CN108541590A - A kind of big white orchid lateral bud histocyte extracting process - Google Patents
A kind of big white orchid lateral bud histocyte extracting process Download PDFInfo
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- CN108541590A CN108541590A CN201810252235.8A CN201810252235A CN108541590A CN 108541590 A CN108541590 A CN 108541590A CN 201810252235 A CN201810252235 A CN 201810252235A CN 108541590 A CN108541590 A CN 108541590A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
- B01D11/0265—Applying ultrasound
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Abstract
The invention discloses a kind of big white orchid lateral bud histocyte extracting process, are related to biological tissue cell abstraction technique field.The present invention includes the following steps：Step 1, plant culture, step 2, sprouting are cultivated, step 3, lateral bud interception, step 4, lateral bud proliferation growth, step 5, the extraction of lateral bud growing point embryonic tissue, step 6, purification by liquid extraction, step 7, extraction component analysis.The present invention is solvent with pure water by taking big white orchid V3 kinds Flowering Plants section stalk bud to carry out tissue cultures, and progress high frequency extraction at 50 60 DEG C is set in the neonatal cell merging ultrasonic equipment of lateral bud cutting.
Description
Technical field
The invention belongs to biological tissue cell abstraction technique fields, more particularly to a kind of big white orchid lateral bud histocyte
Extracting process.
Background technology
General tissue cultures take the terminal bud with meristematic capacity to be cultivated, and growing point production in stem top mostly uses sprout
It is proliferated (bud sprouts) or the mitogenetic seedling mode of protocorm body (PLBs), wherein bud proliferation (bud sprouts) is intended in induction, that is, lower or go
Except terminal bud advantage, so that lateral bud is easy longer, be proliferated sprout by this method.At present traditional water or alcohol are generally taken for sprout
Extraction, no further other technologies extract.
Invention content
The purpose of the present invention is to provide a kind of big white orchid lateral bud histocyte extracting process, by taking big white orchid V3
Kind Flowering Plants section stalk bud carries out tissue cultures, is solvent with pure water, is set with the neonatal cell merging ultrasonic of lateral bud cutting
Standby setting carries out high frequency extraction at room temperature.
In order to solve the above technical problems, the present invention is achieved by the following technical solutions：
The present invention is a kind of big white orchid lateral bud histocyte extracting process, is included the following steps：
Step 1, plant culture：It takes the seed of big white orchid V3 kinds to be put into culture medium and applies enough nutrients and carry out
Plant is cultivated；
Step 2, sprouting are cultivated：It waits for big white orchid V3 kind plant blossoms, intercepts the stem of plant, by the stem of intercepted plant
It is cut into chunks as unit of section and makees sprouting cultivation；
Step 3, lateral bud interception：It takes the lateral bud part of sprouting and carries out cutting leaf；
Step 4, lateral bud proliferation growth：Leaf is cut to the lateral bud of the sprouting of acquisition and makees proliferation growth；
Step 5, the extraction of lateral bud growing point embryonic tissue：After the growing point cell tissue of interception lateral bud cuts section into pieces, with 5
Times amount pure water covering is impregnated, and merging ultrasonic equipment water temperature is set in 50-60 DEG C, power setting 300W, and the time sets 40 minutes,
It carries out stalk bud growing point new life embryonic tissue and extract liquor is obtained by extraction；Extract liquor is evacuated to be obtained by filtration extracted by filtration liquid, mistake
Filter extract liquor goes water removal to obtain thick extract using reduced pressure machine；
Step 6, purification by liquid extraction：Obtained thick extract is impregnated with 5 times of amount pure water coverings, is placed in ultrasonic equipment water temperature
Be set in 50-60 DEG C, power setting 300W, the time sets 40 minutes, is extracted, after extraction the evacuated filtering of extract liquor and
Reduced pressure machine goes to remove water, and repeats 3-5 times to complete that big white orchid growing point extract is obtained by extraction, by the big of acquisition
At -20 DEG C, refrigeration is spare after the freeze-drying of white orchid growing point extract；
Step 7, extraction component analysis：To the extraction component of acquisition carry out total phenol content measurement, determination of total flavonoids and
Total anthocyanidin content measures.
Further, step 2 sprouting is cultivated and includes the following steps：
SS01, the segment of stem is placed in wide-mouth bottle, is disappeared at 75% hydrogen peroxide drop axil, then with 75% alcohol
Poison is finally rinsed 3~5 times with sterile distilled water again；
SS02, the segment of the stem after disinfecting is seeded to bud inducement cultivation base, adjusts illumination and temperature, cultivated 6 days
~10 days to growing sprouting.
