CN114403005A - Ginseng adventitious root induction method and application of extract in cosmetics - Google Patents
Ginseng adventitious root induction method and application of extract in cosmetics Download PDFInfo
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Images
Classifications
-
- 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
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for inducing adventitious roots of ginseng, which comprises the following steps: obtaining tissue of ginseng seedlings and disinfecting; cutting the tissue of the disinfected ginseng seedling into tissue sections, inoculating the tissue sections into a ginseng adventitious root induction culture medium, inducing ginseng adventitious roots, wherein the culture medium for inducing the adventitious roots of the ginseng comprises an SH culture medium with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-50g/L, agar with the concentration of 6-8g/L and indolebutyric acid with the concentration of 0.5-3.5mg/L, can reduce the problem of tissue infection in the prior art of inducing the adventitious roots of the ginseng and fully utilize the nutritional value of the ginseng seedlings, and uses stem or leaf tissues in the ginseng seedlings to induce the adventitious roots of the ginseng, and confirms the conditions of the ginseng adventitious root induction culture medium, and further evaluates the applicability of the ginseng adventitious root as a raw material with antioxidant activity in the field of cosmetics based on abundant bioactive components of ginseng.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for inducing adventitious roots of ginseng and application of an extract in cosmetics.
Background
Ginseng (Panax ginseng) belongs to Panax notoginseng and purslane, has been widely used as a herbal plant in east asia due to its excellent pharmacological action, has been used for over 2000 years, plays an important role in many pharmacopoeias, and has the physiological activity effects of mainly regulating metabolism, blood pressure, immune function, tumor, oxidation and aging.
However, in the breeding process of ginseng, the environmental conditions such as temperature, luminosity and humidity need to be accurately adjusted, and a long time period is needed to obtain ginseng of a certain size, the tissue culture of ginseng using the tissue culture technology can adopt a small amount of tissues to culture a large amount of tissues, and a large amount of nutritive seedlings can be obtained in a short period of time without being limited by uncontrollable factors such as weather or seasons. And the sterile state is kept in the culture process without the treatment of pesticides, and simultaneously, the nutrient components of the maternal tissue can be stably kept, so that the method conforms to the concept of environmental protection and has economic values of reducing labor force and the like.
The ginseng is composed of roots, stems, leaves and berries, wherein the stems and leaves of the ginseng seedlings contain rich nutritional ingredients such as saponin, beta-sitosterol, daucosterol and the like, and have sufficient application value, but most of the existing ginseng adventitious root induction methods adopt the roots of the ginseng to induce the adventitious roots of the ginseng, and on the contrary, the ginseng adventitious root induction methods utilizing the stems or the leaves of the ginseng seedlings are not enough, which is the value of the ginseng seedlings which are not fully utilized; moreover, the ginseng root tissue has a lot of threads and silt on the surface, so that the ginseng root tissue is difficult to clean in the surface disinfection stage and has higher bacterial contamination rate. On the other hand, the use of a high-concentration disinfectant solution for improving the disinfection efficiency causes excessive damage to plant tissues, resulting in withering of the plant tissues. Therefore, the problem of efficiently reducing the bacterial contamination in the tissue culture process of ginseng is a problem to be solved in the tissue culture technology of ginseng.
Meanwhile, although the ginseng adventitious roots currently retain rich bioactive components in ginseng, their applications in the cosmetic field are rare, and oxidative damage is one of the main causes of skin damage and aging. Therefore, the evaluation of antioxidant effect on the ginseng adventitious roots extract opens the applicability of the ginseng adventitious roots extract as a cosmetic raw material having antioxidant effect.
Disclosure of Invention
The invention aims to solve the technical problems that because the surface of ginseng root tissue has a plurality of threads and silt, the ginseng root tissue is difficult to clean and has higher bacterial contamination rate in the surface disinfection stage, on the contrary, if high-concentration disinfection solution is used for improving the disinfection efficiency, the excessive damage of plant tissue is caused, the withering of the plant tissue is caused, and the like And the method is used for solving the defects caused by the prior art.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for inducing adventitious roots of ginseng comprises the following steps:
obtaining tissue of ginseng seedlings and disinfecting;
cutting the sterilized tissue of the ginseng seedling into tissue sections, and inoculating the tissue sections into a ginseng adventitious root induction culture medium to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises an SH culture medium (Schenk and Hildebrandt) with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-35g/L, agar with the concentration of 6-8g/L and indolebutyric acid (IBA) with the concentration of 0.5-3.5 mg/L.
The ginseng adventitious root induction method comprises the following steps of:
washing the surface of the ginseng seedling tissue in tap water, then washing in 70% ethanol solution for 30-60 s, then soaking in 5-15% bleaching agent, shaking for 5-25min at the rotating speed of 70-130rpm, and then washing with sterilized distilled water for 4-5 times in a sterile environment.
