WO2021196079A1 - 可活化能量、抗皱、抗发炎及美白的兰花芽胚组织萃取物 - Google Patents

可活化能量、抗皱、抗发炎及美白的兰花芽胚组织萃取物 Download PDF

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WO2021196079A1
WO2021196079A1 PCT/CN2020/082769 CN2020082769W WO2021196079A1 WO 2021196079 A1 WO2021196079 A1 WO 2021196079A1 CN 2020082769 W CN2020082769 W CN 2020082769W WO 2021196079 A1 WO2021196079 A1 WO 2021196079A1
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extract
orchid
tissue
bud embryo
buds
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PCT/CN2020/082769
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French (fr)
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梁家华
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梁家华
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Priority to PCT/CN2020/082769 priority Critical patent/WO2021196079A1/zh
Priority to AU2020439080A priority patent/AU2020439080A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/898—Orchidaceae (Orchid family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q13/00—Formulations or additives for perfume preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations

Definitions

  • the present invention relates to a plant extraction method, in particular to an extraction method applied to orchid bud embryo tissue extract, and the prepared extract can be used as a raw material for products such as medicines, cosmetics, skin care products, fragrances or human body cleaning products.
  • Taiwan, China has mature planting and tissue culture technologies, including a wide variety of rich and sophisticated tissue culture techniques, virus testing capabilities, and mass production engineering technologies.
  • the agriculture of Taiwan, China currently has the advantages to promote the orchid industry, including unique natural geographical conditions, a diversified variety that is world-renowned, a group of top-notch breeding experts, a super-level agricultural science and technology foundation, and agricultural experimentation and improvement throughout the province. Academic research and other various agricultural technology auxiliary institutions, world-renowned information industry and modern logistics channels, etc.
  • the "tissue culture method” mostly extracts the "apical bud” part with meristematic ability for cultivation; and the production of the “stem apex growth point” part mostly adopts bud proliferation (bud growth) or induced protosphere (PLBs) )
  • the meristem mode in which the buds proliferate, that is, to reduce or remove the dominance of the terminal buds, so that the lateral buds can easily grow out, and the buds are proliferated in this way.
  • orchids have also been widely used in cosmetics and skin care products in recent years as raw materials, which has increased the economic value of orchids, and the demand for orchids is greater; and orchids need to undergo extraction operations to become extracts.
  • appropriate extraction techniques must be used. If the extraction process changes, the extracted ingredients will be different. The whitening function, anti-oxidation and anti-aging effects that the extract can achieve will also be affected. Influence.
  • orchids for cosmetics such as "Orchid extract and its preparation method and application” disclosed in Chinese Invention Patent Application No. CN201510295194.7.
  • the orchid is of the genus Phalaenopsis.
  • the orchid extract is prepared by the following steps: (1) Extraction: The orchid and the solvent are mixed and crushed or broken at a weight ratio of 0.5:1 to 10:1 to obtain orchid pulp.
  • the solvent is water, Alcohol or alcohol aqueous solution, wherein the alcohol is ethanol, propanol, or butanol;
  • solid-liquid separation the orchid pulp extracted in step (1) is subjected to solid-liquid separation, leaving the liquid as the orchid filtrate;
  • Activity division the orchid filtrate separated in step (2) is treated and purified with molecular sieve membrane to obtain orchid extraction sieving liquid;
  • concentration the orchid extraction sieving liquid purified in step (3) is treated with molecular sieve membrane Concentrate, or partially concentrate in a vacuum or cooking method, to obtain an active precipitate and an active liquid active extract.
  • the preparation method of adenophorum adenophorum and spider plant extract is: extract the dried adenophorum or spider plant extract by refluxing with ethanol, combine the filtrate, and concentrate until there is no alcohol to obtain the ethanol extraction concentrate; then dilute with water and sequentially use petroleum ether , Ethyl acetate and water-saturated n-butanol extraction; the n-butanol extract is dissolved in water, filtered, the filtrate is enriched with a macroporous resin for active ingredients, and it is obtained by spray drying.
  • the spot-removing and whitening cosmetics provided by the invention use plant extracts as functional components, and by controlling the content ratio of the adenophora adenophorum extract and the spider plant extract, the spot-removing and whitening cosmetics can be maximized.
  • the first case involves the extraction, solid-liquid separation, activity division, and concentration of the whole orchid; the second case uses the whole orchid or spider plant to dry , Heat reflux extraction, concentration, dilution, dissolution and filtration; the third case is the operation process of using the petals of white phalaenopsis to perform supercritical CO 2 extraction, and then extract the residue with water.
  • the aforementioned prior art uses different parts of orchids for extraction. In the first and second cases, the whole plant is used, and the third case uses only the petal parts.
  • the extraction method, process, equipment and environment-related parameters are different. The composition and content of the extract obtained will also be different.
  • the extraction process in the second and third cases mentioned above involves high temperature steps, which may cause extraction Reduced activity of substances or volatilization of effective substances, resulting in poor whitening, anti-oxidation and anti-aging effects of makeup and maintenance products.
  • the entire orchid is used for extraction, so that all parts of the orchid are not active The inventory is good or bad.
  • the activity of the produced extracts will be limited on average and the quality cannot be improved.
  • the existing technology uses alcohol or alcohol aqueous solution as the solvent for the extraction method, except for the use of equipment In addition to the high cost, volatile gases are generated during the process and there are safety concerns.
  • the stem node part of orchids due to the excellent growth function of the stem node part of orchids, this part usually becomes a growth point.
  • the cells at this growth point have the characteristics of rapid division and differentiation to produce new buds, and the bud axis can be continuously extended. It is as long as an animal’s stem cell regeneration function. For example, it can extract cells from the growth points of orchid stem nodes for extraction.
  • the resulting extract has high active ingredient content and active benefits.
  • the extraction technology related to orchids in the prior art has not yet been targeted for orchid blooming.
  • the stem nodes of the plant are extracted or refined.
  • the inventor of this patent application provides an orchid growth point cutting and extraction technology to solve the aforementioned defects in the prior art to obtain an orchid bud embryo extract with high active ingredient content and good effect.
  • one of the objectives of the present invention is to provide an orchid bud embryo extraction method and its extract, mainly to provide a complete process for the cultivation, cutting and extraction of orchid bud embryos, and only extract and collect the most active ingredients of orchids
  • the regenerative plant cells at the growth point of the stem node are used for extraction.
  • the bud embryo extract produced contains high total polyphenols, total flavonoids and plant hormones. It is added to cosmetics and skin care products to have activating energy, anti-wrinkle, anti-wrinkle, and anti-wrinkle. Inflammation, whitening, anti-oxidation, anti-aging, protection and repair.
  • Another object of the present invention is to provide an orchid bud embryo extraction method and an extract thereof, which are induced and cultivated at the growth point of the stem node of a flowering plant, so that new side buds are grown and regenerated by cutting to obtain new side buds.
  • Plant tissues, this stem node growth point induction cultivation method can obtain a large number of new plant tissues, which can meet the needs of mass production.
  • Another object of the present invention is to provide an orchid bud embryo extraction method and an extract thereof.
  • the extraction method is operated at a non-high temperature to reduce heat loss caused by temperature, and also to avoid low boiling point and effective substance volatilization and retention Growth activity of growth point tissue.
  • a further object of the present invention is to provide an orchid bud embryo extraction method and its extract.
  • the semi-finished product extracted by ultrasonic extraction is then filtered and removed solvent to obtain buds with high activity and stability. Embryo extract.
  • the method for extracting orchid bud embryos disclosed in the present invention includes the following steps: (a) inducing regeneration of new lateral buds: cutting out several segments of the stem buds from the stem node of the flowering plant of the orchid, and performing cultivation operations Make it grow new lateral buds; (b) Cut the stem buds and extract the new lateral buds: After cutting the stem buds and extract the new lateral buds, take out the growth point tissue; (c) Remove the wall membrane: remove the small fragments of the growth point tissue Remove the wall membrane to become new plant tissue; (d) Collect new plant tissue: Collect the new plant bud embryo tissue of the new side buds at the growth point; (a) Ultrasonic extraction: Put the bud embryo tissue into the ultrasonic extraction equipment, and use it at low temperature.
  • the amount of pure water 5 times the weight of the germ tissue is used as the solvent to cover and soak the germ tissue to prepare a semi-finished product extract; and (b) Filtration and solvent removal operations: the semi-finished product extract is subjected to vacuum filtration before reuse The decompression concentrator removes the solvent from the semi-finished product extract to prepare the extract.
  • the whole process is carried out at non-high temperature, which can avoid the low boiling point and the volatilization of effective substances and maintain the growth activity of the bud tissue, and the extracted extract has a high content of total polyphenols and total flavonoids, which can be added to makeup Among the skin care products, it has the functions of activating energy, anti-wrinkle, anti-inflammatory, whitening, anti-oxidation, anti-aging and protection and repair.
  • the use of the above-mentioned extract disclosed in the present invention is to prepare a composition with activating energy, anti-wrinkle, anti-inflammatory, whitening, anti-oxidation, anti-aging, and protective and repairing effects, and this composition can be a medicine, Cosmetics, skin care products, fragrances or body cleaning products.
  • the use of the above-mentioned extract disclosed in the present invention is to prepare a composition that promotes the regeneration of mitochondria of skin cells, and the composition can be medicines, cosmetics, skin care products, fragrances or body cleansing products.
  • Fig. 1 is a schematic flow chart illustrating the method for extracting the growth points of the stem nodes of the flowering orchid plant disclosed in the present invention.
  • Figure 2 is a photomicrograph showing the effect of the extract disclosed in the present invention on the morphology of human skin keratinocytes.
  • Figure 3 is a flow cytometric analysis result diagram illustrating the effect of the extract disclosed in the present invention on the cell cycle of human skin keratinocytes.
  • Figure 4(A) is a photograph of DNA gel electrophoresis, illustrating the effect of the extract disclosed in the present invention on the form of plastid DNA.
  • Fig. 4(B) is a quantification result diagram of DNA gel electrophoresis, illustrating the influence of the extract disclosed in the present invention on the form of plastid DNA.
  • FIG. 5 is a diagram of the results of DNA gel electrophoresis, illustrating the influence of the extract disclosed in the present invention on the type of DNA marker.
  • Figure 6 is a graph showing the results of cell survival analysis, illustrating the effect of the extract disclosed in the present invention on cell survival.
  • FIG. 7(A) is a photograph of fluorescence detection, illustrating the influence of the extract disclosed in the present invention on the production of CPD as a photoproduct.
