CN113768849B - Orchid bud embryo composite microcrystal capsule composition, preparation method thereof and application thereof in aging resistance - Google Patents
Orchid bud embryo composite microcrystal capsule composition, preparation method thereof and application thereof in aging resistance Download PDFInfo
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- CN113768849B CN113768849B CN202010518430.8A CN202010518430A CN113768849B CN 113768849 B CN113768849 B CN 113768849B CN 202010518430 A CN202010518430 A CN 202010518430A CN 113768849 B CN113768849 B CN 113768849B
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- A—HUMAN NECESSITIES
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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Abstract
The orchid bud embryo composite microcrystal capsule body composition comprises the following components: a carrier formed into a shell-like structure; and an extract of orchid bud embryo inlaid in or coated by the shell-like structure, the preparation method of the extract of orchid bud embryo comprises: picking up growing bud embryo tissue from the top bud or side bud of orchid; removing the parietal film of the growing bud embryo tissue; grinding the growing bud embryo tissue into powder at low temperature; covering and soaking the powder with water or ethanol to obtain a mixed solution; vibrating the mixed solution 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. The composition of the invention can prevent the deterioration of the orchid bud embryo extract so as to provide stable efficacy. In addition, the composition of the invention can promote cell detoxification, energy activation, anti-saccharification and anti-aging.
Description
Technical Field
The invention relates to a microcrystal capsule composition, and in particular relates to an orchid bud embryo composite microcrystal capsule composition. In addition, the invention also relates to a preparation method of the composition and application of the composition in promoting cell detoxification, energy activation and aging resistance.
Background
Taiwan has mature orchid planting and tissue culture technology, which includes a great variety and abundance of varieties, pure-maturing tissue culture skills, virus inspection capability, engineering technology for mass production, etc. Moreover, taiwan currently has the advantage of promoting the orchid industry, including unique natural geographic conditions, diversified varieties in the open world, breeding expert groups in technical centers, super-horizontal agricultural technological bases, various agricultural technology auxiliary institutions such as agricultural test improvement and research and development all over the island, information industry and modern logistics channels in the known world and the like.
In taiwan, the annual yield of butterfly orchid is about 1 hundred million 3,294 ten thousand, the annual total amount of the butterfly orchid is about 4 ten million, the butterfly orchid accounts for 20% of the global supply, and the selling areas are throughout europe and america and japan, so that the butterfly orchid needs to be propagated in large quantities by using advanced biotechnology to meet the supply demand. The cultivation and propagation method of orchid is as follows: five methods, namely a sowing propagation method, a pedicel germination propagation method, a broken core germination propagation method, a stem cutting propagation method and a tissue culture method, can be selected according to the variety characteristics and requirements. The tissue culture method is to culture the terminal bud part with meristematic capacity; in addition, the growth point of the top of the stem is usually produced by adopting a bud multiplication (bud growth) or a pseudo-spheroid seedling generation induction mode, so that the bud multiplication can reduce the top bud advantage, the side buds can easily grow out, and the bud is proliferated in the mode.
In addition to ornamental use, orchid has been widely used in recent years as a raw material for products such as cosmetics and maintenance products, so that the economic value of orchid is greatly improved and the demand of orchid is greater. The orchid beards are extracted to be an extract, and then made into cosmetics or maintenance products. Because the extraction sites of orchid are different, proper extraction technology is matched, the extraction flow is changed, the extracted components are different, and the whitening effect, the antioxidation effect or the anti-aging effect can be affected.
Since human skin is often subjected to external environmental influences such as ultraviolet rays, air, or temperature, there is a risk of accelerating skin aging or suffering from skin cancer. In the case of skin aging, extrinsic factors account for 60%, while ultraviolet rays account for up to 60%. In research on skin aging, it is found that ultraviolet rays not only cause melanocytes to be active, so that skin is speckled, but also dermis tissues are degenerated, skin oxidization is accelerated, free radicals are generated, and the free radicals damage fibroblasts, cause collagen degeneration or reduce the content of collagen, or cause elastic fibrous tissues to degenerate and generate aging wrinkles, and further accelerate DNA damage in cells, so that the renewal capacity of skin cells and the replacement speed of old cells by new cells are reduced.
At present, aiming at various skin problems, different cosmetic raw materials are developed, and other additives with special effects are added, wherein the active ingredients of the additives are as follows: anthocyanin, flavonoid, polyphenol, etc. have quite remarkable effects on skin whitening, antioxidation, anti-aging, crease resistance, etc., so that various plant extraction active ingredients are commonly added into a plurality of cosmetic or maintenance commodities on the market. The extraction techniques to be used are different based on different plant species, different parts, or different requirements and effects of the extracted components. Taking orchid as an example, the extraction technology of orchid is also used as an additive for cosmetics or maintenance products in many different ways, and the extraction steps, processes and environmental parameters are also different.