Further, the bud inducement cultivation base is to add 100 grams per liter~150 using MS culture mediums as minimal medium
Grams per liter 6- benzyl aminoadenines.
Further, the light intensity of the illumination is 1000~1500lux, and the temperature is 24-28 DEG C.
Further, the step 4 lateral bud proliferation growth includes the following steps：
S01, it the lateral bud of sprouting is cut into leaf is inoculated on callus tissue culture base, in 20-26 DEG C, 1000~1500lux
Under the conditions of cultivate 20-60 days, evoked callus grows to form protocorm；
S02, protocorm is inoculated on the proliferated culture medium after sterilizing, in 22-26 DEG C, the condition of 1000~1500lux
Lower culture 20-60 days, makes Protocorm Multiplication grow.
Further, the callus tissue culture base is basic culture medium, addition sucrose, agar, 6- benzyls with MS culture mediums
Aminoadenine and auxin analog.
Further, the proliferated culture medium is basic culture medium with MS culture mediums, and adds Thidiazuron, methyl α-naphthyl acetate, work
Property charcoal, cerous nitrate, vitamin, agar powder and white granulated sugar.
Further, it includes taking 1ul orchid growing point extracts that 25ul forint is added that total phenol content, which measures, in the step 6
10 times of dilution reagent oscillation mixing of phenol, stand 5 minutes, add 20ul 7.5%Na2CO3Solution and 50ul secondary ion water,
After room temperature is protected from light 1 hour, the light absorption value under 760nm wavelength is measured.It compares gallic acid standard items and makes calibration curve, conversion
Contained gallic acid milligram number in every gram of extract；
Wherein, step 6 determination of total flavonoids takes 1ul orchid growing point extracts and bis- deionizations of 49ul is added
Water and 3ul 5%NaNO2Reaction 6 minutes, adds 3ul 10%AlCl3Reaction 6 minutes after add 40ul 4%NaOH and
4ul secondary deionized waters vibrate after mixing, stand 15 minutes, the light absorption value under 510m wavelength are measured, with the mark of rutin sophorin
Directrix curve calculates the content of flavonoids in sample；
Wherein, it includes taking 0.5g orchid growing point extracts that total anthocyanidin content, which measures, in the step 6, and 10mL extractions are added
Solvent extraction anthocyanidin is taken, this mixed liquor is uniformly mixed, is protected from light effect 60 minutes；Up to after reaction time point, under the conditions of 4 DEG C
Centrifugation 15 minutes takes its supernatant, measures the light absorption value under 530nm wavelength；Total anthocyanidin content=light absorption value × sample weight ×
484.84×100/34300。
The invention has the advantages that：
1, the present invention captures the regeneration of section stalk bud induction lateral bud plant cell tissue with big white orchid V3 kind Flowering Plants,
The lateral bud of cut regeneration takes out the cell of plant new life, and is extracted by Supersonic and obtain growing point extract.
2. ultrasonic wave water extracting process of the present invention, the shortcomings that breaking through improvement conventional solvent extraction, reduces processing time and molten
Agent usage amount simultaneously obtains high yield, and extracting process under low temperature to operate, and to reduce the heat loss caused by temperature, also can avoid low
Boiling point volatilizees with active principle and the growth activity of holding embryonic stem cell.
3. the extract liquor standard of the present invention is up to total polyphenols 2.5mg/ml.
Certainly, it implements any of the products of the present invention and does not necessarily require achieving all the advantages described above at the same time.
Specific implementation mode
A kind of big white orchid lateral bud histocyte extracting process, includes the following steps：
Step 1, plant culture：It takes the seed of big white orchid V3 kinds to be put into culture medium and applies enough nutrients and carry out
Plant is cultivated；
Step 2, sprouting are cultivated：It waits for big white orchid V3 kind plant blossoms, intercepts the stem of plant, by the stem of intercepted plant
It is cut into chunks as unit of section and makees sprouting cultivation；
Step 3, lateral bud interception：It takes the lateral bud part of sprouting and carries out cutting leaf；
Step 4, lateral bud proliferation growth：Leaf is cut to the lateral bud of the sprouting of acquisition and makees proliferation growth；
Step 5, the extraction of lateral bud growing point embryonic tissue：After the growing point cell tissue of interception lateral bud cuts section into pieces, with 5
Times amount pure water covering is impregnated, and merging ultrasonic equipment water temperature is set in 50-60 DEG C, power setting 300W, and the time sets 40 minutes,
It carries out stalk bud growing point new life embryonic tissue and extract liquor is obtained by extraction；Extract liquor is evacuated to be obtained by filtration extracted by filtration liquid, mistake
Filter extract liquor goes water removal to obtain thick extract using reduced pressure machine；
Step 6, purification by liquid extraction：Obtained thick extract is impregnated with 5 times of amount pure water coverings, is placed in ultrasonic equipment water temperature
Be set in 50-60 DEG C, power setting 300W, the time sets 40 minutes, is extracted, after extraction the evacuated filtering of extract liquor and
Reduced pressure machine goes to remove water, and repeats 4 times to complete that big white orchid growing point extract is obtained by extraction, by the great Bai of acquisition
At -20 DEG C, refrigeration is spare after the freeze-drying of orchid growing point extract；
Step 7, extraction component analysis：To the extraction component of acquisition carry out total phenol content measurement, determination of total flavonoids and
Total anthocyanidin content measures.