  • Fig. 7(B) is a graph showing the results of fluorescence detection and quantification, illustrating the influence of the extract disclosed in the present invention on the production of CPD as a photoproduct.
  • Figure 8 is a graph showing the results of cell senescence analysis, illustrating the effect of the extract disclosed in the present invention on cell senescence.
  • Fig. 9 is a graph showing the results of DNA gel electrophoresis, illustrating the effect of the extract disclosed in the present invention on the RNA expression of the SIRT-1 regulating longevity gene.
  • Fig. 10 is a graph showing the results of real-time quantitative PCR, illustrating the effect of the extract disclosed in the present invention on the RNA expression of the SIRT-1 regulating longevity gene.
  • an orchid bud embryo extraction method provided by the present invention is illustrated by the best-implemented Phalaenopsis V3 (Phalaenopsis Sogo Yukidian V3) variety.
  • the steps are as follows: (a) Inducing regeneration of new lateral buds; (b) cutting off stem buds and extracting new lateral buds; (c) removing wall membranes; (d) collecting new plant tissues; (e) ultrasonic extraction; (f) filtering and solvent removal operations; and (g) ) Is made into germ extract; among them:
  • the regeneration method is to form a new plant embryo through buds and other organs; the new plant tissue is a round shape with the same shape.
  • the solvent covers and soaks the bud embryo tissue and puts it in an ultrasonic device.
  • the water temperature is set at 50 to 60°C
  • the power is set to 300 watts
  • the time is set to 40 minutes.
  • the bud embryo tissue at the growth point is extracted by ultrasonic vibration. , To make semi-finished bud embryo extract.
  • the entire process can be carried out at a non-high temperature, which can avoid the volatilization of low boiling points and effective substances and maintain the growth activity of the growth point tissues.
  • the extracted extract has high content of total polyphenols, total flavonoids and plant hormones. It is added to cosmetics and maintenance products. It has the functions of activating energy, anti-wrinkle, anti-inflammatory, whitening, anti-oxidation, anti-aging, and protecting and repairing.
  • the growth point induction cultivation method can obtain a large number of new plant bud embryo tissues, which can meet the needs of mass production, and has high economic value and high practicability.
  • the orchid bud embryo extraction technology of the present invention is mainly aimed at the growth point of the stem node of the orchid with excellent growth function.
  • the cells that use this growth point have the characteristics of rapid division and differentiation to produce new buds and continuous elongation of the bud axis.
  • Has the characteristics of high vitality and activity, and the regeneration of orchid bud embryos is to form new plant embryos through buds and other organs, just like the regeneration function of animal stem cells; to establish a complete process from growth point cultivation, cutting to extraction .
  • the present invention can be applied to the growth point extraction of Phalaenopsis Sogo Yukidian V3 varieties of large white orchids, and this extraction technique can also be applied to other varieties of orchids to obtain bud embryo extracts with high active ingredients.
  • step (a) to induce regeneration of new lateral buds For the method for cultivating orchid stem node growth points of the present invention, please refer to step (a) to induce regeneration of new lateral buds.
  • the induction method of the present invention is based on the stem node position of the flowering plant. After 4 to 4.5 weeks, new lateral buds can be formed.
  • step (b) to remove the stem buds and extract new lateral buds
  • step (c) to remove the wall membrane. After the new lateral buds are cut off the stem buds, the wall membrane is removed to obtain Round new plant tissue, the present invention only uses this new plant bud embryo tissue for extraction.
  • step (e) ultrasonic extraction For the orchid bud embryo extraction method of the present invention, please refer to step (e) ultrasonic extraction.
  • the new plant tissues obtained above are oscillated at high frequency and constant speed, and the characteristics of density and rank are used to make the ultrasonic vibration instantaneously pressurize the liquid. "And “decompression”, push the medium, produce cavitation. When countless small vacuum bubbles in the extraction liquid burst, instantaneous high temperature and strong impact force are generated, which can separate the components of the material to be extracted and achieve the extraction effect.
  • the method for determining the total flavonoid content of the extract obtained through the foregoing process is as follows: Take 1 ⁇ L of orchid bud embryo extract and add 49 ⁇ L of secondary deionized water and 3 ⁇ L of 5% NaNO 2 solution to react for 6 minutes, then add 3 ⁇ L of 10 % AlCl 3 solution and react for 6 minutes, then add 40 ⁇ L of 4% NaOH solution and 4 ⁇ L of secondary deionized water, shake and mix uniformly, stand for 15 minutes, and measure the absorbance at a wavelength of 510 nm. Calculated based on the standard curve of rutin, the amount of flavonoids contained in each gram of extract is about 4.2 to 4.75 mg.
  • the total polyphenol content of the extract obtained through the foregoing process is determined as follows: (1) 1 ⁇ L of orchid bud embryo extract is mixed with 25 ⁇ L of 10-fold diluted Folin-Ciocalteu's phenol reagent and reacted for 5 minutes, and then 20 ⁇ L of 7.5% Na is added.
  • the extract obtained through the foregoing process contains indole-3-acetic acid, which is a plant auxin, one of the important plant hormones, which is widely present in the leaf buds and young leaves of plants. In the meristem, it can make cells grow and differentiate, and then promote the growth and development of plants.
  • the extract prepared by the present invention uses high-performance liquid chromatography to analyze the components of the orchid growth point extract, and can perform the component analysis of indole-3-acetic acid to establish the index component analysis of the orchid bud embryo extract, and Quantitative analysis of a compound is used as an indicator of orchid bud embryo extract.
  • the index component analysis method of the bud embryo extract prepared by the present invention mainly uses different proportions of pure water and acetonitrile in the mobile phase, the flow rate is 1mL/1min, the total test time is 30 minutes, and the standard product indole-3-
  • the retention time of acetic acid was 23.194min; the test volume of orchid bud embryo extract was 20 ⁇ L, the maximum area was measured at the same retention time (23.194min), and the integral area parameter was 92.75%.
  • the orchid bud embryo extract contains 92.75% of indole-3-acetic acid.
  • indole-3-acetic acid can be used as an indicator of the effectiveness of the orchid bud embryo extract.
  • the present invention mainly provides a complete process for the cultivation, cutting and extraction of orchid bud embryos, and only extracts and collects the regenerated plant tissues at the growth point of the stem node which has the most active ingredients in orchids for extraction.
  • the total polyphenols, total flavonoids and phytohormones of the active ingredients of the extract are high and the quality is excellent. Therefore, compared with the method of extracting the whole orchid or petals in the prior art, the total amount of the extract prepared by the present invention is more The content of phenols, total flavonoids and plant hormones is higher and the quality is better.
  • the extract of the present invention has better content and quality of active ingredients than the extracts of the prior art, the extract of the present invention is added to cosmetics and skin care products to have activating energy, anti-wrinkle, anti-inflammatory, whitening, Anti-oxidation, anti-aging and protection and repair functions.
  • the stem node growth point of the flowering plant is used for induction and cultivation, so that after regenerating new lateral buds, the new plant tissues of the new lateral buds are cut and the stem node growth point induction cultivation method can obtain a large number of new plant buds. Embryo tissue can meet the needs of mass production.
  • the growth point of the stem node of the flowering plant is first cultivated to multiply new lateral buds, and then new plant tissues are cut out.
  • This cutting operation of the stem node growth point can remove other stem and leaf parts, and only use higher ones.
  • the new plant tissue with the growth activity content is extracted, so it has the effect of removing turn and keeping the greens, thereby improving the quality of the extract, and solving the problem that the active content and quality of the orchid extraction technology in the prior art cannot be improved.
  • the bud embryo extraction method of the present invention is operated at a non-high temperature, and the temperature during the extraction process is about 50 to 60°C to reduce heat loss caused by temperature, and also to avoid low boiling point and effective substance volatilization and maintain growth
  • the growth activity of the point tissue can maintain the active content of new plant tissues, and solve the problem of reduced activity of extracts or volatilization of effective substances caused by processing processes such as boiling, steam or heat reflux extraction in the prior art.
  • the present invention uses the ultrasonic water extraction method and only uses reverse osmosis pure water without other solvents. Therefore, it can improve the traditional solvent extraction method's high cost and dangerous extraction process shortcomings, and can reduce the processing time and solvent usage and obtain High yield.
  • the semi-finished product extract produced by ultrasonic extraction of the present invention is then filtered and solvent removed. Because the solvent used in the present invention is non-toxic and sterile pure water, there is no use of alcohols in the prior art Or the safety problem of the extraction method in which the alcohol aqueous solution is the solvent, and the present invention can obtain an extract with high activity and good stability through this filtration and solvent removal operation.
  • the present invention cooperates with the establishment of the "Orchid Bud Germ Extract Index Component Analysis Method" to perform component analysis to determine whether the amount of the plant hormone "indole-3-acetic acid" contained in the extract reaches Standard, and has the effect of establishing the analysis method and index basis of the phytohormone "indole-3-acetic acid” component.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to "tests related to the safety of human skin cells", and the test methods include “cell viability test”, “observation of cell type changes” and “cell cycle”. There are three kinds of tests, and the various test methods are detailed as follows:
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "cell viability test", and the test method is as follows: (1) The human skin keratinocyte strain HaCaT is quickly moved to 37°C from the liquid nitrogen tank Thaw rapidly in a water bath within 40 to 60 seconds, then add the cell cryopreservation solution (cell culture solution containing 10% DMSO) to the cell culture solution, break up the cells and transfer the 25cm 2 cell culture flask to 37 Grow in a 5% CO 2 cell incubator, and change the culture medium every 2 days on average; (2) Aspirate the cell culture medium from the culture flask, wash it once with PBS, and add an appropriate amount of 1xTrypsin.
  • cell cryopreservation solution cell culture solution containing 10% DMSO
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to "cell type change observation", and the test method is as follows: (1) Human skin keratinocytes HaCaT are cultured at a density of 1 ⁇ 10 4 cells/well 96-well plate, and incubate at 37°C and 5% CO 2 incubator for at least 24 hours; (2) Add 1 ⁇ L of orchid bud embryo extract to act on human skin keratinocytes HaCaT for 24 hours. After the reaction time is reached, the microscope (Nikon , TE2000-U, Japan) to observe the cell morphology and take photos for recording. As shown in Figure 2, after 24 hours of treatment of human skin keratinized cells with orchid bud embryo extract (0.05, 0.1 and 0.5 mg/mL), there was no significant difference between the cell morphology and the cell morphology of the control group.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to a "cell cycle test", and the test method is as follows: (1) Cultivate 1 ⁇ 10 4 cells/well density in a 24-well plate for at least 24 hours, and then add 10 ⁇ L of orchid bud embryo extract, react in the incubator. After that, the supernatant was collected into a 15mL centrifuge tube, and then the cells were removed into the centrifuge tube with trypsin-EDTA solution, and the supernatant was centrifuged at 1200 rpm for 5 minutes.