Chinese patent application No. 201510295194.7 discloses an orchid extract, a preparation method and application thereof, wherein the orchid is of the genus Phalaenopsis, and the orchid extract is prepared by the following steps: (1) extraction: mixing orchid with solvent in a weight ratio of 0.5:1 to 10:1 mixing, crushing or breaking wall to obtain orchid pulp, wherein the solvent is water, alcohol or alcohol aqueous solution, and the alcohol is ethanol, propanol or butanol; (2) solid-liquid separation: carrying out solid-liquid separation on the orchid pulp, and leaving liquid orchid filtrate; (3) Activity partitioning: purifying the separated orchid filtrate by a molecular sieve to obtain orchid extraction screening liquid; (4) concentrating: concentrating the purified orchid extraction screening liquid by molecular sieve treatment or vacuum or steaming to obtain active precipitate or active extractive liquid.
Chinese patent publication No. CN105878122A discloses a natural extract cosmetic with freckle removing and whitening effects, which comprises the following effective components of herba lycopi extract and herba lycopi extract, wherein the weight ratio of the herba lycopi extract to the herba lycopi extract is 2-4: 1. the preparation method of the extract comprises the following steps: extracting dried herba Lycopi or herba Lysimachiae Foenumgraeci with ethanol under reflux, mixing filtrates, concentrating until no ethanol smell exists to obtain ethanol extract concentrate; diluting with water, and sequentially extracting with petroleum ether, ethyl acetate and water-saturated n-butanol; dissolving n-butanol extract with water, filtering, collecting the active ingredients with macroporous resin, and spray drying. The freckle-removing and whitening cosmetic disclosed by the patent takes plant extracts as effective components, and the freckle-removing and whitening effects are maximized by controlling the content ratio of the herba lycopi extracts to the herba lycopi extracts.
Taiwan patent application No. 102140137 discloses an extract of petals of butterfly orchid, a preparation method and application thereof, wherein the extract is prepared by the following steps: extracting the petals of the butterfly orchid with supercritical carbon dioxide, thereby obtaining fat-soluble extracts and residues of the petals of the butterfly orchid; and extracting the residue with water to obtain water-soluble extract of petal of Phalaenopsis amabilis, which can be used for whitening skin, improving skin moisture retention, and preventing or delaying skin aging.
In the 1 st case, the whole orchid is extracted, solid-liquid separated, activity divided, concentrated and other processes are carried out; in case 2, the whole herba Lycopi or herba Lysionoti Pauciflori is subjected to drying, hot reflux extraction, concentration, dilution, dissolution, filtration and other steps; in case 3, the petals of the butterfly orchid are used for supercritical carbon dioxide extraction and water extraction. However, the orchid parts used in the above cases are different, the 1 st and 2 nd cases are performed by the whole plant, the 3 rd case is performed by petals only, and the obtained extraction components and contents are different due to different extraction methods, processes, use equipment, or environmental related parameters.
Disclosure of Invention
Whether whole orchid or petal is used for extraction, the content and effect of the extracted active ingredients are still limited. The Chinese patent publication No. CN105878122A and the Taiwan patent application No. 102140137 disclose that the high temperature process is performed, and the chemical structure of the active ingredients is damaged or other possibly active substances volatilize, so that the whitening, antioxidation or anti-aging effects of the subsequently prepared cosmetics or maintainers are affected. Furthermore, the whole orchid is extracted in the past, so that all parts of the orchid can be extracted and obtained no matter whether the activity is improved or the activity is inhibited, and the total activity quality of the obtained extract is limited.
The stem node of the plant usually has growing points to provide excellent growing functions, the cells of the growing points can be rapidly divided and differentiated to generate new buds, the bud axes can be continuously elongated as if the stem cells of animals are regenerated, and the content and the benefit of the obtained active ingredients can be quite high if the stem node growing point cells of the plant are extracted. However, the well-known orchid-related extraction techniques do not extract the stem node sites; the present inventors have thus provided a technique for extracting the growing points of orchid, so as to obtain an extract of orchid bud embryo with high content of active ingredients and good biological effect.
Accordingly, an object of the present invention is to provide an extract product of orchid embryo tissue, which mainly extracts and collects the embryo regeneration cells of orchid with high active ingredient, and the prepared extract has high active ingredient content, and has the effects of promoting cell detoxification, energy activation, anti-saccharification, anti-aging, anti-oxidation, whitening, promoting ATP generation, maintaining telomere function, and protecting and repairing against light injury caused by UVB, and can be added into cosmetics or maintenance products.
It is still another object of the present invention to provide an extract product of orchid embryo tissue, which is operated at a non-high temperature to reduce heat loss due to temperature, avoid volatilization of low boiling point and effective substances, and retain growth active ingredients of the growth point tissue.
Still another object of the present invention is to provide a composite microcrystal capsule containing an extract of orchid embryo tissue, which can protect the extract of orchid embryo tissue, keep the extract of orchid embryo tissue from deterioration and maintain stability for a long time to maintain bioactivity.
It is still another object of the present invention to provide a composite microcrystal capsule containing the extract product of orchid bud embryo tissue, which has a high transdermal penetration capability to effectively deliver the extract product of orchid bud embryo tissue into the skin.
