceβ-galactosidasestaining kit (Cell Signaling Technology, Danvers, MA, USA), and then observe and count the blue aged cells using a microscope.

As shown in Figure 8, it is shown that after UVB irradiation, the β-galactosidase of cells increases significantly; and after UVB irradiation and OG-L, the cells can reduce the activation of β-galactosidase caused by UVB. In addition, 25 μg/mL vitamin C in the positive control group can effectively inhibit β-galactosidase activity, especially β-galactosidase activation 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 functions 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 functions, preventing cells from aging and causing diseases.

Reverse transcription polymerase chain reaction (RT-PCR) was used to conduct experiments to analyze the expression of OG-L in regulating the longevity gene SIRT-1.

Human skin keratinocytes HaCaT were cultured in a 24-well plate at a density of 1.5x10 ⁵ cell/mL, and after 24 hours of cell attachment, the culture medium was removed. Then, OG-L was added for pretreatment for 24 hours, and then 50mJ/cm ² UVB was used to irradiate the cells, and then serum-free culture medium was added and cultured for another 24 hours. Afterwards, after removing the culture medium, the cells were removed with Trypsin, collected with PBS, and centrifuged to remove the supernatant. Then, add 1 mL REzol ^TM C&T and mix evenly for 5 minutes at room temperature to dissolve the cells. Add 200 μL chloroform and mix evenly for 15 seconds, then place it at 4°C to react for 5 minutes to extract RNA. Then, after centrifugation at 12,000 rpm for 10 minutes at 4°C, take the upper transparent layer into a microcentrifuge tube soaked in DEPC water and sterilized. Afterwards, add an equal volume of isopropanol, mix evenly and let stand 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-cold alcohol to wash the RNA, centrifuge at 12,000 rpm for 10 minutes at 4°C and remove the supernatant. Finally, invert the tube to dry in the shade on a sterile workbench to completely evaporate the remaining moisture 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, add 2 μL of RNA to 198 μL of 0.1% DEPC water, mix evenly, and put 100 μL into a micro quartz tube, and measure the absorbance values of OD260nm and OD280nm with a spectrophotometer (BioTek, Synergy ^TM 2, USA).

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

Take 1μL cDNA into a microcentrifuge tube, and add 5μL 10x reaction buffer, 0.8μL 10mMdNTP (200mM each dNTP) and 1μL each of 50mM forward primer (5'-tcgcaactataccccagaacatagaca-3') and reverse primer (5'- ctgttgcaaaggaaccatgaca-3'), Taq DNA polymerase (5unit/μL), and finally add deionized water to bring the total volume to 50μL. After mixing evenly, place it in an automatic temperature cycler (Techne Progene) for PCR reaction. The reaction conditions are as follows: denaturation reaction, 98°C, 3 minutes; followed by denaturation reaction, 94°C, adhesion reaction, 60°C, and synthesis. Reaction, 72, and PCR for 1 minute each, for a total of 35 cycles of PCR. After the reaction is completed, take an appropriate amount of the reaction product and conduct 2% agar gel electrophoresis to analyze the reference gene (β-actin) and target gene. In addition, imaging software (Image J) was used for quantification.

As shown in Figure 9, without UVB damage, the expression amount of SIRT-1 was set at 1.0 (control group), but after UVB irradiation, its expression amount was significantly reduced to 0.2. In addition, after UVB irradiation and OG-L, the expression level 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 regulatory gene TERT>

mTOR is a serine-threonine protein kinase. It is an important factor in aging and cancer. It promotes cells to absorb heat, shuts down the autophagy mechanism, and prevents cell renewal. Therefore, in recent years, studies on inhibiting mTOR have pointed out that By inhibiting the expression of mTOR in aging cells, cells can be regenerated.

The role of telomeres is to maintain the integrity of eukaryotic chromosomes and control the cell division cycle. After cell division, telomeres will stabilize the copied DNA and ensure the integrity of the genetic sequence. However, telomeres shorten every time a cell divides. When telomeres are exhausted, cells initiate apoptosis, so telomeres are considered the best marker of aging. Telomerase can replenish telomeres, extend the number of cell divisions, and slow down the aging rate. 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 conduct experiments to analyze the expression of genes related to the OG-L-regulated rejuvenation gene mTOR and the telomerase-regulated gene TERT.

Human skin keratinocyte cells HaCaT were cultured in a 6-well plate at a density of 1.5x10 ⁵ cell/mL. After 24 hours of culture, OG-L mixed serum-free culture medium was added and incubated for 72 hours. Afterwards, after removing the culture medium, the cells were removed with Trypsin, collected with PBS, and centrifuged to remove the supernatant. Then, add 1 mL REzol ^TM C&T and mix evenly for 5 minutes at room temperature to dissolve the cells. Add 200 μL chloroform and mix evenly for 15 seconds, then place it at 4°C to react for 5 minutes to extract RNA. Then, after centrifugation at 12,000 rpm for 10 minutes at 4°C, take the upper transparent layer into a microcentrifuge tube soaked in DEPC water and sterilized. Afterwards, add an equal volume of isopropanol, mix evenly and let stand 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-cold alcohol to wash the RNA, centrifuge at 12,000 rpm for 10 minutes at 4°C and remove the supernatant. Finally, invert the tube to dry in the shade on a sterile workbench to completely evaporate the remaining moisture 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, add 2 μL of RNA to 198 μL of 0.1% DEPC water, mix evenly, and put 100 μL into a micro quartz tube, and measure the absorbance values of OD260nm and OD280nm with a spectrophotometer (BioTek, Synergy ^TM 2, USA).