  • the orchid bud embryo extract (0.1 and 0.5 mg/mL) has no significant changes in the cell cycle after 24 hours of treatment on the skin keratinocytes; specifically, the orchid bud embryo extract (0.1 and 0.5 mg/mL) After 0.5mg/mL), the ratio of sub-G1 (M1 area) was 5.8 and 6.2%, both of which were less than 10%.
  • the present invention also establishes related test methods, including "mitochondrial regeneration test", “skin collagen production test”, “inhibition of inflammatory factor nitric oxide activity test”, “inhibition of tyrosinase” Activity test”, “DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test”, “protection of DNA damage caused by ultraviolet and oxidative damage”, “protection of fragmented DNA effect test”, “repair Cell viability test of skin cells under the action of ultraviolet light, “Cyclobutane pyrimidine dimers (CPD) production test for detecting DNA damage", “Senescence-associated ⁇ -galactosidase , SA- ⁇ -gal) activity test” and “SIRT-1 gene mRNA expression test”, respectively, are described in detail as follows:
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "mitochondrial regeneration test", and the test method is as follows: (1) Human skin keratinocytes HaCaT at a density of 1 ⁇ 10 4 cells/well Cultivate in a 24-well plate, incubate in a 37°C and 5% CO 2 incubator for at least 24 hours, add different concentrations of orchid growth point extracts, and place in the incubator for 24 hours; (2) After the reaction time is reached, remove The culture medium was washed with PBS, and paraforaldehyde was added to fix the cells. The cells were treated with 1% TritonX100 for 5 minutes.
  • Relative mass of mitochondria (Mitoview intensity/Hoechst 33342 intensity) X 100%
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "skin collagen production test".
  • the test method is as follows: (1) 3T3L-1 cells are cultured at a density of 2 ⁇ 10 5 cells/mL. After 24 hours in the centimeter plate, remove the supernatant and add serum-free culture medium containing different concentrations of orchid bud embryo extracts for 48 hours. After washing with PBS, scrape off the cells, centrifuge at 1200 rpm for 5 minutes, and then remove the supernatant.
  • Collagen is an important factor for maintaining skin elasticity and anti-wrinkle. Its synthesis is affected by age. Therefore, if the synthesis of collagen can be promoted in a timely manner, it can prevent the skin from forming wrinkles and losing elasticity. According to this result, after the action of orchid bud embryo extract (0.05, 0.1 and 0.5mg/mL), it can promote the production of collagen in fibroblasts by about 4.7 ⁇ 0.4%, 21.1 ⁇ 2.1% and 35.0 ⁇ 0.1%. This result shows that the orchid bud embryo extract has an excellent effect on promoting collagen production.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "inflammation factor nitric oxide activity test", and the test method is as follows: Take 2 ⁇ L of orchid bud embryo extract (0.05, 0.1, 0.5 and 1.0 mg/mL ) Separately add 98 ⁇ L of 25 mM sodium nitroprusside (SNP) SNP to react for 120 minutes, and then add 100 ⁇ L of Griess reagent to react for 10 minutes, and measure the absorbance at 546 nm with an enzyme immunoassay.
  • SNP sodium nitroprusside
  • SNP itself is a kind of nitric oxide donor. It reacts with oxygen to form nitrite, which then reacts with Griess reagent and turns into a pink solution with a specific absorbance at a wavelength of 546nm. If the sample can inhibit the rate of nitric oxide generation and reduce the production of nitrous acid, its absorbance value will decrease; the lower the absorbance value, the stronger the sample's ability to scavenge nitric oxide free radicals. According to the above analysis results, the orchid bud embryo extract (0.05, 0.1, 0.5 and 1.0 mg/mL) has 22.0 ⁇ 1.4%, 25.0 ⁇ 1.3%, 34.7 ⁇ 1.1% and 63.4 ⁇ 1.9% in scavenging nitric oxide free radicals, respectively.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "inhibition of tyrosinase activity test".
  • the test method is as follows: Take 2 ⁇ L of the orchid bud embryo extract of different concentrations into a 96-well plate, and add 18 ⁇ L of Mix with DMSO, then add 25 ⁇ L of 100unit lentinus edodes tyrosinase and react for 10 minutes at room temperature. Then 155 ⁇ L of 2.5mML-DOPA was added, and the absorbance was measured with an enzyme immunoassay at a wavelength of 492nm.
  • the orchid bud embryo extract (0.05, 0.1, 0.5 and 1.0 mg/mL) inhibited tyrosinase activity by 12.8 ⁇ 1.5%, 28.5 ⁇ 2.2%, 46.2 ⁇ 2.5% and 75.2 ⁇ 1.8%, respectively.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test", and the test method is as follows: the orchid bud embryo extract is configured to different concentrations , Respectively add 90 ⁇ L of freshly prepared 100 ⁇ M DPPH free radical ethanol solution and add them to the 96-well plate for reaction, and detect the absorbance at 517 nm with an enzyme immunoassay.
  • the test standard product is AA, and the formula of DPPH free radical scavenging rate is as follows:
  • DPPH radical scavenging rate [(1- 517nm absorbance value of the sample to be tested)/ 517nm absorbance value of the control group] X100%
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to "protection against DNA damage caused by ultraviolet and oxidative damage", and the test method is as follows: (1) DNA plastid pUC119 (25 ⁇ g, 0.5 ⁇ g/ ⁇ l, Takara, Japan) was diluted with PBS at a ratio of 1:8 and treated separately: 1control group, 2UVB (20mJ/cm 2 ) and H 2 O 2 (1mM)+FeSO 4 (0.5mM) group, 3UVB (20mJ/cm 2 ) and H 2 O 2 (1mM)+FeSO 4 (0.5mM)+Orchid bud embryo extract group; (2) Take 2 ⁇ L of pUC119 each in a microcentrifuge tube, and use 1, 2 and 3 Groups were treated separately and acted at 37°C for 1 hour; (3) After adding loadingdye and mixing, electrophoresis in 0.8% agarose colloid for 30 minutes, and then analyzed by electrophoresis film image capture system;
  • the plastid is originally a ring structure (supercoil form, S-form). When it is damaged by oxidation and ultraviolet rays, it will form an open form (O-form) or a linear form (L-form). structure.
  • the plastid pUC119 is a DNA plastid with an S-form structure. After oxidation and UVB damage, the plastid forms L-form and O-form, and even fragments when it is severely damaged.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "protective fragmented DNA effect test", and the test method is as follows: (1) Add the diluted 2 ⁇ L DNA Mw Standard Marker into a 0.5mL microtube and perform each Do not treat: 1Control group, 2UV and H 2 O 2 action group, 3UV and H 2 O 2 + extract action group; (2) After acting at 37°C for 1 hour, add loading dye and mix with agar gel Nucleic acid electrophoresis analysis. As shown in Figure 5, the DNA marker is a strip of DNA (undamaged DNA, the first strip), and the DNA will be arranged according to different molecular weights.
  • the strip DNA will become fragments (the second strip), and after the orchid bud embryo extract (0.05, 0.1 and 0.5 mg/mL), the DNA will still remain a clear strip.
  • the orchid bud embryo extract has the effect of protecting DNA at 0.05mg/mL. This result once again confirmed that the orchid bud embryo extract has the effect of protecting DNA from oxidation and UVB damage.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "cell viability test of repairing skin cells by ultraviolet rays".
  • the test method is as follows: (1) Human skin keratinocytes are divided into 1 ⁇ 10 4 cells/ The hole density was cultured in a 96-well plate, and cultured in a 37°C and 5% CO 2 incubator for at least 24 hours, and processed separately: 1Control group-remove the cell supernatant, replace the fresh culture medium without serum, culture 4 After hours, wash with PBS and place in fresh serum-free culture medium, continue to incubate for 4 hours for viability analysis; 2UV irradiation group-remove the cell supernatant, replace the serum-free fresh culture medium, and place it in a cell incubator for culture After 4 hours, remove the supernatant and wash with PBS and drain it.
  • UV irradiation + extract group remove cells Mix the extracts with fresh serum-free culture medium separately. After adding the culture for 4 hours, remove the supernatant and wash with PBS and drain it. After UV irradiation, immediately add the extract-containing culture medium.
  • Serum fresh culture medium continue to incubate for 4 hours for viability analysis; (2) When the reaction time is reached, remove the old culture medium, wash once with PBS, and replace with new culture medium, add 10 ⁇ L of MTT solution to react, After reacting at 37°C and 5% CO 2 for 4 hours, the culture solution was removed, 100 ⁇ L of DMSO was added to dissolve the formazan precipitate, and finally the absorbance was measured at a wavelength of 570 nm (BioTek, Synergy TM 2, USA).
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "test for the production of cyclic pyrimidine dimers to detect DNA damage", and the test method is as follows: (1) 1 ⁇ 10 5 cells/mL are cultured in The 24-well plate should be treated separately for at least 24 hours: 1Control group, 2UV irradiation group; 3UV irradiation+extract group-extract after 4 hours of UV irradiation, replace the serum-free culture medium and incubate for 4 hours; (2) Remove the culture medium and wash with PBS, fix the cells with ice methanol, then use Triton X-100, add 2M HCl solution for 1 hour, then fill the cell gap with 1% BSA, and then shake with CPD primary antibody at 37°C React for 1 hour.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to "Aging-related Galactosidase Activity Test", and the test method is as follows:
  • “Aging” is an irreversible state of the human body. When cells divide and multiply for multiple generations, they may be overstimulated by the outside world, leading to growth stagnation and aging. Senescence-related galactosidase is overexpressed in senescent cells, so it can be used as one of the indicators of cell senescence. As shown in Figure 8, after UVB irradiation, the galactosidase in the cells will accumulate rapidly, while the vitamin C in the control group can effectively inhibit the galactosidase activity. Similarly, cells treated with the extract (0.1 and 0.5 mg/mL) can also inhibit the activation of galactosidase caused by UVB.
  • the extract produced by the orchid bud embryo extraction technology of the present invention is subjected to the "SIRT-1 gene mRNA expression test", and the test method is as follows:
  • RNA will precipitate at the bottom of the microcentrifuge tube to form a transparent white precipitate.