The method for inducing adventitious roots of ginseng comprises the steps of inoculating the tissue section with the ginseng adventitious root induction culture medium, and culturing the tissue section at 25 +/-2 ℃ in the dark for 3-6 weeks to induce the adventitious roots of ginseng, wherein the length of the tissue section is 0.5-1 cm;
the ginseng adventitious root induction culture medium comprises an SH culture medium with the concentration of 12.15g/L, sucrose with the concentration of 30g/L, agar with the concentration of 7g/L and indolebutyric acid with the concentration of 2 mg/L.
The method for inducing adventitious roots of ginseng is characterized in that the pH value of the ginseng adventitious root induction culture medium is 5.6-5.9.
In the method for inducing adventitious roots of ginseng, the SH culture medium with the concentration of 6.075-24.3g/L can be replaced by MS (Murashige and Skoog) culture medium with the concentration of 2.2-6.6g/L or B5 culture medium with the concentration of 1.605-4.815 g/L;
the induction culture medium also comprises one or more of 2, 4-dichlorophenoxyacetic acid with the concentration of 0.1-3mg/L, 6-furfurylaminopurine with the concentration of 0.01-1mg/L, 1-naphthylacetic acid with the concentration of 1-5mg/L and 6-benzylaminopurine with the concentration of 0.001-1 mg/L.
The method for inducing the adventitious roots of the ginseng further comprises the following specific steps of carrying out subculture on the adventitious roots of the ginseng:
cutting the ginseng adventitious root into tissue sections of 30-150mm multiplied by 30-150mm by using a sterilization knife, transferring the tissue sections into a subculture medium, performing solid proliferation culture every 3-6 weeks, repeating the solid proliferation culture for 1-2 times, and performing liquid medium culture, wherein the solid proliferation culture comprises an SH culture medium with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-50g/L, agar with the concentration of 6-8g/L, and indolebutyric acid with the concentration of 0.5-3.5 mg/L; the liquid proliferation culture comprises SH culture medium with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-35g/L and indolebutyric acid with the concentration of 0.5-3.5 mg/L.
In the method for inducing adventitious roots of ginseng, the liquid proliferation culture is performed by adding the adventitious roots of ginseng with the inoculation amount of 0.5-10g/L into the liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 70-100rpm and the temperature of 25 +/-2 ℃ in a dark condition.
The method for inducing adventitious roots of ginseng further comprises the step of extracting an extract of adventitious roots of ginseng from the subcultured adventitious roots of ginseng, and the method specifically comprises the following steps:
washing the ginseng adventitious roots with distilled water twice, drying by adopting a drying or freeze-drying method, crushing, and sieving by using a 60-100-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 70% -80% of ethanol according to a material-liquid ratio of 1:10-100, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtered filtrate under reduced pressure until ethanol is removed to obtain the ginseng adventitious root extract.
In the method for inducing adventitious roots of ginseng, the powder of the adventitious roots of ginseng is preferably obtained by sieving the ginseng with a 80-mesh sieve;
the ginseng adventitious root tissue powder is mixed with 70% ethanol according to the material-liquid ratio of 1: 20.
The extract of ginseng adventitious root can be used in cosmetics.
The technical scheme provided by the method for inducing the adventitious roots of the ginseng has the following technical effects:
the tissue infection problem in the existing method for inducing the adventitious roots of the ginseng by using the roots of the ginseng can be reduced, the nutritive values of stem and leaf tissues of ginseng seedlings are fully utilized, the stem or leaf tissues of the ginseng seedlings are used for inducing the adventitious roots of the ginseng, the conditions of a culture medium for inducing the adventitious roots of the ginseng are confirmed, the obtained adventitious root tissues are subjected to preparation of the extract of the adventitious roots of the ginseng, and meanwhile, the applicability of the extract of the adventitious roots of the ginseng as a raw material with antioxidant activity in the field of cosmetics is further evaluated based on rich bioactive components of the ginseng.
Drawings
FIG. 1 is a diagram of ginseng adventitious roots induced in the present invention;
FIG. 2 is a graph of DPPH radical scavenging rate of an extract from adventitious roots of Panax ginseng;
FIG. 3 is a graph of ABTS free radical clearance of ginseng adventitious root extract.
Detailed Description
In order to make the technical means, the inventive features, the objectives and the effects of the invention easily understood and appreciated, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the specific drawings, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments.
All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.