Mix 3 μg RNA with an appropriate amount of DEPC water, add 1 μL Oligo(dT) ₁₈ primer, incubate at 70°C for 2 minutes and quickly move to ice. Then, add 4 μL 10x MMLV RT buffer solution, 1 μL dNTP mixture (10mM of each dNTP), 0.5 μL recombinant RNase inhibitor (1 unit/mL), and 1 μL MMLV reversetranscr iptase (5 unit) respectively, so that the total volume is 20 μL. After mixing the mixture evenly, incubate at 42°C for 1 hour, and then heat at 94°C for 5 minutes to remove the MMLV reverse transcriptase activity to terminate the reaction and complete cDNA template preparation. Finally, add 80 μL DEPC water and store it at -20°C for later use.

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

As shown in Figure 10, OG-L down-regulates mTOR gene expression and up-regulates 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 they cannot be used to limit the scope of the present invention; therefore, any simple equivalent changes and modifications made based on the patent scope of the present invention and the content of the invention specification, All are still within the scope of the patent of this invention.

Claims (9)

1. An orchid bud embryo composite microcrystal capsule composition, which is characterized by comprising:

a carrier, forming a shell-like structure, wherein: the carrier comprises hydrogenated soy lecithin and d-alpha-tocopheryl polyethylene glycol 1000 succinate; and

the orchid bud embryo extract is inlaid in or coated by the shell-shaped structure;

the preparation method of the orchid bud embryo extract comprises the following steps:

picking up the bud embryo tissue of the growing point from the terminal bud or the lateral bud of the orchid;

removing the parietal membrane of the growing point bud embryo tissue;

grinding the bud embryo tissue of the growing point into powder at a low temperature;

soaking the powder in water to obtain a mixed solution;

vibrating the mixed liquor at a low temperature by using ultrasonic vibration extraction equipment at a total energy of 300-600W;

centrifuging the mixture to obtain a supernatant; and

removing the solvent of the supernatant to obtain the orchid bud embryo extract.

2. The orchid bud embryo composite microcrystal capsule composition according to claim 1, wherein: the weight percent of the hydrogenated soy lecithin is 50 to 99% and the weight percent of the d-alpha-tocopheryl polyethylene glycol 1000succinate is 1 to 50% based on the total weight of the carrier.

3. The orchid bud embryo composite microcrystal capsule composition according to claim 1, wherein: the orchid is butterfly orchid.

4. The orchid bud embryo composite microcrystal capsule composition according to claim 1, wherein: the shaking time in the step of shaking the mixed liquor is 60 minutes.

5. The orchid bud embryo composite microcrystal capsule composition according to claim 1, wherein: the wall film removing step includes:

the bud embryo tissue of the growing point is frozen rapidly by liquid nitrogen.

6. The orchid bud embryo composite microcrystal capsule composition according to claim 1, wherein: the step of growing the bud embryo tissue comprises the following steps:

cutting the growing point bud embryo tissue from which the wall film is removed into blocks;

adding liquid nitrogen to solidify the tissue block; and

the tissue mass is ground into the powder.

7. Use of the composite microcrystalline capsule composition according to claim 1, wherein: is used for preparing a composition for resisting saccharification, aging, oxidation, whitening, promoting ATP generation, maintaining telomere function, and protecting and repairing photodamage caused by UVB.

8. The preparation method of the orchid bud embryo composite microcrystal capsule composition is characterized by comprising the following steps:

picking up the bud embryo tissue of the growing point from the terminal bud or the lateral bud of the orchid;

removing the parietal membrane of the growing point bud embryo tissue;

grinding the growing bud embryo tissue into powder;

soaking the powder in water to obtain a mixed solution;

vibrating the mixed liquor at a low temperature by using ultrasonic vibration extraction equipment at a total energy of 300-600W;

centrifuging the mixture to obtain a supernatant;

removing the solvent from the supernatant to obtain an extract of orchid bud embryos;

mixing the orchid bud embryo extract with a carrier material, dispersing the carrier material to form a colloidal complex, wherein: the carrier material comprises hydrogenated soy lecithin and d-alpha-tocopheryl polyethylene glycol 1000 succinate; and

oscillating the colloid compound by using a high-power ultrasonic oscillator to obtain the microcrystal capsule composition containing the orchid bud embryo extract.

9. The method for preparing the orchid bud embryo composite microcrystal capsule composition according to claim 8, which is characterized in that: the operation temperature of the colloid compound shaking step is 45 ℃, and the shaking time is 30 minutes.