  • the supernatant was removed, the precipitate was gently rinsed with 200 ⁇ l of ice 75% alcohol, and centrifuged at 12,000 rpm at 4° C. for 5 minutes, and then the supernatant was removed. Leave the RNA pellet to dry for 15 minutes, then dissolve the RNA pellet in an appropriate amount of DEPC-treated water and store it at -80°C. Finally, the purity of RNA was measured by spectrophotometer at OD 260 and OD 280.
  • RNA Take 3 ⁇ g of RNA and mix with an appropriate amount of DEPC-treated water, add 1 ⁇ L Oligo(dT)18 primer and place it at 70°C for 2 minutes, then quickly move to ice, then add 4 ⁇ L 10x MMLV RT buffer and 1 ⁇ L dNTP mixture respectively (Each dNTP 10mM), 0.5 ⁇ L recombinant ribonuclease inhibitor (1unit/ml), 1 ⁇ L MMLV reverse transcriptase (5unit), to make the total volume 20 ⁇ L. After mixing them uniformly and acting at 42°C for 1 hour, heat at 94°C for 5 minutes to remove the MMLV reverse transcriptase activity and terminate the reaction. The preparation of the first strand of cDNA template is completed, and then 80 ⁇ l of DEPC-treated water is added. , Stored at -20°C for later use.
  • the reaction conditions are as follows: pre-denaturation reaction, 98°C, 3 minutes as a cycle; followed by denaturation reaction 94°C, adhesion reaction 60°C, and synthesis reaction 72°C. Each 1 minute polymerase chain reaction, the total reaction process carried out a total of 35 cycles. After the reaction is completed, take an appropriate amount of the reaction product and analyze it by 2% agarose gel electrophoresis.
  • SIRT-1 is a longevity gene. After years of research, it was found that promoting the performance of SIRT-1 can effectively protect cells from repairing and maintaining normal functions, avoiding diseases caused by cell aging.
  • RT-PCR observes the final product of amplification, also known as the end-point, which requires gel electrophoresis. It can be analyzed, and may be affected by image processing and background value to cause experimental error; real-time quantitative PCR is to add fluorescent agent, start to collect the signal during the amplification period, and analyze the relative relationship between the amplification factor and the fluorescent signal to calculate the target gene The relative content.
  • the present invention provides a technology for cutting and extracting orchid bud embryos.
  • the complete process from cultivation, cutting and extraction utilizes the regeneration characteristics of orchid stem node growth point cells that will continue to divide and differentiate, and has high vitality and activity.
  • the characteristics of this product can be carried out under non-high temperature, which can avoid the low boiling point and the volatilization of effective substances and maintain the growth activity of the growth point tissue, and the prepared germ extract contains high total polyphenols, total flavonoids and plant hormones and good quality .
  • Used in cosmetics and maintenance products can activate energy, anti-wrinkle, anti-inflammatory, whitening, anti-oxidation, anti-aging, protection and repair, etc.
  • the stem node growth point induction cultivation method can meet the needs of mass production, and cooperate with filtration and removal Solvent operation can obtain extracts with high activity and stability, and the orchid active extracts with high practicability and high economic value can be obtained, so as to make the whole industry practical and cost-effective.

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Abstract

一种兰花芽胚组织萃取物的萃取方法，步骤有：超声波萃取；以及过滤及去除溶剂。由于整体在非高温下进行，可避免低沸点与有效物质挥发及保持芽胚组织的生长活性，所得的萃取物的总多酚及总黄酮含量高，添加于化妆保养品中，具活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复等功效。

Description

可活化能量、抗皱、抗发炎及美白的兰花芽胚组织萃取物 技术领域
本发明有关植物萃取方法，特别是指应用于兰花芽胚组织萃取物的萃取方法，所制成的萃取物可作为医药品、化妆品、保养品、香氛或人体清洁用品等产品的原料者。
背景技术
中国台湾拥有成熟的种植与组织培养技术，其中包括品种繁多丰富、纯熟的组织培养技巧、病毒检验能力及量产工程技术等。此外，中国台湾农业目前拥有推动兰花产业的优势条件，包括得天独厚的天然地理条件、傲视全球的多元化品种、技术顶尖的育种专家群、超水平的农业科技基础、遍布全省的农业试验改良及学研等各类农业技术辅助机构、闻名世界的信息产业与现代化的物流通路等。
在中国台湾，全年蝴蝶兰产量约1亿3,294万株，外销总量约4千万株，占全球供应量的20％，销售地区遍及欧洲、日本与美国，因此需要应用精进的生物科技技术，进行大量繁殖以因应供货需求。而兰花的培育繁殖方法，最常见的有“播种繁殖法”、“花梗催芽繁殖法“、“断心催芽繁殖法”、“切茎繁殖法”及“组织培养法”等五种，可按照培殖品种的特性及需求选用适当繁殖方法进行培育。其中“组织培养法”大多撷取具有分生能力的“顶芽”部位来进行培养；而“茎顶生长点”部位的生产多采用芽体增殖(芽长芽)或诱导拟原球体(PLBs)分生苗方式，其中芽体增殖亦即减低或去除顶芽优势，使侧芽容易长出来，以此方式增殖芽体。
兰花除了可供观赏用外，近年来亦广泛用于化妆品及保养品等产品中作为原料，使得兰花的经济价值提升，兰花的需求量更大；而兰花需经过萃取作业成为萃取物后，再作为化妆品及保养品的添加物。而因萃取兰花的部位 不同，则要配合适当的萃取技术，萃取流程如有变化，所制成的萃取物成份也会不同，萃取物可达到的美白功能、抗氧化及抗老化效果也会受到影响。
由于人类皮肤经常受到紫外线、空气与温度等环境因素的影响，而有加速皮肤老化现象及罹患皮肤癌的风险。以皮肤老化来说，外因性因素占据60％，而其中紫外线又占了老化原因高达60％。于影响皮肤老化的研究中发现，紫外线不只会造成黑色素细胞的活跃性，导致皮肤斑点的出现，也会使真皮组织产生退化现象，并且加速皮肤的氧化，产生游离自由基，这些自由基会造成纤维母细胞破坏、胶原蛋白变性和含量减少以及弹性纤维组织退化而产生老化皱纹，更会加速细胞内DNA伤害，使得皮肤细胞更新能力变差，新细胞取代旧细胞的速度变慢。