In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1, obtaining a ginseng seedling stem tissue or a ginseng seedling leaf tissue, washing the surface of the ginseng seedling stem tissue or the ginseng seedling leaf tissue in tap water, then washing the surface of the ginseng seedling stem tissue or the ginseng seedling leaf tissue in 70% ethanol solution for 30s to 60s, then soaking the ginseng seedling stem tissue or the ginseng seedling leaf tissue in 5% bleaching agent, shaking the ginseng seedling stem tissue or the ginseng seedling leaf tissue at the speed of 70rpm for 5min, and then washing the ginseng seedling stem tissue or the ginseng seedling leaf tissue with sterilized distilled water for 4 to 5 times in an aseptic environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing the tissue sections in the dark at the temperature of 25 +/-2 ℃ for 3-6 weeks to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises an SH culture medium (Schenk and Hildebrandt) with the concentration of 6.075g/L, sucrose with the concentration of 15g/L, agar with the concentration of 6g/L and indolebutyric acid (IBA) with the concentration of 0.5 mg/L;
carrying out subculture on the ginseng adventitious roots:
cutting adventitious roots of ginseng into tissue segments of 30-150mm multiplied by 30-150mm by a sterilization knife, transferring the tissue segments into a subculture medium, performing solid proliferation culture every 3-6 weeks, repeating for 1-2 times, and performing liquid proliferation culture, wherein the solid proliferation culture comprises an SH culture medium with the concentration of 6.075g/L, sucrose with the concentration of 15g/L, agar with the concentration of 6g/L and indolebutyric acid with the concentration of 0.5mg/L, and the liquid proliferation culture comprises an SH culture medium with the concentration of 6.075g/L, sucrose with the concentration of 15g/L and indolebutyric acid with the concentration of 0.5 mg/L;
adding the ginseng adventitious roots with the inoculation amount of 0.5g/L into a liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 70rpm and the temperature of 25 +/-2 ℃ under the condition of keeping out of the sun;
extracting the ginseng adventitious root extract from the subcultured ginseng adventitious root:
washing the adventitious roots of the ginseng with distilled water twice, drying and crushing the ginseng by adopting a drying or freeze-drying method, and sieving the ginseng with a 60-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 70% ethanol according to a material-liquid ratio of 1:10, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtrate under reduced pressure until ethanol is removed to obtain Ginseng radix adventitious root extract.
Example 2, obtaining a stem tissue or a leaf tissue of a ginseng seedling, washing the surface of the stem tissue or the leaf tissue of the ginseng seedling in tap water, then washing the surface in a 70% ethanol solution for 30s to 60s, then soaking the stem tissue or the leaf tissue of the ginseng seedling in a 10% bleaching agent, shaking the tissue at a speed of 100rpm for 15min, and then washing the tissue with sterilized distilled water for 4 to 5 times in an aseptic environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing the tissue sections in the dark for 3-6 weeks at the temperature of 25 +/-2 ℃ to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises SH culture medium (Schenk and Hildebrandt) with the concentration of 12.15g/L, cane sugar with the concentration of 20g/L, agar with the concentration of 7g/L and indolebutyric acid (IBA) with the concentration of 2 mg/L;
carrying out subculture on the ginseng adventitious roots:
cutting adventitious roots of ginseng into tissue segments of 30-150mm multiplied by 30-150mm by a sterilization knife, transferring the tissue segments into a subculture medium, performing solid proliferation culture every 3-6 weeks, repeating for 1-2 times, and performing liquid proliferation culture, wherein the solid proliferation culture comprises an SH culture medium with the concentration of 12.15g/L, sucrose with the concentration of 20g/L, agar with the concentration of 7g/L and indolebutyric acid with the concentration of 2mg/L, and the liquid proliferation culture comprises an SH culture medium with the concentration of 12.15g/L, sucrose with the concentration of 20g/L and indolebutyric acid with the concentration of 2 mg/L;
adding the ginseng adventitious roots with the inoculation amount of 5g/L into a liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 85rpm and the temperature of 25 +/-2 ℃ under the condition of keeping out of the sun;
extracting the ginseng adventitious root extract from the subcultured ginseng adventitious root:
washing the ginseng adventitious roots with distilled water twice, drying by adopting a drying or freeze-drying method, crushing, and sieving by using a 80-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 75% ethanol according to the material-liquid ratio of 1:55, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtrate under reduced pressure until ethanol is removed to obtain Ginseng radix adventitious root extract.