The invention provides an orchid bud embryo composite microcrystal capsule body composition, which comprises the following components: a carrier formed in a shell-like structure; and the orchid bud embryo extract is inlaid in or coated by the shell-shaped structure, and 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 film of the growing bud embryo tissue; grinding the growing bud embryo tissue into powder at low temperature; covering and soaking the powder with water or ethanol to obtain a mixed solution; vibrating the mixed solution 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.
The orchid embryo extract in the microcrystal capsule composition is prepared under a non-high temperature process, can avoid volatilization of low boiling point and effective substances and keep the growth activity of embryo tissues, has high total polyphenol and total flavone content, and has the effects of promoting cell detoxification, energy activation, saccharification resistance, aging resistance, oxidation resistance, whitening, ATP generation promotion, telomere maintenance and protection and repair of photodamage caused by UVB when being added into cosmetics or maintenance products. However, the invention protects the orchid bud embryo extract through the shell-like structure formed by the carrier, thereby providing the orchid bud embryo extract with long-time stability without deterioration, and further having no damage to the effects of promoting cell detoxification, energy activation, anti-saccharification, anti-aging, anti-oxidation, whitening, promoting ATP generation, maintaining telomere function and protecting and repairing against light damage caused by UVB. Thus, the microcrystalline capsule composition of the present invention may be prepared into a composition for promoting skin cell detoxification and skin cell energy activation, anti-glycation, anti-aging, anti-oxidation, whitening, promoting ATP generation, maintaining telomere function, and protecting and repairing against photodamage caused by UVB, and the composition may be a pharmaceutical, cosmetic, care, fragrance, or human cleaning product, but is not limited thereto.
Drawings
FIG. 1 is a photograph showing the change in appearance of the extract of orchid embryo after being left for different days at different ambient temperatures.
FIG. 2 is a statistical graph of the free radical scavenging experiments to demonstrate the relative free radical scavenging activity of the orchid embryo extract after various days of exposure to various ambient temperatures.
FIG. 3 is a transmission electron micrograph showing the appearance of a orchid bud embryo composite microcrystal capsule composition.
FIG. 4 is a high performance liquid chromatography chart illustrating the stability of the orchid embryo extract in water of the orchid embryo extract and orchid embryo composite microcrystalline capsule composition.
FIG. 5 is a graph showing autophagy results of the orchid bud embryo composite microcrystal vesicle composition.
FIG. 6 is a graph of the results of ROS reactive oxide experiments to illustrate the inhibition of ROS reactive oxide induction by a orchid bud embryo complex microcrystalline vesicle composition.
FIG. 7 is a graph of the results of an ATP content experiment to illustrate the promotion of ATP content in skin cells by the orchid bud embryo composite microcrystal vesicle composition.
FIG. 8 is a graph of SA- β -gal experimental results to demonstrate the retardation of skin cell aging by the orchid bud embryo complex microcrystalline vesicle composition.
FIG. 9 is a photograph of a PCR electrophoresis to illustrate the effect of the orchid bud embryo composite microcrystal capsule composition on SIRT-1 gene expression.
Fig. 10 is a graph of qPCR experiment results to illustrate the effect of the orchid bud embryo composite microcrystal vesicle composition on mTOR, TOP1, TPP1, etc. gene expression.
Detailed Description
In order to make the above and/or other objects, effects and features of the present invention more comprehensible, preferred embodiments accompanied with figures are described in detail below:
one embodiment of the present invention provides an orchid bud embryo composite microcrystal capsule composition comprising: a carrier and an extract of orchid bud embryo.
The carrier is formed into a shell structure, and examples thereof may be hydrogenated soybean lecithin or d-alpha-tocopheryl polyethylene glycol 1000succinate, but are not limited thereto. In a preferred embodiment, the carrier comprises hydrogenated soy lecithin and d-alpha-tocopheryl polyethylene glycol 1000succinate, and the weight percent of hydrogenated soy lecithin is 50 to 99% and the weight percent of d-alpha-tocopheryl polyethylene glycol 1000succinate is 1 to 50% based on the total weight of the carrier.
The extract of the orchid bud embryo is inlaid in or coated by the shell-like structure. In a preferred embodiment, the orchid bud embryo extract is 2% to 10% by weight based on the total weight of the composite microcrystal capsule composition. In another preferred embodiment, the total concentration of the orchid bud embryo extract and the carrier is 0.1 to 20mM by volume mole based on the total volume of the composite microcrystal capsule composition. The preparation method of the orchid bud embryo extract is described in detail below:
first, the tissue of the bud embryo of the growing point is extracted from the terminal bud or the lateral bud of the orchid, and the example of the orchid is, but not limited to, the butterfly orchid.
Next, the parietal membrane of the growing bud embryo tissue is removed. In a preferred embodiment, the tissue of the growing bud is flash frozen with liquid nitrogen to remove the parietal film.