因此市售的化妆品及保养品大多针对前述皮肤问题，开发出不同的化妆品原料，再配合添加其它具特殊效果的添加物或萃取物，其中添加植物萃取物的活性成份如花青素、类黄酮、多酚类等，对于皮肤的美白、抗氧化、抗老化或防皱等功效相当显着，因此市面上许多化妆或保养商品常见有各类植物活性成份添加其中。然而，因不同的植物、不同部位、或所制成萃取物成份要求与功效不同，则需应用的萃取技术皆不同，以兰花而言，虽同样作为化妆品及保养品的添加原料，但其萃取技术应用有许多方式，且萃取作业的步骤、流程与环境参数亦有差异。
目前，现有技术应用于化妆品的兰花的萃取技术，如中国发明专利申请号第CN201510295194.7号所揭露的“兰花萃取物及其制备方法和应用”，所述兰花为蝴蝶兰属，所述兰花萃取物由下列步骤所制备而得：(1)萃取：将所述兰花与溶剂以重量比例为0.5：1至10：1，进行混合粉碎或者破壁得到兰花浆，所述溶剂为水、醇类或醇类水溶液，其中所述醇类为乙醇、丙醇、或丁醇；(2)固液分离：将步骤(1)萃取的兰花浆进行固液分离，留下液态为兰花滤液；(3)活性划分：将步骤(2)所分离的兰花滤液以分子筛薄膜处理纯化，得到兰花萃取筛分液；(4)浓缩：将步骤(3)纯化的兰花萃取筛分液以分子筛薄膜处理浓缩，或者真空或蒸煮方式进行部分浓缩，得到 活性沉淀物与具活性的液态活性萃取液。
另一种应用于化妆品的兰花的萃取技术，如中国发明专利公开号第CN105878122A号所揭露的“一种具有怯斑美白效果的天然提取物化妆品”，其包括功效成分泽兰提取物和石吊兰提取物，其中，所述泽兰提取物和石吊兰提取物的重量比为2至4：1。泽兰提取物和石吊兰提取物的制备方法为：将干燥的泽兰或石吊兰用乙醇热回流提取，合并滤液，浓缩至无醇味得到乙醇提取浓缩液；再用水稀释，依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取；正丁醇萃取物用水溶解，过滤，滤液用大孔树脂富集活性成分，喷雾干燥即得。该发明提供的祛斑美白化妆品以植物提取物为功效成分，且通过控制泽兰提取物和石吊兰提取物的含量比值，可最大化去斑美白。
更有一种应用于化妆品的兰花的萃取技术，如中国台湾发明专利申请号第102140137号所揭露的“白花蝴蝶兰花瓣的萃取物及其制备方法与用途”，其藉由下列步骤的方法而制得：以超临界CO 2来萃取白花蝴蝶兰花瓣，藉此而得到一白花蝴蝶兰花瓣的脂溶性萃取物以及一残余物；以及以水来萃取残余物，俾以得到一白花蝴蝶兰花瓣的水溶性萃取物，可用于促进皮肤美白、提升皮肤的保湿能力以及预防或推迟皮肤老化。
上述萃取技术均可以取得兰花的萃取物，其中第一案例是以整株兰花施以萃取、固液分离、活性划分及浓缩等流程；第二案例则以整株泽兰或石吊兰施以干燥、热回流提取、浓缩、稀释、溶解及过滤等步骤；第三案例即利用白花蝴蝶兰的花瓣进行超临界CO 2萃取后，再以水来萃取残余物的作业流程者。前述现有技术利用兰花进行萃取的部位皆不同，第一、二案例以整株进行，第三案例则仅使用花瓣部位，且又因萃取方法、流程、使用设备及环境相关参数均不同，所取得的萃取物成份与含量也会不一样。
无论以整株兰花或花瓣部位进行萃取，所制成的萃取物活性成份含量及效果仍会受到限制；此外，前述第二、三案例的萃取过程中有透过高温进行的步骤，恐造成萃取物的活性减低或有效物质挥发的情形，以致化妆及保养产品美白功能、抗氧化及抗老化的效果不佳；再者，现有技术中以整株兰花 进行萃取作业，使得兰花所有部位无论活性存量好坏，于同时萃取的情形下，制成的萃取物的活性平均下来质量将会受到限制，无法提升；另外，现有技术以醇类或醇类水溶液为溶剂的萃取方式，除了使用设备成本高外，过程中有挥发性气体产生而有安全上的疑虑。
另一方面，由于兰花的茎节部位具有优异的生长机能，通常此部位会成为一生长点，此一生长点的细胞拥有快速地分裂和分化以产生新芽的特性，并可使芽轴不断伸长，犹如动物的干细胞再生功能，如能撷取兰花茎节生长点细胞进行萃取，所得的萃取物活性成份含量与活性效益高，然而现有技术中兰花相关的萃取技术尚未见有针对兰花开花株的茎节部位进行萃取或提炼等技术。
因此，本专利申请的发明人提供一种兰花生长点切割及萃取技术，以解决前述现有技术中存在的缺陷，以获得活性成份含量高且效果佳的兰花芽胚萃取物。
发明内容
于是，本发明的之一目的即在提供一种兰花芽胚的萃取方法及其萃取物，主要提供兰花芽胚的培育、切割及萃取的完整流程，且仅撷取并收集兰花最具有活性成份的茎节生长点的再生植物细胞来进行萃取作业，所制成的芽胚萃取物的总多酚、总黄酮及植物激素含量高，添加于化妆保养品产品中，具有活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复等功效。
本发明的另一目的即在提供一种兰花芽胚的萃取方法及其萃取物，以开花株的茎节生长点部位进行诱导培育，使长出再生新侧芽后，且切割取得新侧芽的新生植物组织，此茎节生长点诱导培育方式可获得大量的新生植物组织，而可符合大量生产的需求。
本发明的再一目的即在提供一种兰花芽胚的萃取方法及其萃取物，萃取方法于非高温下操作，以减少温度所造成的热损失，亦可避免低沸点与有效物质挥发及保持生长点组织的生长活性。
本发明的更一目的即在提供一种兰花芽胚的萃取方法及其萃取物，经超声波萃取所制成的半成品萃取液，再进行过滤及去除溶剂作业，可获得活性及稳定性高的芽胚萃取物。
有鉴于此，本发明所揭露兰花芽胚的萃取方法，其步骤为：(a)诱导再生新侧芽：将兰花的开花株的茎节部位的梗芽切出若干段梗芽，并进行培育作业使之长出新侧芽；(b)切除梗芽并撷取新侧芽：将梗芽切除并撷取新侧芽后，取出生长点组织；(c)去除壁膜：将各小片段的生长点组织去除壁膜，成为新生植物组织；(d)收集新生植物组织：收集生长点新侧芽的新生植物芽胚组织；(a)超声波萃取：将芽胚组织放入超声波萃取设备，于低温下，以芽胚组织重量5倍量的纯水为溶剂将芽胚组织覆盖浸泡，以制成半成品萃取液；及(b)过滤及去除溶剂作业：将半成品萃取液经由抽真空的过滤作业后，再利用减压浓缩机将半成品萃取液进行去除溶剂作业，以制成萃取物。
藉由上述步骤，整个流程于非高温下进行，可避免低沸点与有效物质挥发及保持芽胚组织的生长活性，且所制成的萃取物的总多酚及总黄酮含量高，添加于化妆保养品产品中，具有活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复等功效。
此外，本发明所揭露上述制成萃取物的用途为用于制备具有活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复功效的组合物，而此组合物可为医药品、化妆品、保养品、香氛或人体清洁用品。
另外，本发明所揭露上述制成萃取物的用途为用于制备具促进皮肤细胞粒线体再生的组合物，而此组合物可为医药品、化妆品、保养品、香氛或人体清洁用品。
附图说明
图1为流程示意图，说明本发明所揭露兰花开花株茎节生长点的萃取方法。
图2为显微镜照片图，说明本发明所揭露的萃取物对人类皮肤角质株化 细胞型态的影响。
图3为流式细胞分析结果图，说明本发明所揭露的萃取物对人类皮肤角质株化细胞的细胞周期影响。
图4(A)为DNA胶体电泳照片图，说明本发明所揭露的萃取物对质体DNA型态的影响。
图4(B)为DNA胶体电泳量化结果图，说明本发明所揭露的萃取物对质体DNA型态的影响。
图5为DNA胶体电泳结果图，说明本发明所揭露的萃取物对DNA marker型态的影响。
图6为细胞存活分析结果图，说明本发明所揭露的萃取物对细胞存活的影响。
图7(A)为荧光侦测照片图，说明本发明所揭露的萃取物对光产物CPD生成的影响。
图7(B)为荧光侦测量化结果图，说明本发明所揭露的萃取物对光产物CPD生成的影响。
图8为细胞衰老分析结果图，说明本发明所揭露的萃取物对细胞衰老的影响。
图9为DNA胶体电泳结果图，说明本发明所揭露的萃取物对调控长寿基因SIRT-1的RNA表现的影响。
图10为实时定量PCR结果图，说明本发明所揭露的萃取物对调控长寿基因SIRT-1的RNA表现的影响。
具体实施方式
请参照图1，本发明所提供的一种兰花芽胚的萃取方法，以最佳实施的蝴蝶兰V3(大白兰花V3，Phalaenopsis Sogo Yukidian V3)品种作说明，其步骤依序为：(a)诱导再生新侧芽；(b)切除梗芽并撷取新侧芽；(c)去除壁膜；(d)收集新生植物组织；(e)超声波萃取；(f)过滤及去除溶剂作 业；及(g)制成芽胚萃取物；其中：
步骤(a)：将兰花的开花株的茎节部位的梗芽，于梗芽侧边进行切割，以茎节为单位切出若干段梗芽，各梗芽内所切割的部位将自行愈合，消毒后种入进行培育作业，使之长出新侧芽；培育作业以苹果、马铃薯、香蕉、胡萝卜为培养基；培养温度为15至20℃；培养时间约为4至4.5周。
步骤(b)：将梗芽切除并撷取新侧芽后，于两片新侧芽交接的位置往下0.1cm的部位的生长点组织，切成小片段。
步骤(c)：将各小片段的生长点组织去除壁膜，成为新生植物组织，其再生的途径为经由芽等器官形成新生植物胚胎；新生植物组织呈形状一致的圆形。
步骤(d)：收集生长点新侧芽的新生植物芽胚组织。
步骤(e)：将收集的新生植物芽胚组织秤取100公克，放入超声波萃取设备，于低温下，以芽胚组织重量5倍量的反渗透纯水或灭过菌的去离子水为溶剂将芽胚组织覆盖浸泡，置入超声波设备内，水温设定于50至60℃，功率设定为300瓦，时间设定为40分钟，以超声波震荡对生长点的芽胚组织进行萃取作业，以制成半成品芽胚萃取液。
步骤(f)：先将半成品芽胚萃取液经由抽真空的过滤作业；再利用减压浓缩机以45℃加热，以旋转速度80转/分钟，将半成品芽胚萃取液减压浓缩，进行去除溶剂作业；且重复此一去除溶剂作业1至4次，以完整萃取。
步骤(g)：制成的芽胚萃取物具有总多酚及总黄酮等成份；所制成的萃取物，约1.51公克，萃取率约1.51％；所制成的芽胚萃取物内含总多酚，每克萃取物中所含没食子酸(gallic acid)毫克数约为4.5至5.28；所制成的萃取物，以芸香苷(rutin)的标准曲线来计算，每克芽胚萃取物中含类黄酮化合物的量约为4.2至4.75mg；所制成的萃取物，可于冷冻干燥后于温度-20℃下保存冷藏；且所制成的芽胚萃取物可应用于医药品、化妆品、保养品、香精、香水或人体清洁用品等产品的原料。
藉由上述步骤，可提供针对兰花芽胚的培育、切割及萃取的完整流程， 使整个流程于非高温下进行，可避免低沸点与有效物质挥发及保持生长点组织的生长活性，且所制成的萃取物的总多酚、总黄酮及植物激素含量高，添加于化妆保养产品中，具有活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复等功效，同时此茎节生长点诱导培育方式可获得大量的新生植物芽胚组织，而可因应大量生产的需求，并且具有高经济价值与高实用性。
为进一步了解本发明技术特征、运用技术手段及所预期达成的功效，兹将本发明使用方式加以说明叙述如下：
实施例1