Example 3, obtaining a stem tissue or a leaf tissue of a ginseng seedling, washing the surface of the stem tissue or the leaf tissue of the ginseng seedling in tap water, then washing the surface in a 70% ethanol solution for 30s to 60s, then soaking the stem tissue or the leaf tissue of the ginseng seedling in a 15% bleaching agent, shaking the tissue at a speed of 130rpm for 25min, and then washing the tissue with sterilized distilled water for 4 to 5 times in an aseptic environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing the tissue sections in the dark at the temperature of 25 +/-2 ℃ for 3-6 weeks to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises SH culture medium (Schenk and Hildebrandt) with the concentration of 24.3g/L, cane sugar with the concentration of 50g/L, agar with the concentration of 8g/L and indolebutyric acid (IBA) with the concentration of 3.5 mg/L;
carrying out subculture on the ginseng adventitious roots:
cutting adventitious roots of ginseng into tissue sections of 30-150mm multiplied by 30-150mm by a sterilization knife, transferring the tissue sections into a subculture medium, performing solid proliferation culture every 3-6 weeks, repeating for 1-2 times, and performing liquid proliferation culture, wherein the solid proliferation culture comprises an SH culture medium with the concentration of 24.3/L, sucrose with the concentration of 50g/L, agar with the concentration of 8g/L and indolebutyric acid with the concentration of 3..5mg/L, and the liquid proliferation culture comprises an SH culture medium with the concentration of 24.3g/L, sucrose with the concentration of 50g/L and indolebutyric acid with the concentration of 3.5 mg/L;
adding the ginseng adventitious roots with the inoculation amount of 10g/L into a liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 100rpm and the temperature of 25 +/-2 ℃ under the condition of keeping out of the sun;
extracting the ginseng adventitious root extract from the subcultured ginseng adventitious root:
washing the adventitious roots of the ginseng with distilled water twice, drying and crushing the ginseng by adopting a drying or freeze-drying method, and sieving the ginseng with a 100-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 80% ethanol according to the material-liquid ratio of 1:100, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtrate under reduced pressure until ethanol is removed to obtain Ginseng radix adventitious root extract.
Example 4, obtaining a stem tissue or a leaf tissue of a ginseng seedling, washing the surface of the stem tissue or the leaf tissue of the ginseng seedling in tap water, then washing the surface in a 70% ethanol solution for 30s to 60s, then soaking the stem tissue or the leaf tissue of the ginseng seedling in a 15% bleaching agent, shaking the tissue at a speed of 130rpm for 25min, and then washing the tissue with sterilized distilled water for 4 to 5 times in an aseptic environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing the tissue sections in the dark at the temperature of 25 +/-2 ℃ for 3-6 weeks to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises an MS culture medium with the concentration of 4.4g/L, cane sugar with the concentration of 35g/L, agar with the concentration of 8g/L and indolebutyric acid (IBA) with the concentration of 3.5 mg/L;
carrying out subculture on the ginseng adventitious roots:
cutting adventitious roots of ginseng into tissue segments of 30-150mm multiplied by 30-150mm by a sterilization knife, transferring the tissue segments into a subculture medium, performing solid proliferation culture every 3-6 weeks, repeating for 1-2 times, and performing liquid proliferation culture, wherein the solid proliferation culture comprises an MS culture medium with the concentration of 4.4g/L, sucrose with the concentration of 35g/L, agar with the concentration of 8g/L and indolebutyric acid with the concentration of 3.5mg/L, and the liquid proliferation culture comprises an MS culture medium with the concentration of 4.4g/L, sucrose with the concentration of 35g/L and indolebutyric acid with the concentration of 3.5 mg/L;
adding the ginseng adventitious roots with the inoculation amount of 10g/L into a liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 100rpm and the temperature of 25 +/-2 ℃ under the condition of keeping out of the sun;
extracting the ginseng adventitious root extract from the subcultured ginseng adventitious root:
washing the adventitious roots of the ginseng with distilled water for 2 times, drying by adopting a drying or freeze-drying method, crushing, and sieving by using a 100-mesh sieve to obtain tissue powder of the adventitious roots of the ginseng;
mixing the ginseng adventitious root tissue powder with 70% ethanol according to a material-liquid ratio of 1:100, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtrate under reduced pressure until ethanol is removed to obtain Ginseng radix adventitious root extract.