Then, the growing bud embryo is ground into powder at low temperature. In a preferred embodiment, the tissue of the growing bud embryo with the wall film removed is cut into blocks, then liquid nitrogen is added to solidify the tissue blocks, and finally the tissue blocks are ground into powder. In order to avoid the temperature generated by the grinding tool or the grinding process affecting the active ingredients obtained later, the grinding tool (such as a tissue grinding bowl and a tissue grinder) can be preset at-80 ℃ for cooling.
Subsequently, 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 liquor is oscillated at a low temperature with a total energy of 300 to 600W by using an ultrasonic oscillation extraction apparatus. The step is to extract the effective components from the embryo tissue of the growing bud into the solvent by shaking. In a preferred embodiment, the shaking time is 60 minutes. In another preferred embodiment, each time of shaking for 3 minutes, the rest is performed for 2 minutes until the total shaking time reaches 60 minutes (20 times total shaking). In a further preferred embodiment, the oscillation temperature is 50 to 60 ℃.
Finally, the mixture is centrifuged to obtain a supernatant, and the solvent of the supernatant is removed to obtain the orchid embryo extract. In a preferred embodiment, the mixture is centrifuged at 15,000rpm for 15 minutes at 4℃to obtain a supernatant. In another preferred embodiment, the supernatant is vacuum filtered to obtain an extract, and the solvent of the extract is removed by a vacuum concentrator and a freeze dryer to obtain the extract of the orchid embryo.
In addition, another embodiment of the invention provides a preparation method of the orchid bud embryo composite microcrystal capsule body composition, which is described in detail below:
after the orchid bud embryo extract according to upper Wen Qude, the orchid bud embryo extract is mixed with a carrier raw material, and the carrier raw material is dispersed to form a colloidal complex. In a preferred embodiment, the carrier material is dissolved in a solvent, the solvent is removed by a spin-on reduced pressure concentrator to form a lipid film, and then the extract of orchid sprout is added to the lipid film to disperse the lipid into a colloidal complex.
Then, the colloid compound is oscillated by a high-power ultrasonic oscillator to obtain the orchid bud embryo compound microcrystal capsule composition. In a preferred embodiment, the shaking time is 30 minutes and the shaking temperature is 45 ℃.
The above embodiments will be illustrated by the following examples:
example 1: preparation of orchid bud embryo extract and stability test
And picking up the bud embryo tissue of the growing point from the top bud or the side bud of the orchid. Secondly, a rapid liquid nitrogen freezing ultrasonic oscillation method is adopted, and the specific operation is as follows: rapidly freezing bud embryo tissue with liquid nitrogen to remove wall film, and presetting a tissue grinding bowl and a tissue grinder at-80deg.C to cool; then, cutting the tissue with the wall film removed into blocks by using a cutter, putting the blocks into a grinding pot, adding a little liquid nitrogen to solidify the tissue blocks, and grinding the tissue blocks into powder; then, placing the tissue powder into ultrasonic oscillation extraction equipment, and covering and soaking the tissue powder by pure water or ethanol; then, the mixed solution of the solvent and the powder is vibrated for 60 minutes (vibrating for 3 minutes and resting for 2 minutes until vibrating time reaches 60 minutes) at low temperature with total energy of 300 to 600W; after centrifugation at 15,000rpm for 15 minutes at 4 ℃, the supernatant was collected and vacuum filtered to make an extract. Finally, the solvent of the extract was removed using a reduced pressure concentrator (Rotary evaporator R-2000) and a freeze dryer (Eco nomic freeze dryer CT 5020D) to obtain an extract of orchid embryo (hereinafter used interchangeably with symbol "OG").
For stability of Jie Lan flower bud embryo extract, the appearance was observed after being placed at 25℃or 50℃for 7 days, respectively, and the color Lab value was measured with a color difference meter (MET-CM 6).
As shown in fig. 1 and table 1, the appearance and color Lab values were not significantly changed after OG was left at 25 ℃ for 7 days; in contrast, after 7 days at 50℃the appearance became deep and the L value was reduced from 22.3 to 20.1, the a value was increased from 3.4 to 4.7 and the b value was changed from 3.4 to 4.2.
Table 1, appearance change of OG after different days at different ambient temperatures
DPPH- (2, 2-diphenyl-1-picrylhydrol) is a stable free radical and a purple DPPH ethanol solution is used to generate a specific light absorption value under the irradiation of 517nm wavelength. If DPPH and free radicals react with the sample, the absorbance decreases; therefore, the ability of the sample to scavenge DPPH radical can be known from the change in absorbance. Specifically, the lower the absorbance, the stronger the DPPH radical scavenging ability of the sample.
Here, each concentration was set to be different before and after OG was set at 50℃for 7 days, and 90. Mu.L of freshly prepared 100. Mu.M DPPH. Ethanol solution was added to each concentration and reacted in a 96-well plate. Next, the absorbance at 517nm of the reaction solution was measured by an enzyme immunoassay.
As shown in fig. 2, OG has a radical scavenging capacity of 82.3% before being placed at 50 ℃; the radical scavenging capacity after 7 days at 50℃was 73.7%.