Further, step 2 sprouting is cultivated and includes the following steps：
SS01, the segment of stem is placed in wide-mouth bottle, is disappeared at 75% hydrogen peroxide drop axil, then with 75% alcohol
Poison is finally rinsed 3~5 times with sterile distilled water again；
SS02, the segment of the stem after disinfecting is seeded to bud inducement cultivation base, adjusts illumination and temperature, cultivated 6 days
~10 days to growing sprouting.
Further, the bud inducement cultivation base is to add 100 grams per liter~150 using MS culture mediums as minimal medium
Grams per liter 6- benzyl aminoadenines.
Further, the light intensity of the illumination is 1000~1500lux, and the temperature is 24-28 DEG C.
Further, the step 4 lateral bud proliferation growth includes the following steps：
S01, it the lateral bud of sprouting is cut into leaf is inoculated on callus tissue culture base, in 20-26 DEG C, 1000~1500lux
Under the conditions of cultivate 20-60 days, evoked callus grows to form protocorm；
S02, protocorm is inoculated on the proliferated culture medium after sterilizing, in 22-26 DEG C, the condition of 1000~1500lux
Lower culture 20-60 days, makes Protocorm Multiplication grow.
Further, the callus tissue culture base is basic culture medium, addition sucrose, agar, 6- benzyls with MS culture mediums
Aminoadenine and auxin analog.
Further, the proliferated culture medium is basic culture medium with MS culture mediums, and adds Thidiazuron, methyl α-naphthyl acetate, work
Property charcoal, cerous nitrate, vitamin, agar powder and white granulated sugar.
Further, it includes taking 1ul orchid growing point extracts that 25ul forint is added that total phenol content, which measures, in the step 6
10 times of dilution reagent oscillation mixing of phenol, stand 5 minutes, add 20ul 7.5%Na2CO3 solution and 50ul secondary ions
Water measures the light absorption value under 760nm wavelength after room temperature is protected from light 1 hour.It compares gallic acid standard items and makes calibration curve,
Convert contained gallic acid milligram number in every gram of extract；
Wherein, step 6 determination of total flavonoids takes 1ul orchid growing point extracts and bis- deionizations of 49ul is added
Water and 3ul 5%NaNO2Reaction 6 minutes, adds 3ul 10%AlCl3Reaction 6 minutes after add 40ul 4%NaOH and
4ul secondary deionized waters vibrate after mixing, stand 15 minutes, the light absorption value under 510m wavelength are measured, with the mark of rutin sophorin
Directrix curve calculates the content of flavonoids in sample；
Wherein, it includes taking 0.5g orchid growing point extracts that total anthocyanidin content, which measures, in the step 6, and 10mL extractions are added
Solvent extraction anthocyanidin is taken, this mixed liquor is uniformly mixed, is protected from light effect 60 minutes；Up to after reaction time point, under the conditions of 4 DEG C
Centrifugation 15 minutes takes its supernatant, measures the light absorption value under 530nm wavelength；Total anthocyanidin content=light absorption value × sample weight ×
484.84×100/34300。
In the description of this specification, the description of reference term " one embodiment ", " example ", " specific example " etc. means
Particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are contained at least one implementation of the present invention
In example or example.In the present specification, schematic expression of the above terms may not refer to the same embodiment or example.
Moreover, particular features, structures, materials, or characteristics described can be in any one or more of the embodiments or examples to close
Suitable mode combines.