Example 5, obtaining a stem tissue or a leaf tissue of a ginseng seedling, washing the surface of the stem tissue or the leaf tissue of the ginseng seedling in tap water, then washing the surface in a 70% ethanol solution for 30s to 60s, then soaking the stem tissue or the leaf tissue of the ginseng seedling in a 15% bleaching agent, shaking the tissue at a speed of 130rpm for 25min, and then washing the tissue with sterilized distilled water for 4 to 5 times in an aseptic environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing the tissue sections in the dark at the temperature of 25 +/-2 ℃ for 3-6 weeks to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises a B5 culture medium with the concentration of 3.21g/L, cane sugar with the concentration of 35g/L, agar with the concentration of 8g/L and indolebutyric acid (IBA) with the concentration of 3.5 mg/L;
carrying out subculture on the ginseng adventitious roots:
cutting adventitious roots of ginseng into tissue segments of 30-150mm × 30-150mm by using a sterilization knife, transferring the tissue segments into a subculture medium, performing solid proliferation culture every 3-6 week period, repeating the solid proliferation culture for 1-2 times, and performing liquid proliferation culture, wherein the solid proliferation culture comprises a B5 culture medium with the concentration of 3.21g/L, sucrose with the concentration of 35g/L, agar with the concentration of 8g/L and indolebutyric acid with the concentration of 3.5mg/L, and the liquid proliferation culture comprises a B5 culture medium with the concentration of 3.21g/L, sucrose with the concentration of 35g/L and indolebutyric acid with the concentration of 3.5 mg/L;
adding the ginseng adventitious roots with the inoculation amount of 10g/L into a liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 100rpm and the temperature of 25 +/-2 ℃ under the condition of keeping out of the sun;
extracting the ginseng adventitious root extract from the subcultured ginseng adventitious root:
washing the adventitious roots of the ginseng with distilled water twice, drying and crushing the ginseng by adopting a drying or freeze-drying method, and sieving the ginseng with a 100-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 70% ethanol according to a material-liquid ratio of 1:100, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtrate under reduced pressure until ethanol is removed to obtain Ginseng radix adventitious root extract.
Example 6: obtaining a ginseng seedling stem tissue or a ginseng seedling leaf tissue, washing the surface of the ginseng seedling stem tissue or the ginseng seedling leaf tissue in tap water, then washing the surface of the ginseng seedling stem tissue or the ginseng seedling leaf tissue in 70% ethanol solution for 30-60 s, then soaking the washed surface in 10% bleaching agent, shaking the washed surface for 15min at the speed of 100rpm, and then washing the washed surface for 4-5 times by using sterilized distilled water in a sterile environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing the tissue sections in the dark at the temperature of 25 +/-2 ℃ for 3-6 weeks to induce the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises SH culture medium (Schenk and Hildebrandt) with the concentration of 12.15g/L, cane sugar with the concentration of 20g/L, agar with the concentration of 7g/L, indolebutyric acid with the concentration of 2mg/L, 2, 4-dichlorophenoxyacetic acid with the concentration of 1.55mg/L, 6-furfurylaminopurine with the concentration of 0.55mg/L, 1-naphthylacetic acid with the concentration of 2.5mg/L, and 6-benzylaminopurine with the concentration of 0.505 mg/L;
carrying out subculture on the ginseng adventitious roots:
cutting adventitious roots of Ginseng radix into tissue segments of 30-150mm × 30-150mm with a sterilizing knife, transferring into subculture medium, performing solid proliferation culture every 3-6 weeks, repeating for 1-2 times, and performing liquid proliferation culture, wherein the solid proliferation culture comprises SH medium (Schenk and Hildebrandt) with concentration of 12.15g/L, sucrose with concentration of 20g/L, agar with concentration of 7g/L, indolebutyric acid with concentration of 2mg/L, 2, 4-dichlorophenoxyacetic acid with concentration of 1.55mg/L, 6-furfurylaminopurine with concentration of 0.55mg/L, 1-naphthylacetic acid with concentration of 2.5mg/L, and 6-benzylaminopurine with concentration of 0.505mg/L, and the liquid proliferation culture comprises SH medium (Schenk and Hildebrandt) with concentration of 12.15g/L, Sucrose with the concentration of 20g/L, indolebutyric acid with the concentration of 2mg/L, 2, 4-dichlorophenoxyacetic acid with the concentration of 1.55mg/L, 6-furfurylaminopurine with the concentration of 0.55mg/L, 1-naphthylacetic acid with the concentration of 2.5mg/L and 6-benzylaminopurine with the concentration of 0.505 mg/L;
adding the ginseng adventitious roots with the inoculation amount of 5g/L into a liquid culture medium, and culturing for 3-5 weeks at the rotation speed of 85rpm and the temperature of 25 +/-2 ℃ under the condition of keeping out of the sun;
extracting the ginseng adventitious root extract from the subcultured ginseng adventitious root:
washing the ginseng adventitious roots with distilled water twice, drying by adopting a drying or freeze-drying method, crushing, and sieving by using a 80-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 70% ethanol according to a material-liquid ratio of 1:20, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtrate under reduced pressure until ethanol is removed to obtain Ginseng radix adventitious root extract.