Example 2: preparation of orchid bud embryo composite microcrystal capsule composition and related test
d-alpha-tocopheryl polyethylene glycol 1000succinate (d-alpha tocopheryl polyethylene glycol 1000succinate, tpgs) is a vitamin E derivative, is safe and has excellent oral biocompatibility with an effective active ingredient. TPGS is currently used in drug delivery systems for liposomes to increase liposome stability. The formation of microcells from TPGS has been studied in the past as a carrier for anticancer drugs. There have also been studies in the past on the addition of TPGS to polymeric carriers as carriers for controlled release of anticancer drugs (Cao & Feng, 2008). Thus, TPGS is an excellent efficacy adjunct to the microcrystalline capsule composition of this example, and also has anti-aging and antioxidant activity itself.
Specifically, this example prepared a orchid embryo composite microcrystal capsule composition from hydrogenated soy lecithin (hydrogenated soybean lecithin, HL) and d-alpha-tocopheryl polyethylene glycol 1000succinate, the detailed procedure is as follows: adding HL and TPGS into a solvent according to an experimental design proportion for dissolution (the weight percentage of the HL is 50-99 percent and the weight percentage of the TPGS is 1-50 percent based on the total HL and TPGS), and removing the solvent by using a rotary reduced pressure concentrator to form a lipid film on the wall of the flask; then, OG is added to interact with the film, so that lipid is dispersed to form a colloid compound; finally, the gel complex was oscillated at 45℃for 30 minutes using a high-power ultrasonic oscillator to obtain a orchid embryo composite microcrystal capsule composition (hereinafter, used interchangeably with the symbol "OG-L") having a volume molar concentration of 0.1 to 20mM.
Observing the particle structure state of the OG-L by a penetration electron microscope (transmission electron microscope, TEM), attaching the OG-L on a carbon-plated copper mesh on a polyvinyl formal support film (formvar film) by using liposome dispersion liquid drops before observation, standing for 2 minutes to enable sample particles to be embedded on the copper mesh, and wiping by using wiping paper to remove redundant samples; and then dropwise adding 0.5wt% of uranyl acetate to dye a sample on a copper mesh, standing for 2 minutes, wiping to remove excessive dye by wiping, storing in a drying oven for 12 hours, and observing the morphological structure of OG-L by using a penetration electron microscope.
As shown in FIG. 3, OG-L forms a colloidal particle complex with a particle size of 50 to 400 nm.
OG-L particle size and interfacial potential were measured using a dynamic light scattering particle size and interfacial potential analyzer (Dynamic Light Scattering, DLS, model Brookhaven 90Plus Particle Size Analyzer, available from Brookhaven inc., u.s.a.). The analysis results were: the average particle size (average particle size, A.P.S.) of OG-L was 286.13.+ -. 10.17nm, the distribution coefficient (polydispersity index, pd.I) was 0.217.+ -. 0.01, the average interface potential (average zeta potential, A.Z.P.) was-7.13.+ -. 4.51mV, and the OG binding ratio (combined percentage, C.P.) was 94.31.+ -. 3.76% (OG concentration ratio in OG-L).
OG was quantitatively analyzed using a high performance liquid chromatograph (high performance liquid chromatography, HPLC), using a pump model L-7100, an auto-injector model L-7200, an ultraviolet light detector (UV-vis) model L-4200, all available from Hitachi (Japan), and a column model Microsorb-MV 100-5C18 (250mm x 4.6mm i.d.,5 μm particle size).
As shown in fig. 4, the change of the OG active ingredient concentration in the OG aqueous solution and the OG-L aqueous solution with time is illustrated; it can be seen that the OGs in OG-L are stable in water and easy to store compared to pure OGs. In other words, OG-L can improve the problem of unstable activity of OG in water.
Example 3: skin cell autophagy assay
Autophagy is the coating of aged organelles or protein aggregates and the delivery to a solution for resolution into small molecules. If the cells cannot resist the damage caused by the oxidative pressure, the cells will be in an aging state.
Human skin keratinocyte HaCaT cells were 1.5x10 in density 5 The cells/mL were cultured in 96-well plates and grown in a 5% carbon dioxide incubator at 37℃for more than 24 hours. Thereafter, OG-L reaction was added and after the reaction time was reached, the supernatant was removed and washed with PBS. Next, the mixture was passed through a reactor of 20mJ/cm 2 After UVB irradiation, fresh medium containing 0.1mM hydrogen peroxide was added for 1 hour. Then, autophagosome detection reagent working solution was added and placed in an incubator for reaction for 15 minutes to 1 hour, and then the cells were washed 3 to 4 times using a wash buffer. Finally, quantitative PBS was added and absorbance was measured with 360nm excitation light and 520nm emission light (BioTek, synergy TM 2，USA)。
As shown in FIG. 5, the autophagy of human skin keratinocyte HaCaT can be induced only under the action of oxidation inducer (UVB and hydrogen peroxide), but the autophagy can be improved under the action of the oxidation inducer together with OG-L to increase the oxidation pressure resistance and toxin expelling ability of the cells. Taken together, OG-L promotes autophagy of skin cells against oxidative stress.