Present invention disclosed above preferred embodiment is only intended to help to illustrate the present invention.There is no detailed for preferred embodiment
All details are described, are not limited the invention to the specific embodiments described.Obviously, according to the content of this specification,
It can make many modifications and variations.These embodiments are chosen and specifically described to this specification, is in order to preferably explain the present invention
Principle and practical application, to enable skilled artisan to be best understood by and utilize the present invention.The present invention is only
It is limited by claims and its full scope and equivalent.
Claims (8)
1. a kind of big white orchid lateral bud histocyte extracting process, which is characterized in that include the following steps：
Step 1, plant culture：It takes the seed of big white orchid V3 kinds to be put into culture medium and applies enough nutrients and carry out plant
Culture；
Step 2, sprouting are cultivated：It waits for big white orchid V3 kind plant blossoms, the stem of plant is intercepted, by the stem of intercepted plant to save
It cuts into chunks for unit and makees sprouting cultivation；
Step 3, lateral bud interception：It takes the lateral bud part of sprouting and carries out cutting leaf；
Step 4, lateral bud proliferation growth：Leaf is cut to the lateral bud of the sprouting of acquisition and makees proliferation growth；
Step 5, the extraction of lateral bud growing point embryonic tissue：After the growing point cell tissue of interception lateral bud cuts section into pieces, measured with 5 times
Pure water covering is impregnated, and merging ultrasonic equipment water temperature is set in 50-60 DEG C, power setting 300W, and the time sets 40 minutes, carries out
Extract liquor is obtained by extraction in stalk bud growing point new life embryonic tissue；Extract liquor is evacuated to be obtained by filtration extracted by filtration liquid, filtering extraction
Liquid is taken to go water removal to obtain thick extract using reduced pressure machine；
Step 6, purification by liquid extraction：Obtained thick extract is impregnated with 5 times of amount pure water coverings, merging ultrasonic equipment water temperature setting
In 50-60 DEG C, power setting 300W, the time sets 40 minutes, is extracted, the evacuated filtering of extract liquor and decompression after extraction
Thickener goes to remove water, and repeats 3-5 times to complete that big white orchid growing point extract is obtained by extraction, by the big white orchid of acquisition
At -20 DEG C, refrigeration is spare after the freeze-drying of flower growth point extract；
Step 7, extraction component analysis：Total phenol content measurement, determination of total flavonoids and total flower are carried out to the extraction component of acquisition
Green cellulose content measures.
2. a kind of big white orchid lateral bud histocyte extracting process according to claim 1, which is characterized in that the step
2 sproutings, which are cultivated, to be included the following steps：
SS01, the segment of stem is placed in wide-mouth bottle, at 75% hydrogen peroxide drop axil, then with 75% alcohol disinfecting, most
It is rinsed 3~5 times with sterile distilled water again afterwards；
SS02, the segment of the stem after disinfecting is seeded to bud inducement cultivation base, adjusts illumination and temperature, culture 6 days~10
It is to growing sprouting.
3. a kind of big white orchid lateral bud histocyte extracting process according to claim 2, which is characterized in that the bud lures
It is the addition grams per liter 6- benzyl aminoadenines of 100 grams per liters~150 using MS culture mediums as minimal medium to lead culture medium.
4. a kind of big white orchid lateral bud histocyte extracting process according to claim 2, which is characterized in that the illumination
Light intensity be 1000~1500lux, the temperature be 24-28 DEG C.
5. a kind of big white orchid lateral bud histocyte extracting process according to claim 1, which is characterized in that the step
The proliferation growth of 4 lateral buds includes the following steps：
S01, it the lateral bud of sprouting is cut into leaf is inoculated on callus tissue culture base, in 20-26 DEG C, the condition of 1000~1500lux
Lower culture 20-60 days, evoked callus grows to form protocorm；
S02, protocorm is inoculated on the proliferated culture medium after sterilizing, is trained under conditions of 22-26 DEG C, 1000~1500lux
It supports 20-60 days, Protocorm Multiplication is made to grow.
6. a kind of big white orchid lateral bud histocyte extracting process according to claim 5, which is characterized in that the callus
Tissue culture medium (TCM) is basic culture medium, addition sucrose, agar, 6- benzyls aminoadenine and auxin analog with MS culture mediums.
7. a kind of big white orchid lateral bud histocyte extracting process according to claim 5, which is characterized in that the proliferation
Culture medium is basic culture medium with MS culture mediums, and adds Thidiazuron, methyl α-naphthyl acetate, activated carbon, cerous nitrate, vitamin, agar powder
And white granulated sugar.