Comparative example 1: washing the surface of the ginseng root tissue in tap water, then cleaning the ginseng root tissue in 70% ethanol solution for 30-60 s, then soaking the ginseng root tissue in 5% bleaching agent, shaking for 5min at the speed of 70rpm, and then washing the ginseng root tissue with sterilized distilled water for 4-5 times in a sterile environment;
then, after cutting the ginseng root tissue into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing for 3-6 weeks in the dark at the temperature of 25 +/-2 ℃, wherein the ginseng root tissue inoculation culture medium comprises MS culture medium with the concentration of 4.4g/L, cane sugar with the concentration of 30g/L and agar with the concentration of 7 g/L.
Comparative example 2: washing the surface of the ginseng root tissue in tap water, then cleaning the ginseng root tissue in 70% ethanol solution for 30-60 s, then soaking the ginseng root tissue in 10% bleaching agent, shaking the tissue at the speed of 100rpm for 15min, and then washing the tissue with sterilized distilled water for 4-5 times in a sterile environment;
then, after cutting the ginseng root tissue into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing for 3-6 weeks in the dark at the temperature of 25 +/-2 ℃, wherein the ginseng root tissue inoculation culture medium comprises MS culture medium with the concentration of 4.4g/L, cane sugar with the concentration of 30g/L and agar with the concentration of 7 g/L.
Comparative example 3: washing the surface of the ginseng root tissue in tap water, then cleaning the ginseng root tissue in 70% ethanol solution for 30-60 s, then soaking the ginseng root tissue in 15% bleaching agent, shaking the tissue at the speed of 130rpm for 25min, and then washing the tissue with sterilized distilled water for 4-5 times in a sterile environment;
then, after cutting the ginseng root tissue into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing for 3-6 weeks in the dark at the temperature of 25 +/-2 ℃, wherein the ginseng root tissue inoculation culture medium comprises MS culture medium with the concentration of 4.4g/L, cane sugar with the concentration of 30g/L and agar with the concentration of 7 g/L.
Comparative example 4: obtaining stem tissue or leaf tissue of ginseng seedling, washing the surface of ginseng root tissue in tap water, then washing in 70% ethanol solution for 30-60 s, then soaking in 10% bleaching agent, shaking at 100rpm for 15min, and then washing with sterilized distilled water for 4-5 times in sterile environment;
then, cutting the stem tissue or leaf tissue of the ginseng seedling into tissue sections with the length of 0.5-1cm, inoculating the tissue sections to a ginseng adventitious root induction culture medium with the pH value range of 5.6-5.9, and culturing for 3-6 weeks at the temperature of 25 +/-2 ℃ in a dark place, wherein the culture medium comprises an MS culture medium with the concentration of 4.4g/L, sucrose with the concentration of 30g/L and agar with the concentration of 7 g/L.
(1) The experiment of the bacterial contamination rate after the surface of the ginseng explant is disinfected:
most of the existing methods for inducing the adventitious roots of ginseng adopt the roots of ginseng and then induce the adventitious roots, however, in the process of sterilizing the surface of the ginseng roots, because of a plurality of threads on the surface of the ginseng roots, the soil and sand remained on the surface are not easy to clean and the infection rate after the sterilization treatment is higher, so that the improvement of the surface sterilization efficiency is one of the problems to be solved in the process of culturing the ginseng tissues, in the invention, in order to reduce the bacterial contamination rate in the process of sterilizing the ginseng explants, the ginseng seedling stems or the ginseng seedling leaves are used for surface sterilization and comparison;
the bacterial contamination rates of the stems or leaves of the ginseng seedlings in the comparative examples 1, 2 and 3 and the six examples are compared, and the results are shown in the table 1;
the calculation formula of the bacterial contamination rate is as follows:
| experimental group | The bacterial contamination rate% |
| Comparative example 1 | 97.62 |
| Comparative example 1 | 95.14 |
| Comparative example 3 | 89.17 |
| Example 1 | 62.14 |
| Example 2 | 54.48 |
| Example 3 | 36.24 |
| Example 4 | 40.89 |
| Example 5 | 37.56 |
| Example 6 | 53.27 |
Table 1: infection rate results after surface sterilization of different parts of ginseng explants
As can be seen from the results of table 1, comparative example 2, and comparative example 3 were 97.62%, 95.14%, and 89.17% in terms of the contamination ratio, respectively, and example 1, example 2, example 3, example 4, example 5, and example 6 were 62.14%, 54.48%, 36.24%, 40.89%, 37.56%, and 53.27% in terms of the contamination ratio, respectively, indicating that the contamination ratio was low in all examples compared to comparative example 1, which means that the surface sterilization method using the stem or leaf of ginseng seedling can bring about a more efficient sterilization result than the surface sterilization method using the root of ginseng.