Example 4: cell active oxide content assay
To assess whether OG-L reduced ROS reactive oxide production by hydrogen peroxide-induced skin cells, the reactive oxide content was quantitatively analyzed with the fluorescent label DCFH2DA (2 ',7' -dichlorofluorescin diacetate), detailed procedure as follows:
human skin keratinocyte HaCaT cells were 1.5x10 in density 5 The cells/mL were cultured in 96-well plates and grown in a 5% carbon dioxide incubator at 37℃for more than 24 hours. Thereafter, OG-L reaction was added and after the reaction time was reached, the supernatant was removed and washed with PBS. Next, fresh medium containing 0.1mM hydrogen peroxide was added for 1 hour, then the medium was removed, and after adding fresh medium containing 10. Mu.M DCFH2DA for one hour under light protection, the medium was removed. Finally, quantitative PBS was added and absorbance was measured at 502nm for excitation light and at 524nm for emission light (BioTek, synergy) TM 2，USA)。
As shown in fig. 6, the ROS reactive oxide content of the cells without hydrogen peroxide was set to 100.0±3.1%; the relative content of the reactive oxide of the cellular ROS is 121.0+/-6.1% under the action of hydrogen peroxide only; under the action of hydrogen peroxide and OG-L, the relative content of reactive oxide of ROS in cells is 113.0 +/-4.2%. This indicates that OG-L protects cells from 66.3% oxidative damage. In conclusion, the cells can reduce oxidative damage induced by the hydrogen peroxide oxidation inducer after the cells are acted by OG-L, so that the cells are protected from damage caused by oxidative stress.
Example 5: adenosine triphosphate activation test
ATP is a kind of nucleotide, which is a "molecular cargo" for cell energy transfer, stores and transfers chemical energy, so ATP is a direct source of life movement energy, and the energy required by the human body comes almost from ATP, for example: heart beating, muscle movement, and different cells perform different biological functions. On the contrary, the organs and tissues of the human body lack of ATP are sequentially struck, and physiological phenomena such as heart failure, muscle soreness, or easy fatigue occur.
Human skin keratinocyte HaCaT density 1.5x10 5 The cells/mL were grown in 24-well plates and grown in a 5% carbon dioxide incubator at 37℃for more than 24 hours. Then, OG-L was added to the mixture to effect the mixture for 24 hours, and the mixture was placed in an incubator to effect the mixture for another 24 hours. After the reaction time, the culture solution and the sample are removed, PBS is added for cleaning, and then a new culture solution is replaced. Next, after 30 minutes of action by adding lysies buffer, centrifugation was performed at 1,500rpm for 2 minutes, after which 50. Mu.L of the supernatant was taken into a white 96-well plate, and absorbance was measured using ATP determination kit (Molecular Probes, eugene, OR, USA) (BioTek, synergy) T M2, USA), intracellular ATP content (μmol/g-cell) was assessed.
As shown in FIG. 7, after the action of OG-L, the ATP content of the cells was higher than that of the cells without OG-L (control group).
Example 6: anti-glycation test >
Aging is an irreversible phenomenon of the human body, and after cell division for many generations, the cell is possibly excessively stimulated by the outside to cause growth arrest and aging. Senesence-associated beta-galactose (SA-beta-gal) is overexpressed in senescent cells and is one of the indicators of cellular Senescence.
Human skin keratinocyte HaCaT cells were 1.5x10 in density 5 The cells/mL were cultured on a 24-well plate, and after 24 hours cell attachment, the culture broth was removed. Pretreatment with OG-L was carried out for 24 hours, followed by 50mJ/cm 2 The cells were irradiated with UVB and incubated with serum-free medium for a further 24 hours. After the reaction time, stained with senescence β -galactosidase staining kit (Cell Signaling Technology, danvers, MA, USA) and then observed with a microscope and counted for blue aged cells.
As shown in FIG. 8, it was shown that the beta-galactose of the cells was greatly increased after UVB irradiation; and the cells can reduce the activation of beta-galactose caused by UVB after UVB irradiation and OG-L. In addition, positive control group 25 μg/mL vitamin C was effective in inhibiting β -galactose activity, particularly UVB-induced β -galactose activation.
Example 7: long life gene SIRT-1 expression test
Although aging is an irreversible phenomenon in the human body, activation of some genes maintains cellular functions and promotes health of individuals, and these genes are called "longevity genes", SIRT-1 being one of typical representative examples. Previous studies have demonstrated that up-regulating SIRT-1 expression is effective in protecting cells from repair and maintaining normal function, and preventing disease caused by aging of cells.
The expression of the OG-L regulatory longevity gene SIRT-1 was analyzed by performing experiments using reverse transcription polymerase chain reaction (reverse transcription-PCR, RT-PCR).