本发明的兰花芽胚萃取技术，主要针对兰花的茎节部位具有优异生长机能的生长点部位，利用此一生长点的细胞拥有快速分裂和分化以产生新芽并可使芽轴不断伸长的特性，拥有高生命力及活性的特质，且兰花芽胚其再生的方式为经由芽等器官形成新生植物胚胎，犹如动物干细胞的再生功能；藉此建立从生长点的培育、切割到萃取的一整套流程。再者，本发明除可应用于大白兰花Phalaenopsis Sogo Yukidian V3品种的生长点萃取外，其他品种的兰花亦都可适用此一萃取技术，以获得活性成份高的芽胚萃取物。
本发明的兰花茎结生长点培育方法，请参照步骤(a)诱导再生新侧芽，本发明的诱导方式依开花株的茎节部位，先切出若干段梗芽，再进行培育繁殖作业，约4至4.5周后，即能制成新侧芽。
本发明的兰花芽胚切割撷取方法，请参照步骤(b)切除梗芽并撷取新侧芽及步骤(c)去除壁膜，将新侧芽切除梗芽后，再除去壁膜，则可得圆形的新生植物组织，本发明只使用此一新生植物芽胚组织进行萃取。
本发明的兰花芽胚萃取方法，请参照步骤(e)超声波萃取，将前述所取的新生植物组织，利用超声波高频恒速震荡、疏密有秩的特性，将超声波振动对液体瞬间造成“增压”及“减压”，推动介质，产生空穴效应(cavitation)。当萃取液中无数细小的真空气泡爆裂时，产生瞬间高温及强大冲击力，可将待萃取物的成份分离，而达到萃取效果。
经前述流程所获得的萃取物，其总黄酮含量测定方法如下：取1μL兰 花芽胚萃取物并加入49μL二次去离子水及3μL的5％NaNO 2溶液反应6分钟后，再加入3μL的10％AlCl 3溶液并反应6分钟后再加入40μL的4％NaOH溶液及4μL二次去离子水震荡混合均匀后，静置15分钟后，在波长510nm下测吸光值。以芸香苷的标准曲线来计算，每克萃取物中含类黄酮化合物的量约为4.2至4.75mg。
经前述流程所获得的萃取物，其总多酚含量测定方法如下：(1)1μL兰花芽胚萃取物混合25μL的10倍稀释Folin-Ciocalteu’s phenol reagent反应5分钟后，再加入20μL的7.5％Na 2CO 3溶液，最后加入ddH 2O使总体积为100μL，混合均匀后反应15分钟后，于波长510nm下测定吸光值(BioTek，Synergy TM2，USA)；(2)利用没食子酸标准品制作的校正曲线y＝0.0225x+0.097(R 2＝0.993)，再对照萃取物的吸光值；(3)换算每克萃取物中所含没食子酸毫克数，得到萃取物的总酚含量，其中1g萃取物相当于含有5.5±1.6mg没食子酸。
经前述流程所获得的萃取物，其总黄酮含量测定方法如下：(1)1μL兰花生长点萃取物，依序混合3μL的5％NaNO 2溶液、3μL的AlCl 3溶液和40μL的NaOH溶液，再加入ddH 2O使总体积为100μL，混合均匀后反应15分钟后，于波长510nm下测定吸光值(BioTek，Synergy TM2，USA)；(2)利用芸香苷标准品制作的标准曲线y＝0.0032x+0.032(R 2＝0.999)后，再对照萃取物的吸光值；(3)换算每克萃取物中所含芸香苷毫克数，得到萃取物的总黄酮含量，其中1g萃取物相当于含5.0±0.3mg芸香苷。
经前述流程所获得的萃取物，含有吲哚-3-乙酸(indole-3-acetic acid)，此为植物生长素，为重要的植物激素之一，其广泛存在于植物的叶芽和嫩叶等分生组织中，可使细胞生长和细胞分化，进而促进植物的生长与发育。本发明所制成的萃取物利用高效能液相层析法分析兰花生长点萃取物的成分，可进行吲哚-3-乙酸的成分分析，以建立兰花芽胚萃取物的指标成分分析，并以一化合物进行定量分析，藉此做为兰花芽胚萃取物的指标依据。
本发明所制成芽胚萃取物的指标成分分析方法，主要于移动相使用不同 比例的纯水和乙晴，流速为1mL/1min，总试验时间30分钟，测得标准品吲哚-3-乙酸滞留时间为23.194min；兰花芽胚萃取物测试体积为20μL，于相同滞留时间(23.194min)测得最大面积，积分面积参数为92.75％。藉由此一指标成分分析，可得知兰花芽胚萃取物中含有92.75％的吲哚-3-乙酸，往后可以吲哚-3-乙酸作为兰花芽胚萃取物效能成分的指标依据。
本发明的兰花开芽胚萃取方法，具有下列功效：
一、本发明主要提供针对兰花芽胚的培育、切割及萃取的完整流程，且仅撷取并收集兰花最具有活性成份的茎节生长点的再生植物组织，来进行萃取作业，所制成的萃取物活性成分的总多酚、总黄酮及植物激素含量高，品质优，故相较于现有技术中将整株兰花或花瓣进行萃取的方法，本发明所制成的萃取物的总多酚、总黄酮及植物激素含量较高，品质较好。
二、因本发明的萃取物具有优于现有技术的萃取物的活性成份含量及质量，因此将本发明的萃取物添加于化妆保养品产品中，具有活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复等功效。
三、本发明以开花株的茎节生长点部位进行诱导培育，使长出再生新侧芽后，且切割取得新侧芽的植物新生组织，此茎节生长点诱导培育方式可获得大量的新生植物芽胚组织，而可因应大量生产的需求。
四、本发明于开花株的茎节生长点部位先进行培育以大量繁殖新侧芽后，再切割出新生植物组织，此茎节生长点切割作业可去除其它茎梗叶部位，只使用具有较高生长活性含量的新生植物组织进行萃取，故具有去芜存菁的效果，进而可提升萃取物质量，解决现有技术中的兰花萃取技术活性含量及质量无法提升的问题。
五、本发明的芽胚的萃取方法，是在非高温下操作，其萃取过程中温度约50至60℃，以减少温度所造成的热损失，亦可避免低沸点与有效物质挥发及保持生长点组织的生长活性，故可维持新生植物组织的活性含量，解决现有技术中以煮沸、蒸气或热回流提取等加工流程中所造成萃取物的活性减低或有效物质挥发的问题。
六、本发明以超声波水萃取方法，且只使用反渗透纯水，未有其它溶剂，故能改良传统溶剂萃取方法成本高且萃取过程危险的缺点，又可减少处理时间和溶剂使用量并得到高产率。
七、本发明经超声波萃取所制成的半成品萃取液，再进行过滤及去除溶剂作业，因本发明所使用的溶剂为无毒无菌的纯水，因此不会有现有技术中使用醇类或醇类水溶液为溶剂的萃取方式的安全性问题，且本发明经此过滤及去除溶剂作业，可获得活性高及稳定性佳的萃取物。
八、本发明在获得萃取物后，配合建立“兰花芽胚萃取物的指标成分分析方法”可进行成份分析，以得知萃取物中所含植物激素“吲哚-3-乙酸”量是否达到标准，而具有建立植物激素“吲哚-3-乙酸”成分的分析方法及指标依据的效果。
实施例2
本发明的兰花芽胚萃取技术所制成的萃取物，进行与“人体皮肤细胞安全性相关试验”，其试验方式包括有“细胞存活度试验”、“细胞型态变化观察”及“细胞周期试验”三种，各种试验方法详述如下：
本发明的兰花芽胚萃取技术所制成的萃取物，进行“细胞存活度试验”，其试验方式如下：(1)从液态氮桶中将人类皮肤角质株化细胞株HaCaT迅速移至37℃水浴箱内使其在40至60秒内急速解冻，随即将细胞冷冻保存液(含10％DMSO的细胞培养液)加入细胞培养液，将细胞打散并移置25cm 2细胞培养瓶，于37℃、5％CO 2细胞培养箱中生长，平均每隔2天更换一次培养液；(2)将培养瓶的细胞培养液吸去，以PBS清洗一次，再加入适量的1xTrypsin。待细胞剥落后，离心去除上层液，接着加入培养液，把细胞均匀打散。取100μl细胞液到微量离心管，加入同体积的trypan blue均匀混合。最后将混合液吸至血球计数器(hemacytometer)在显微镜下计算细胞数目；(3)将人类皮肤角质株化细胞以1x10 4个细胞/孔密度培养在96孔盘，并在37℃及5％CO 2培养箱中培养至少24小时；(4)加入不同浓度的萃取物以及在指定的时间作用，达反应时间，移除旧的培养液，以PBS清洗一 次，并换上新的培养液，加入10μL的MTT(3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)溶液反应，于37℃、5％CO 2反应4小时后移除培养液，加入100μL的DMSO溶解formazan沉淀物，最后在波长570nm下测定吸光值(BioTek，Synergy TM2，USA)。其中，将人类皮肤角质株化细胞HaCaT分别作用0、0.05、0.1和0.5mg/mL兰花芽胚萃取物作用24小时后，细胞存活度分别为100±2.2％、108.5±1.2％、105.0±2.9％、127.0±3.2％。此结果显示兰花芽胚萃取物浓度小于0.5mg/mL时，细胞存活度大于100％以上。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“细胞型态变化观察”，其试验方式如下：(1)将人类皮肤角质株化细胞HaCaT以1x10 4个细胞/孔密度培养在96孔盘，并在37℃及5％CO 2培养箱中培养至少24小时；(2)加入1μL兰花芽胚萃取物作用人类皮肤角质株化细胞HaCaT24小时，达反应时间后，在显微镜(Nikon，TE2000-U，Japan)下观察细胞型态，并拍照记录。如图2所示，人类皮肤角质株化细胞经兰花芽胚萃取物(0.05、0.1和0.5mg/mL)作用24小时后，细胞型态与控制组的细胞型态无明显差异。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“细胞周期试验”，其试验方式如下：(1)将1x10 4个细胞/孔密度培养在24孔盘中至少24小时，之后加入10μL兰花芽胚萃取物，在培养箱中反应。之后收集上清液至15mL离心管，再以trypsin-EDTA溶液将细胞取下至离心管中，与上清液一并离心1200rpm、5分钟。移除上清液，加入300μL的PBS，缓慢震荡，并逐滴加入700μL绝对酒精固定细胞，再移至微量离心管，置于4℃冰箱中储存；(2)流式细胞仪上机前，细胞在4℃下以1200rpm离心5分钟，移除上清液，依序加入445μL的PBS、5μL的RNase(10mg/mL)、50μL的10％Triton X-100，将细胞的RNA破坏分解后，在37℃反应30分钟，以1200rpm离心5分钟，移除上清液，加入400μL的PBS混合均匀，再加入5μL的PI(5mg/mL)，在4℃避光反应5分钟，以过滤膜过滤；(3)利用流式细胞分析仪(flow cytometer，FACScan)，配合Winmdi计算机软件来分析细胞周期分布比例。 如图3所示，兰花芽胚萃取物(0.1和0.5mg/mL)作用于皮肤角质株化细胞24小时后，细胞周期无显着变化；具体地说，经兰花芽胚萃取物(0.1和0.5mg/mL)作用后sub-G1(M1区域)比例为5.8和6.2％，皆小于10％。
实施例3