(2) Ginseng adventitious root induction experiment:
cutting the sterile ginseng seedling stem or leaf tissue obtained in the step (1) into 0.5-1.5cm multiplied by 0.5-1.5cm by using a sterilization scalpel, then inoculating the cut tissue into an adventitious root culture medium, culturing for 36 weeks at the temperature of 25 +/-2 ℃ in the dark, inoculating 10 seedlings into each culture dish, paralleling 3 seedlings, and calculating the adventitious root induction rate of the ginseng according to the formula:
as is clear from table 2 and fig. 1, the induction rate of the adventitious roots in the ginseng under the adventitious roots condition is 0% in comparative example 4, and the induction rates of the adventitious roots in the ginseng in examples 1, 2, 3, 4, 5, and 6 are 46%, 81%, 67%, 64%, 42%, and 14%, respectively, and the results show that the adventitious roots in the ginseng can be induced in all of the examples of the present invention, and that the color of the ginseng is milky white in examples 1 to 6 after culturing for 3 to 6 weeks.
| Experimental group | Induction rate of adventitious root% |
| Comparative example 4 | 0 |
| Example 1 | 46 |
| Example 2 | 81 |
| Example 3 | 67 |
| Example 4 | 64 |
| Example 5 | 42 |
| Example 6 | 14 |
Table 2: adventitious root induction rate and induction state of ginseng
(3) Evaluation of antioxidant efficacy:
1) determination of DPPH radical scavenging Capacity:
DDPH free radical is a very stable free radical, and the color of 1, 1-diphenyl-2-picrylhydrazino reagent is reduced from purple to yellow with the increase of the amount of antioxidant, so that the antioxidant capacity of the antioxidant can be quantified, and the determination method comprises adding 500 μ L of ginseng adventitious root extract into a 24-well plate, adding 500 μ L of 0.2mM DPPH solution into the well into which all experimental samples have been added, and determining the light absorption value at 517nm after reacting for 30min in a dark condition;
the concentration of the solution in the embodiments 1, 2, 3, 4, 5 and 6 is 0.1%;
the positive control group is 5 mug/L of L-ascorbic acid;
the negative control group is blank;
the DPPH free radical clearance is calculated by the formula:
the results of DPPH radical scavenging at 0.1% concentration in examples 1-6 are shown in FIG. 2, and DPPH radical scavenging at 0.1% concentration in examples 1-6 is 62.54%, 91.25%, 87.25%, 86.24%, 76.42%, 84.53%, respectively, i.e., examples 1-6 all effectively scavenge DPPH radicals, indicating that examples 1-6 all have good antioxidant activity.
2) ABTS + clearance assay:
ABTS is oxidized into green ABTS + under the action of a proper oxidant, the generation of ABTS + is inhibited in the presence of the oxide, and the total antioxidant capacity of the sample can be measured and calculated by measuring the absorbance of ABTS at 734 nm.
Adding 0.2ml of potassium persulfate solution with the concentration of 2.6mmol/L into a test tube, then adding 0.2ml of ABTS solution with the concentration of 7.4mmol/L, storing for 12h at 4 ℃ to prepare ABTS stock solution, enabling the light absorption value of the ABTS solution to be 0.700 +/-0.02 at 734nm to prepare ABTS working solution, adding 0.1ml of experimental sample into 1.9ml of ABTS working solution, mixing uniformly, reacting for 6min in a dark place, and then measuring the light absorption value at 734 nm;
the concentration of the solution in the embodiments 1, 2, 3, 4, 5 and 6 is 0.1%;
the positive control group is 5 mug/L of L-ascorbic acid;
the negative control group is blank;
the formula for calculating the ABTS free radical clearance is as follows:
the results of the removal of ABTS free radicals at 0.1% concentration in examples 1-6 are shown in FIG. 3, and the removal of ABTS free radicals at 0.1% concentration in examples 1-6 are 85.36%, 93.65%, 97.35%, 91.19%, 92.52%, 87.54%, respectively, i.e., examples 1-6 all effectively remove ABTS free radicals. It is demonstrated that examples 1-6 all have good antioxidant activity.
In conclusion, the method for inducing the adventitious roots of ginseng according to the present invention can reduce the problem of tissue infection in the conventional method for inducing the adventitious roots of ginseng roots, fully utilize the nutritive value of the ginseng seedlings, induce the adventitious roots of ginseng using the stem or leaf tissues of the ginseng seedlings, and confirm the conditions of the culture medium for inducing the adventitious roots of ginseng. Then, an extract of ginseng adventitious roots was prepared. Finally, the applicability of ginseng adventitious roots extract as a raw material having antioxidant activity in the field of cosmetics has been further advanced based on the abundant bioactive components of ginseng.