Human skin keratinocyte HaCaT cells were 1.5x10 in density 5 The cells/mL were cultured on a 24-well plate, and after 24 hours cell attachment, the culture broth was removed. Next, OG-L was added for pretreatment for 24 hours, followed by 50mJ/cm 2 The cells were irradiated with UVB and then incubated for a further 24 hours with serum-free medium. After removal of the culture, the cells were removed with Trypsin, and the cells were collected with PBS and the supernatant removed by centrifugation. Then, 1mL REzol was added TM C&T and the cells were lysed by mixing homogeneously for 5 min at room temperature. 200. Mu.L of chloroform was added thereto and mixed uniformly for 15 seconds, followed by reaction at 4℃for 5 minutes to extract RNA. Then, after centrifugation at 12,000rpm for 10 minutes at 4℃the upper transparent layer was taken into a microcentrifuge tube which was soaked with DEPC water and sterilized. Thereafter, an equal volume of isopropanol was added and mixed well and allowed to stand at 4℃for 5 minutes to precipitate RNA. Further, the mixture was centrifuged at 12,000rpm for 10 minutes at 4℃and the supernatant was removed. After washing RNA with 200. Mu.L of 75% glacial alcohol, it was centrifuged at 12,000rpm for 10 minutes at 4℃and the supernatant was removed. Finally, the remaining moisture in the centrifuge tube was evaporated completely by inverted drying in the shade in a sterile operating station, and 100 μl of 0.1% depc water was added to dissolve RNA in water and stored at-80 ℃. In addition, after taking 2. Mu.L of RNA to 198. Mu.L of 0.1% DEPC water, 100. Mu.L was mixed uniformly and the absorbance at OD260nm and OD280nm was measured by spectrobrightness in a micro-quartz tube (BioTek, synergy) TM 2，USA)。
After mixing 3. Mu.g RNA with an appropriate amount of DEPC water, 1. Mu.L Oligo (dT) was added 18 The primer was allowed to act at 70℃for 2 minutes and quickly transferred to ice. Next, 4. Mu.L of 10xMMLV RT buffer solution, 1. Mu.L of dNTP mix (10 mM for each dNTP), 0.5. Mu. L recombinant RNase inhibitor (1 unit/ml), and 1. Mu. L MMLV reverse transcriptase (5 unit) were added, respectively, to make the total volume 20. Mu.L. Uniformly mixing the mixed solution, allowing to act at 42deg.C for 1 hr, heating at 94deg.C for 5 min, and removing MMLV reverseThe reaction was terminated by the se trans-script activity to complete the preparation of cDNA template. Finally, 80. Mu.L of DEPC water was added and stored at-20℃until use.
mu.L of cDNA was taken into a microcentrifuge tube, 5. Mu.L of 10x reaction buffer, 0.8. Mu.L of 10mM dNTPs (200 mM each) and 1. Mu.L of 50mM forward (5'-tcgcaactatacccagaacatagaca-3') and reverse (5'-ctgttgcaaaggaaccatgaca-3'), taq DNA polymerase (5 units/. Mu.L) primers each were added, and finally deionized water was added to bring the total volume to 50. Mu.L. After mixing uniformly, placing in an automatic temperature cycle machine (Techne Progene) to carry out PCR reaction under the following reaction conditions: denaturation reaction (98 ℃ C.) for 3 min; next, denaturation reaction, 94 ℃, adhesion reaction, 60 ℃, synthesis reaction, 72, and polymerase chain reaction for 1 minute each were carried out for a total of 35 cycles. After the reaction, a proper amount of reaction products are taken for 2% agarose gel electrophoresis analysis of the reference gene (beta-actin) and the target gene. Further, quantification was performed by Image software (Image J).
As shown in FIG. 9, SIRT-1 expression was 1.0 (control group) without UVB injury, and was significantly reduced to 0.2 after UVB irradiation. In addition, after UVB irradiation and OG-L function, the expression amount of SIRT-1 can be obviously improved to 0.6, which means that OG-L can effectively regulate and control skin cells to be protected from damage of UV to a longevity gene SIRT-1.
Example 8: cell rejuvenation gene mTOR and telomerase regulatory gene TERT expression test >
mTOR is a serine-threonine protein kinase, an important factor in aging and canceration, which promotes cells to absorb heat and close autophagy mechanism to prevent cell turnover, so in recent years, research on inhibition of mTOR expression indicates that cells can be regenerated by inhibiting mTOR expression in aging cells.
Telomeres function to maintain eukaryotic chromosomal integrity and control the cell division cycle, and after cell division, telomeres stabilize the replicated DNA, ensuring genetic sequence integrity. However, after each cell division, telomeres shorten; when telomeres are depleted, cells initiate apoptosis, so telomeres are the best marker of aging. Telomerase can supplement telomeres, prolong cell division times, and slow aging speed. TOP1 and TPP1 are telomere protection proteins, and promotion of expression of the telomere protection proteins can promote protection of telomeres.
Here, the expression of the OG-L regulatory rejuvenation gene mTOR and telomerase regulatory gene TERT-related genes was analyzed by real-time quantitative PCR (qPCR) experiments.