针对兰花芽胚的活性评估，本发明亦建立相关测试方法，包括“粒线体再生试验”、“皮肤胶原蛋白生成试验”、“抑制发炎因子一氧化氮活性试验”、“抑制酪胺酸酶活性试验”、“DPPH(1,1-diphenyl-2-picrylhydrazyl)自由基清除试验”、“保护因紫外线及氧化伤害而造成的DNA损伤试验”、“保护片段化DNA效应试验”、“修护皮肤细胞经紫外线作用的细胞存活度试验”、“侦测DNA损伤的环丁嘧啶二聚体(cyclobutane pyrimidine dimers，CPD)生成量试验”、“衰老有关的半乳糖苷酶(Senescence-associatedβ-galactosidase，SA-β-gal)的活性试验”、“SIRT-1基因的mRNA表现试验”，兹分别详细说明如下：
本发明的兰花芽胚萃取技术所制成的萃取物，进行”粒线体再生试验”，其试验方式如下：(1)人类皮肤角质株化细胞HaCaT以1×10 4个细胞/孔的密度培养于24孔盘，在37℃及5％CO 2培养箱中培养至少24小时，加入不同浓度兰花生长点萃取物处理，置入培养箱作用24小时；(2)达反应时间后，移除培养液，加入PBS清洗，加入paraforaldehyde固定细胞，使用1％TritonX100作用5分钟，接着使用PBS清洗细胞，加入mitoview染剂及用Hoechst 33342 staining solution染细胞核(作为定量细胞数)，使用荧光免疫分析仪(BioTek，Synergy TM2，USA)测定粒线体(绿色，激发光：490nm，发射光：523nm)及细胞核荧光(蓝色，激发光：346nm，发射光：460nm)表现；(3)粒线体表现计算公式如下：
粒线体相对质量＝(Mitoview强度/Hoechst 33342强度)X100％
人类粒线体在体内有许多功能，最重要的功能为产生能量；然而，粒线体会随着年龄增加逐渐减少，因此粒线体质量与健康和老化有极大关系。依此结果，人类皮肤角质株化细胞HaCaT经兰花芽胚萃取物(0.05、0.1、0.5 和1.0mg/mL)作用24小时后，粒线体质量分别为102.2±1.2％、105.7±0.2％、110.5±2.1％和124.9±1.7％。此结果显示兰花芽胚萃取物可促进人类皮肤角质株化细胞的粒线体再生。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“皮肤胶原蛋白生成试验”，其试验方式如下：(1)将3T3L-1细胞以2×10 5个细胞/mL密度培养在3公分盘中24小时后，移除上清液再加入含有不同浓度兰花芽胚萃取物的无血清培养液培养48小时，PBS清洗后，刮除细胞，离心1200rpm、5分钟，再去除上清液；(2)利用Sircol soluble collagne assay kit进行胶原蛋白测定，为先将100μL细胞液混合1mL的sircol dye reagent，再在室温下均匀摇晃30分钟，再离心12000rpm、10分钟，直接倒掉上清液，再加入750μL ice-cold acid-salt wash reagent，再离心12000rpm、10分钟，将dyereagent完全去除干净。之后加入250μL的alkali reagent混合均匀，取100μL至96孔盘，在555nm下以酶免疫分析仪测吸光值。
胶原蛋白为保持肌肤弹性及抗皱的重要因子，其合成量受到年龄的影响，因此若能适时促进胶原蛋白的合成量，可避免肌肤生成皱纹、失去弹力光泽的情形。依此结果，经过兰花芽胚萃取物(0.05、0.1和0.5mg/mL)作用后，可促进纤维母细胞中胶原蛋白的生成量约4.7±0.4％、21.1±2.1％及35.0±0.1％。此结果显示兰花芽胚萃取物对于促进胶原蛋白生成量具有优异的效果。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“抑制发炎因子一氧化氮活性试验”，其试验方式如下：取2μL兰花芽胚萃取物(0.05、0.1、0.5和1.0mg/mL)分别加入98μL的25mM硝普钠(sodium nitroprusside，SNP)SNP反应120分钟，之后加入100μL的Griessreagent反应10分钟，以酶免疫分析仪检测546nm吸光值。
SNP本身是氧化氮捐献体的一种，与氧反应会形成亚硝酸(nitrite)，亚硝酸再与Griess reagent反应会变成粉红色溶液，在波长546nm具有特定吸光值。若样品能抑制氧化氮生成的速率而减少亚硝酸产生，其吸光值会 降低；吸光值愈低，表示样品清除氧化氮自由基的能力越强。依上述分析结果，兰花芽胚萃取物(0.05、0.1、0.5和1.0mg/mL)清除氧化氮自由基能力分别为22.0±1.4％、25.0±1.3％、34.7±1.1％和63.4±1.9％。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“抑制酪胺酸酶活性试验”，其试验方式如下：将不同浓度兰花芽胚萃取物取2μL至96孔盘中，加入18μL的DMSO混合，再加入25μL的100unit香菇酪胺酸酶并在室温下反应10分钟。之后加入155μL的2.5mML-DOPA，在492nm的波长以酶免疫分析仪测定吸光值。
依此结果，兰花芽胚萃取物(0.05、0.1、0.5和1.0mg/mL)抑制酪胺酸酶活性分别为12.8±1.5％、28.5±2.2％、46.2±2.5％和75.2±1.8％。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“DPPH(1,1-diphenyl-2-picrylhydrazyl)自由基清除试验”，其试验方式如下：将兰花芽胚萃取物配置成不同浓度，分别加入90μL新鲜配置的100μM的DPPH自由基乙醇溶液后一起添加至96孔盘中反应，以酶免疫分析仪检测517nm吸光值。试验标准品为AA，DPPH自由基清除率公式如下：
DPPH自由基清除率＝[(1-待测样品的517nm吸光值)/控制组的517nm吸光值]X100％
依此结果，兰花芽胚(0.05、0.1、0.5和1.0mg/mL)清除DPPH自由基能力分别为19.6±1.2％、30.5±0.3％、50.0±0.2％和82.6±2.1％。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“保护因紫外线及氧化伤害而造成的DNA损伤试验”，其试验方式如下：(1)将DNA质体pUC119(25μg，0.5μg/μl，Takara，Japan)以1:8比例用PBS稀释，进行各别处理：①控制组、②UVB(20mJ/cm 2)与H 2O 2(1mM)+FeSO 4(0.5mM)作用组、③UVB(20mJ/cm 2)与H 2O 2(1mM)+FeSO 4(0.5mM)+兰花芽胚萃取物作用组；(2)各取2μL的pUC119于微量离心管后，以①、②和③组别分别处理，并分别在37℃作用1小时；(3)加入loadingdye混合后，在0.8％agarose胶体进行电泳30分钟后以电泳胶片影像撷取系统分析；(5)分析S-formDNA 和L-form DNA的比例。质体本为环状结构(supercoil form，S-form)，其受到氧化伤害及紫外线伤害时，会形成开放性(open form，O-form)或是直线型(linear form，L-form)的结构。质体pUC119为一段S-form结构的DNA质体，其经过氧化及UVB伤害后，质体会形成L-form、O-form，受到剧烈伤害时甚至会片段化。如图4(A)与4(B)所示，质体DNA经UVB和氧化伤害(H 2O 2+FeSO 4)作用后(第二条带)，DNA形成片段化，超螺旋结构比例仅为1.6％，而经过兰花芽胚萃取物(0.1mg/mL)作用后(第四条带)，超螺旋结构比例为22.5％。此结果显示兰花芽胚点萃取物具有保护DNA质体不受UVB及氧化伤害的效能。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“保护片段化DNA效应试验”，其试验方式如下：(1)将稀释的2μL的DNA Mw Standard Marker加入0.5mL微量管，进行各别处理：①控制组、②UV与H 2O 2作用组、③UV与H 2O 2+萃取物作用组；(2)于37℃下作用1小时后，加入loading dye混合，以琼脂胶凝胶核酸电泳分析。如图5所示，DNA marker为一片段DNA条状带(未经伤害的DNA，第一条状带)，DNA会依照不同分子量排列。一旦受到氧化及UVB伤害后，条状DNA会变成碎片(第二条状带)，而经过兰花芽胚萃取物(0.05、0.1和0.5mg/mL)作用后，DNA仍保持明显的条状带，亦即兰花芽胚萃取物于0.05mg/mL具有保护DNA效能。此结果再次证实兰花芽胚萃取物具有保护DNA不受氧化及UVB伤害的效果。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“修护皮肤细胞经紫外线作用的细胞存活度试验”，其试验方式如下：(1)将人类皮肤角质细胞以1x10 4个细胞/孔密度培养在96孔盘，并在37℃及5％CO 2培养箱中培养至少24小时，进行分别处理：①控制组-移除细胞上清液，更换无血清的新鲜培养液，培养4小时后，以PBS清洗置入无血清的新鲜培养液，继续培养4小时，进行存活度分析；②UV照射组-移除细胞上清液，更换无血清的新鲜培养液，放置细胞培养箱中培养4小时后，移除上清液以PBS清洗并抽干，进行UV照射后，立即加入无血清的新鲜培养液，继续培养4小时，进行存 活度分析；③UV照射+萃取物组：移除细胞的上清液，将萃取物各别混合于无血清的新鲜培养液，加入培养4小时后，移除上清液以PBS清洗并抽干，分别进行UV照射后，立即加入含萃取物的无血清新鲜培养液，继续培养4小时，进行存活度分析；(2)达反应时间，移除旧的培养液，以PBS清洗一次，并换上新的培养液，加入10μL的MTT溶液反应，于37℃、5％CO 2反应4小时后移除培养液，加入100μL的DMSO溶解formazan沉淀物，最后在波长570nm下测定吸光值(BioTek，Synergy TM2，USA)。
由于化妆保养产品使用时最先接触皮肤角质层，故本试验选用人类皮肤角质株化细胞进行的理由于此。如图6所示，经UVB(20mJ/cm 2)照射后，细胞存活度约80％，而经过兰花芽胚萃取物(0.05、0.1和0.5mg/mL)作用后，细胞存活度为92.1％、100.6％和132.9％；亦即，兰花芽胚萃取物在0.05mg/mL便具有保护细胞免于UVB伤害的效能。此结果显示兰花芽胚萃取物具有保护细胞免于UVB伤害的效能。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“侦测DNA损伤的环丁嘧啶二聚体生成量试验”，其试验方式如下：(1)将1x10 5个细胞/mL培养于24孔盘至少24小时，进行各别处理：①控制组、②UV照射组；③UV照射+萃取物组-萃取物作用4小时后进行UV照射，更换不含血清培养液培养4小时；(2)移除培养液以PBS清洗，以冰甲醇固定细胞，再以Triton X-100作用，加入2M的HCl溶液作用1小时，再以1％BSA填补细胞间隙，再以CPD一级抗体于37℃摇晃反应1小时，去除一级抗体后，再加入二级抗体避光反应30分钟，以PBS清洗并在激发光504nm，发射光524nm下侦测荧光表现；(3)再加入Hoechst 33342(10mg/mL)进行细胞核染色，以PBS清洗后再以激发光355nm，发射光460nm下侦测荧光表现，并在荧光显微镜下观察拍照。