8. a kind of big white orchid lateral bud histocyte extracting process according to claim 1, which is characterized in that the step
It includes 10 times of dilution reagent oscillation mixing for taking 1ul orchid growing point extracts that 25ul forint phenol is added that total phenol content, which measures, in 6,
5 minutes are stood, 20ul 7.5%Na are added2CO3Solution and 50ul secondary ion water measure after room temperature is protected from light 1 hour
Light absorption value under 760nm wavelength.It compares gallic acid standard items and makes calibration curve, contained nutgall in the every gram of extract that converts
Sour milligram number；
Wherein, step 6 determination of total flavonoids take 1ul orchid growing point extracts and be added 49ul secondary deionized waters and
3ul 5%NaNO2Reaction 6 minutes, adds 3ul 10%AlCl3Reaction adds 40ul 4%NaOH and 4ul bis- after 6 minutes
Secondary deionized water oscillation after mixing, stands 15 minutes, the light absorption value under 510m wavelength is measured, with the standard curve of rutin sophorin
To calculate the content of flavonoids in sample；
Wherein, it includes taking 0.5g orchid growing point extracts that total anthocyanidin content, which measures, in the step 6, and it is molten that 10mL extractions are added
Agent extracts anthocyanidin, this mixed liquor is uniformly mixed, and is protected from light effect 60 minutes；Up to after reaction time point, centrifuged under the conditions of 4 DEG C
15 minutes, its supernatant is taken, measures the light absorption value under 530nm wavelength；Total anthocyanidin content=light absorption value × sample weight ×
484.84×100/34300。
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Cited By (5)
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| CN111281938A (en) * | 2018-12-07 | 2020-06-16 | 嘉药学校财团法人嘉南药理大学 | Orchid bud embryo tissue extract capable of activating energy, resisting wrinkle, resisting inflammation and whitening |
| CN111281904A (en) * | 2018-12-07 | 2020-06-16 | 嘉药学校财团法人嘉南药理大学 | Cinnamomum kanehirae Hayata embryo tissue extract capable of activating energy, healing wound and protecting and repairing |
| WO2021196079A1 (en) * | 2020-04-01 | 2021-10-07 | 梁家华 | Energy-activating, anti-wrinkle, anti-inflammatory, and whitening orchid embryo tissue extract |
| CN113768849A (en) * | 2020-06-09 | 2021-12-10 | 鸥媄吉国际生物科技有限公司 | Orchid bud embryo composite microcrystalline capsule composition, preparation method thereof and application of orchid bud embryo composite microcrystalline capsule composition in promoting cell detoxification, energy activation and anti-aging |
| TWI794607B (en) * | 2020-05-22 | 2023-03-01 | 鷗媄吉國際生物科技有限公司 | Microspherulite composition composed of orchid germ extract, and its manufacturing method and use for cell detoxification, energy activation, and aging resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111281938A (en) * | 2018-12-07 | 2020-06-16 | 嘉药学校财团法人嘉南药理大学 | Orchid bud embryo tissue extract capable of activating energy, resisting wrinkle, resisting inflammation and whitening |
| CN111281904A (en) * | 2018-12-07 | 2020-06-16 | 嘉药学校财团法人嘉南药理大学 | Cinnamomum kanehirae Hayata embryo tissue extract capable of activating energy, healing wound and protecting and repairing |
| CN111281904B (en) * | 2018-12-07 | 2022-02-08 | 嘉药学校财团法人嘉南药理大学 | Cinnamomum kanehirae Hayata embryo tissue extract capable of activating energy, healing wound and protecting and repairing |
| WO2021196079A1 (en) * | 2020-04-01 | 2021-10-07 | 梁家华 | Energy-activating, anti-wrinkle, anti-inflammatory, and whitening orchid embryo tissue extract |
| TWI794607B (en) * | 2020-05-22 | 2023-03-01 | 鷗媄吉國際生物科技有限公司 | Microspherulite composition composed of orchid germ extract, and its manufacturing method and use for cell detoxification, energy activation, and aging resistance |
| CN113768849A (en) * | 2020-06-09 | 2021-12-10 | 鸥媄吉国际生物科技有限公司 | Orchid bud embryo composite microcrystalline capsule composition, preparation method thereof and application of orchid bud embryo composite microcrystalline capsule composition in promoting cell detoxification, energy activation and anti-aging |
| CN113768849B (en) * | 2020-06-09 | 2023-11-10 | 鸥媄吉国际生物科技有限公司 | Orchid bud embryo composite microcrystal capsule composition, preparation method thereof and application thereof in aging resistance |
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Application publication date: 20180918 |