Human skin keratinocyte HaCaT cells were 1.5x10 in density 5 cell/mL was cultured in a 6-well plate, and after 24 hours of culture, OG-L mixed serum-free medium was added thereto for 72 hours. After removal of the culture, the cells were removed with Trypsin, and the cells were collected with PBS and the supernatant removed by centrifugation. Then, 1mL REzol was added TM C&T and the cells were lysed by mixing homogeneously for 5 min at room temperature. 200. Mu.L of chloroform was added thereto and mixed uniformly for 15 seconds, followed by reaction at 4℃for 5 minutes to extract RNA. Then, after centrifugation at 12,000rpm for 10 minutes at 4℃the upper transparent layer was taken into a microcentrifuge tube which was soaked with DEPC water and sterilized. Thereafter, an equal volume of isopropanol was added and mixed well and allowed to stand at 4℃for 5 minutes to precipitate RNA. Further, the mixture was centrifuged at 12,000rpm for 10 minutes at 4℃and the supernatant was removed. After washing RNA with 200. Mu.L of 75% glacial alcohol, it was centrifuged at 12,000rpm for 10 minutes at 4℃and the supernatant was removed. Finally, the remaining moisture in the centrifuge tube was evaporated completely by inverted drying in the shade in a sterile operating station, and 100 μl of 0.1% depc water was added to dissolve RNA in water and stored at-80 ℃. In addition, after taking 2. Mu.L of RNA to 198. Mu.L of 0.1% DEPC water, 100. Mu.L was mixed uniformly and the absorbance at OD260nm and OD280nm was measured by spectrobrightness in a micro-quartz tube (BioTek, synergy) TM 2，USA)。
After mixing 3. Mu.g RNA with an appropriate amount of DEPC water, 1. Mu.L Oligo (dT) was added 18 The primer was allowed to act at 70℃for 2 minutes and quickly transferred to ice. Next, 4. Mu.L of 10x MMLV RT buffer solution, 1. Mu.L of dNTP mix (10 mM for each dNTP), 0.5. Mu. L recombinant RNase inhibitor (1 unit/mL), and 1. Mu. L MMLV reverse transcr iptase (5 unit) were added, respectively, to make the total volume 20. Mu.L. Mixing the above mixed solution, allowing to act at 42deg.C for 1 hr, heating at 94deg.C for 5 min, removing MMLV reverse transcriptase activity, and stopping reactionAnd (5) preparing a cDNA template. Finally, 80. Mu.L of DEPC water was added and stored at-20℃until use.
mu.L of cDNA was taken into a microcentrifuge tube, and qPCR analysis was performed by adding OmicsGreen 5 XqPCR masterMix (ROX) (Omics Bio) and 5. Mu.L of each 50mM forward and reverse primer were mixed uniformly. The reaction was carried out in a qPCR machine (MyGo Pro) under the following conditions: denaturation reaction (98 ℃ C.) for 3 min; subsequently, a polymerase chain reaction comprising denaturation reaction 94 ℃, adhesion reaction (annealing), synthesis reaction (extension), and 72 ℃ for 1 minute each was carried out for 25 to 35 cycles in total. Data usage 2 -ΔΔCt Expression to calculate the relative expression magnitude of the genes.
As shown in FIG. 10, OG-L down regulates mTOR gene expression and up regulates gene expression of telomere protection proteins such as TOP1 and TPP1, indicating that OG-L has anti-aging potential.
However, the above description is only of the preferred embodiments of the present invention, and should not be taken as limiting the scope of the invention; therefore, all such simple and equivalent changes and modifications as made by the claims and the disclosure of the present invention fall within the scope of the patent coverage 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.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1704046A (en) * | 2004-05-28 | 2005-12-07 | 财团法人工业技术研究院 | Liposome and its preparation process |
| CN108541590A (en) * | 2018-03-26 | 2018-09-18 | 上海数儒生物科技有限公司 | A kind of big white orchid lateral bud histocyte extracting process |
| CN108685808A (en) * | 2017-04-10 | 2018-10-23 | 大江生医股份有限公司 | Using orchid callus extract in the purposes for preparing a skin care compositions |
| CN110559682A (en) * | 2019-09-23 | 2019-12-13 | 北京天然纳美健康科技有限公司 | Plant extract nanoparticle and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1704046A (en) * | 2004-05-28 | 2005-12-07 | 财团法人工业技术研究院 | Liposome and its preparation process |
| CN108685808A (en) * | 2017-04-10 | 2018-10-23 | 大江生医股份有限公司 | Using orchid callus extract in the purposes for preparing a skin care compositions |
| CN108541590A (en) * | 2018-03-26 | 2018-09-18 | 上海数儒生物科技有限公司 | A kind of big white orchid lateral bud histocyte extracting process |
| CN110559682A (en) * | 2019-09-23 | 2019-12-13 | 北京天然纳美健康科技有限公司 | Plant extract nanoparticle and preparation method and application thereof |
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