过量的紫外线照射引起DNA损伤的原因在于其能诱发DNA同条链内相邻的嘧啶碱基产生CPD光产物，使DNA空间结构发生变化，从而阻碍DNA复制、转录进而影响蛋白质的生物功能。换言之，经过UVB照射后细胞受到伤害而 碎裂并产生光产物堆积，CPD为一种光产物，可利用荧光侦测的特性进行试验。如图7(A)与7(B)所示，皮肤细胞经兰花芽胚萃取物(0.1和0.5mg/mL)作用再经UVB照射后，仅有些许光产物产生。亦即，兰花芽胚萃取物于0.1mg/mL便具有保护细胞免于UVB伤害的效能。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“衰老有关之半乳糖苷酶的活性试验”，其试验方式如下：
将1x10 5个细胞/mL人类皮肤角质株化细胞HaCaT培养在24孔盘中经24小时待细胞贴附后，移除培养液。加入萃取物处理24小时后，先以100mJ/cm 2UVB照射细胞，再加入不含血清的培养液培养24小时。最后使用衰老有关的半乳糖苷酶染色套组染色，并利用显微镜观察与计数呈蓝色的衰化细胞。
“衰老”为人体的不可逆状态，当细胞分裂繁殖多代后可能会受外界过度刺激而导致生长停滞进而衰老。衰老有关的半乳糖苷酶会于衰老细胞中过度表现，因此可作为细胞衰老的指标之一。如图8所示，经UVB照射后，细胞中的半乳糖苷酶会迅速积累，而对照组维他命C则能有效抑制半乳糖苷酶活性。同样地，经萃取物(0.1和0.5mg/mL)作用的细胞亦能抑制因UVB引起的半乳糖苷酶活化。
本发明的兰花芽胚萃取技术所制成的萃取物，进行“SIRT-1基因的mRNA表现试验”，其试验方式如下：
将1x10 5个细胞/mL人类皮肤角质株化细胞HaCaT培养在6孔盘中24小时后，加入萃取物处理24小时。接着，先用100mJ/cm 2UVB照射细胞，再加入不含血清的培养液培养24小时。之后，收集上清液至15ml离心管后，依序以PBS清洗6孔盘，以1.5倍trypsin-EDTA溶液将细胞取下至离心管中，离心1200rpm、5分钟。在移除上清液后，将剩余细胞液移至1.5ml微量离心管中，再次离心与移除上清液。然后，加入1ml Rezol TM C&T溶液至微量离心管内并于25℃反应5分钟将细胞溶解。加入200μl氯仿至细胞破裂物混合均匀并置于冰上5分钟以萃取RNA。在4℃中以12,000rpm离心、15分钟后，取上层液至一经DEPC处理之水处理过的微量离心管，并加入等体 积的冰异丙醇混合均匀反应10分钟后，在4℃离心12,000rpm、15分钟，RNA便在微量离心管底部沉淀形成透明白色沉淀物。移除上清液，以200μl的冰75％酒精轻轻润洗沉淀物，并在4℃下以12,000rpm离心、5分钟后，移除上清液。静置干燥RNA沉淀物15分钟，之后将RNA沉淀物溶在适量的经DEPC处理的水，储存于-80℃。最后以分光亮度计于OD 260及OD 280测量取得RNA纯度。
取3μg的RNA与适量经DEPC处理的水混合后，加入1μL Oligo(dT)18引子并置于70℃作用2分钟后迅速移至冰上，再分别加入4μL 10x MMLV RT缓冲液、1μL dNTP混合物(每种dNTP 10mM)、0.5μL重组核糖核酸酶抑制剂(1unit/ml)、1μL MMLV反转录酶(5unit)，使总体积为20μL。将其混合均匀并在42℃下作用1小时后，于94℃加热5分钟以去除MMLV反转录酶活性而终止反应，便完成第一股cDNA模板的制备，之后加入80μl经DEPC处理的水，保存于-20℃备用。
取1μL的cDNA至微量离心管中，加入5μL 10x反应缓冲液、0.8μL10mM dNTP(每种dNTP 200mM)及各1μL的50mM上游引子(5’-tcgcaactatacccagaacatagaca-3’)、下游引子(5’-ctgttgcaaaggaaccatgaca-3’)、Taq DNA聚合酶(5unit/μL)，最后加入去离子水使总体积达50μL。均匀混合混合物后，置于自动温度循环机进行PCR反应，反应条件如下：前变性反应，98℃、3分钟为一个循环；继之进行变性反应94℃、黏合反应60℃、合成反应72℃，各1分钟的聚合酶链反应，总反应过程共进行35个循环。反应完毕后，取适量反应产物以2％琼脂凝胶电泳分析。
衰老虽然是人体的不可逆状态，但有些基因的活化可维持细胞功能，以促进个体健康，这些基因即称为长寿基因。SIRT-1为一种长寿基因，经多年研究证实后，发现促进SIRT-1的表现可有效保护细胞修护并维持正常功能，免于细胞走向衰老引发的疾病。
对上述反应产物利用凝胶电泳以对各组别的参照基因β-actin与目标 基因分析，再利用影像软件image J定量。如图9所示，未经过UVB伤害的控制组，其SIRT-1明显表达，但经过UVB照射后，其表现明显降低。而经过萃取物(0.1和0.5mg/mL)作用的细胞则可明显提升SIRT-1的表现，表示萃取物可有效保护细胞免于UVB对长寿基因SIRT-1的伤害。
实时定量PCR与上述RT-PCR虽同为观察基因表现，但两者最大的不同在于：RT-PCR观察的是扩增最终产物，又称终端(end-point)，须藉由凝胶电泳才可进行分析，可能受到图像处理和背景值影响产生实验误差；实时定量PCR则是加入荧光剂，在扩增期间即开始收集讯号，并分析扩增倍数与荧光讯号的相对关系来计算出目标基因的相对含量。
取10μL的cDNA至微量离心管中，并加入OmicsGreen 5x qPCR masterMix及5μL的50mM上游引子(5’-tcgcaactatacccagaacatagaca-3’)与下游引子(5’-ctgttgcaaaggaaccatgaca-3’)均匀混合。之后，置于实时定量机器进行实时定量PCR反应，反应条件如下：前变性反应，98℃、3分钟为一个循环；继之进行变性反应94℃、黏合反应60℃、合成反应72℃，各1分钟的聚合酶链反应，总反应过程共进行25至35个循环。数据使用2 -ΔΔCt的表示来计算基因的相对表达量值。
如图10所示，以未经过UVB伤害的控制组作为基准，而经过UVB照射后，SIRT-1相对表现降低。但经过萃取物(0.1和0.5mg/mL)作用的细胞则可提升SIRT-1的相对表现，表示此萃取物可有效保护细胞免于UVB对长寿基因SIRT-1的伤害。
综合上述，本发明是提供一种针对兰花芽胚切割及萃取技术，从培育、切割及萃取的完整流程，其利用兰花茎节生长点细胞会不断分裂和分化的再生特性，拥有高生命力及活性的特质，在非高温下进行，可避免低沸点与有效物质挥发及保持生长点组织的生长活性，且所制成的芽胚萃取物的总多酚、总黄酮及植物激素含量高、质量佳，使用于化妆保养产品中，可活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复等功效，且茎节生长 点诱导培育方式可因应大量生产的需求，又配合过滤及去除溶剂作业可获得活性及稳定性高的萃取物，据以获致实用性高与经济价值高的兰花活性萃取物，俾使整体确具产业实用性及成本效益。
虽然本发明已以实施例揭露如上，然其并非用以限定本发明，任何所属技术领域中具有通常知识者，在不脱离本发明的精神和范围内，当可作些许之更动与润饰，故本发明的保护范围当视后附之申请专利范围所界定者为准。

Claims (10)

  1. 一种兰花芽胚组织萃取物的用途，是用于制备具活化能量、抗皱、抗发炎、美白、抗氧化、抗老化及保护修复功效的组合物。
  2. 如权利要求1所述的兰花芽胚组织萃取物的用途，其特征在于：，所述兰花芽胚组织萃取物的制备方法包含：
    (a)超声波萃取：将所述兰花芽胚组织放入超声波萃取设备，于低温下，以所述兰花芽胚组织重量5倍量的纯水为溶剂将所述兰花芽胚组织覆盖浸泡，以制成半成品萃取液；以及
    (b)过滤及去除溶剂作业：将所述半成品萃取液经由抽真空的过滤作业后，再利用减压浓缩机将所述半成品萃取液进行去除溶剂作业，以制成萃取物。
  3. 如权利要求1所述的兰花芽胚组织萃取物的用途，其特征在于：，所述兰花为蝴蝶兰V3品种。
  4. 如权利要求1所述的兰花芽胚组织萃取物的用途，其特征在于：所述兰花芽胚组织为生长点新侧芽的新生植物组织，所述生长点新侧芽的新生植物组织的取得方法包含：
    (i)诱导再生新侧芽：将所述兰花的开花株的茎节部位的梗芽切出多段梗芽，并进行培育作业使之长出新侧芽；
    (ii)切除梗芽并撷取新侧芽：将梗芽切除并撷取新侧芽后，取出生长点组织；
    (iii)去除壁膜：将各小片段的所述生长点组织去除壁膜，成为所述新生植物组织；以及
    (iv)收集新生植物芽胚组织：收集所述生长点新侧芽的新生植物芽胚组织。
  5. 如权利要求1所述的兰花芽胚组织萃取物的用途，其特征在于：，每克所述萃取物中没食子酸的含量为4.5至5.28mg，且以芸香苷的标准曲 线来计算，每克所述萃取物中类黄酮化合物的含量为4.2至4.75mg。
  6. 一种兰花芽胚组织萃取物的用途，是用于制备具促进皮肤细胞粒线体再生的组合物。
  7. 如权利要求6所述的兰花芽胚组织萃取物的用途，其特征在于：所述兰花芽胚组织萃取物的制备方法包含：
    (a)超声波萃取：将所述兰花芽胚组织放入超声波萃取设备，于低温下，以所述兰花芽胚组织重量5倍量的纯水为溶剂将所述兰花芽胚组织覆盖浸泡，以制成半成品萃取液；以及
    (b)过滤及去除溶剂作业：将所述半成品萃取液经由抽真空的过滤作业后，再利用减压浓缩机将所述半成品萃取液进行去除溶剂作业，以制成萃取物。
  8. 如权利要求6所述的兰花芽胚组织萃取物的用途，其特征在于：所述兰花为蝴蝶兰V3品种。
  9. 如权利要求6所述的兰花芽胚组织萃取物的用途，其特征在于：所述兰花芽胚组织为生长点新侧芽的新生植物组织，所述生长点新侧芽的新生植物组织的取得方法包含：
    (i)诱导再生新侧芽：将所述兰花的开花株的茎节部位的梗芽切出多段梗芽，并进行培育作业使之长出新侧芽；
    (ii)切除梗芽并撷取新侧芽：将梗芽切除并撷取新侧芽后，取出生长点组织；
    (iii)去除壁膜：将各小片段的所述生长点组织去除壁膜，成为所述新生植物组织；以及
    (iv)收集新生植物芽胚组织：收集生长点新侧芽的新生植物组织。
  10. 如权利要求6所述的兰花芽胚组织萃取物的用途，其特征在于：每 克所述萃取物中没食子酸的含量为4.5至5.28mg，且以芸香苷的标准曲线来计算，每克所述萃取物中类黄酮化合物的含量为4.2至4.75mg。
PCT/CN2020/082769 2020-04-01 2020-04-01 可活化能量、抗皱、抗发炎及美白的兰花芽胚组织萃取物 WO2021196079A1 (zh)

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