Specific embodiments of the invention have been described above. It is to be understood that the invention is not limited to the particular embodiments described above, in that devices and structures not described in detail are understood to be implemented in a manner common in the art; various changes or modifications may be made by one skilled in the art within the scope of the claims without departing from the spirit of the invention, and without affecting the spirit of the invention.
Claims (10)
1. A method for inducing adventitious roots of ginseng is characterized by comprising the following steps:
obtaining tissue of ginseng seedlings and disinfecting;
cutting the sterilized tissue of the ginseng seedling into tissue segments, inoculating the tissue segments into a ginseng adventitious root induction culture medium, and inducing the ginseng adventitious root, wherein the ginseng adventitious root induction culture medium comprises an SH culture medium with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-50g/L, agar with the concentration of 6-8g/L, and indolebutyric acid with the concentration of 0.5-3.5 mg/L.
2. The method for inducing adventitious roots of ginseng according to claim 1, wherein the tissue of ginseng seedling is tissue of stem of ginseng seedling or tissue of leaf of ginseng seedling, and the sterilization process is as follows:
washing the stem tissue or leaf tissue of the ginseng seedling in tap water, cleaning in 70% ethanol solution for 30-60 s, soaking in 5-15% bleaching agent, shaking at 70-130rpm for 5-25min, and washing with sterilized distilled water under sterile environment for 4-5 times.
3. The method according to claim 2, wherein the tissue section has a length of 0.5 to 1cm, and the adventitious roots of ginseng are induced by inoculating the tissue section into the ginseng adventitious root induction medium and culturing the tissue section at 25 ± 2 ℃ in the dark for 3 to 6 weeks;
the ginseng adventitious root induction culture medium comprises an SH culture medium with the concentration of 12.15g/L, sucrose with the concentration of 30g/L, agar with the concentration of 7g/L and indolebutyric acid with the concentration of 2 mg/L.
4. The method of claim 3, wherein the pH of the ginseng adventitious root induction medium is in the range of 5.6 to 5.9.
5. The method for inducing adventitious roots of ginseng according to any one of claims 1 to 4, wherein the SH medium at a concentration of 6.075-24.3g/L is replaced with MS medium at a concentration of 2.2-6.6g/L or B5 medium at a concentration of 1.605-4.815 g/L;
the induction culture medium also comprises one or more of 2, 4-dichlorophenoxyacetic acid with the concentration of 0.1-3mg/L, 6-furfurylaminopurine with the concentration of 0.01-1mg/L, 1-naphthylacetic acid with the concentration of 1-5mg/L and 6-benzylaminopurine with the concentration of 0.001-1 mg/L.
6. The method of claim 5, further comprising subculturing the ginseng adventitious roots by the following steps:
cutting the ginseng adventitious root into tissue sections of 30-150mm multiplied by 30-150mm by using a sterilization knife, transferring the tissue sections into a subculture medium, performing solid proliferation culture every 3-6 week period, repeating the solid proliferation culture for 1-2 times, and then performing a liquid proliferation culture medium, wherein the solid proliferation culture comprises an SH culture medium with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-50g/L, agar with the concentration of 6-8g/L, and indolebutyric acid with the concentration of 0.5-3.5 mg/L; the liquid proliferation culture comprises SH culture medium with the concentration of 6.075-24.3g/L, sucrose with the concentration of 15-35g/L and indolebutyric acid with the concentration of 0.5-3.5 mg/L.
7. The method according to claim 6, wherein the ginseng adventitious roots are cultured in the liquid medium at a rotation speed of 70 to 100rpm at a temperature of 25 ± 2 ℃ for 3 to 5 weeks in the absence of light, in an amount of 0.5 to 10 g/L.
8. The method of claim 6 or 7, further comprising preparing an extract of ginseng adventitious roots from the ginseng adventitious roots after subculture, the method comprising:
washing the ginseng adventitious roots with distilled water twice, drying by adopting a drying or freeze-drying method, crushing, and sieving by using a 60-100-mesh sieve to obtain ginseng adventitious root tissue powder;
mixing the ginseng adventitious root tissue powder with 70% ethanol according to a material-liquid ratio of 1:10-100, stirring and extracting, and filtering to obtain a filtrate;
concentrating the filtered filtrate under reduced pressure until ethanol is removed to obtain the ginseng adventitious root extract.
9. The method for inducing adventitious roots of ginseng according to claim 8, wherein the powder of the tissue of the adventitious roots of ginseng is obtained after sieving preferably with a 80-mesh sieve;
the ginseng adventitious root tissue powder is mixed with 70% ethanol according to the material-liquid ratio of 1: 20.
10. Use of the ginseng adventitious roots extract according to any one of claims 8 and 9 in cosmetics.
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