CN112075341B - Method for rapidly culturing gynostemma pentaphylla total saponins - Google Patents
Method for rapidly culturing gynostemma pentaphylla total saponins Download PDFInfo
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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
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01H4/001—Culture apparatus for tissue culture
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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Abstract
The invention provides a method for rapidly culturing gynostemma pentaphylla total saponins, which comprises the following steps: s1, taking gynostemma pentaphylla young stems, carrying out light-proof subculture on an MS semi-solid culture medium, and selecting callus which is continuously subcultured for 4 times and grows vigorously and has loose texture and coral-shaped cell mass; s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake culture, adding citric acid and a yeast extract when the culture is carried out for 4-5 days, adding malic acid and methyl jasmonate when the culture is carried out for 12-15 days, and harvesting gynostemma pentaphylla suspension cells on the 20 th-26 th day. The invention can rapidly culture the gynostemma pentaphylla total saponin by a cell suspension culture method, and can further improve the content of the gynostemma pentaphylla total saponin.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for rapidly culturing gynostemma pentaphylla total saponins.
Background
The gynostemma pentaphylla, also called a Gynostemma pentaphylla, is a plant of the genus gynostemma of the family Cucurbitaceae, 13 species are all about 13 species worldwide, 11 species are 2 varieties in China, 7 species are special in China, and the gynostemma pentaphylla is mainly distributed in southeast Asia and regions of the south of Yangtze river of China. Since the 70 s of the 20 th century, a wide range of studies have been initiated around the world on gynostemma pentaphylla, the major active ingredients of which are total saponins, flavonoids and saccharides.
83 kinds of monomer saponins with ginsenoside basic structure have been separated from gynostemma pentaphylla, which are called as "gynostemma pentaphylla total saponins", six kinds of them are identical with Rb1, rb3, rd, F2, rg3 and ginsenoside K, and the other 77 kinds have ginsenoside basic structure. The gynostemma pentaphylla total saponin has bitter taste, cold property and heat and toxic materials clearing away. The main effects are as follows: reducing blood lipid, lowering cholesterol, lowering blood pressure, enhancing coronary artery and cerebral blood flow, resisting DNA variation, and enhancing immunity, and can be used for preventing and treating arteriosclerosis, obesity, cancer and chronic hepatitis. In recent years, japanese scientist researches find that the content of 4 saponins in gynostemma pentaphylla exceeds that of ginseng, has obvious effects of tonifying qi and reducing blood sugar, and has obvious effects on the aspects of sugar metabolism, arteriosclerosis prevention, heart protection, organism immunity enhancement and the like. Gynostemma pentaphylla has been fully verified for preventing and treating hyperlipidemia, hypertension, cardiovascular and cerebrovascular diseases, diabetic complications and the like. The gynostemma pentaphylla has sweet taste because of containing a plurality of saponins which are the same as those of ginseng. The gynostemma pentaphylla is a multifunctional health care medicine with the function of ginseng and without side effect, and is called as a 'crown of four health care products in the world'.
The production of secondary metabolites by plant cell culture techniques is one of the effective means for increasing the content of target components in natural plants. The cell suspension culture refers to a tissue culture system for culturing single cells and small cell clusters in a liquid culture medium which is continuously stirred or rocked, is a culture mode of non-adherence-dependent cells, and has the advantages of high propagation speed, large culture scale, uniform and consistent plant cell culture, stable quality and the like. The total saponins of gynostemma pentaphylla can be purposefully increased by cultivating and producing the total saponins of gynostemma pentaphylla by a plant cell cultivating technology, and meanwhile, the cultivating period is shortened, but in order to further finish the market popularization of the total saponins of gynostemma pentaphylla, the total saponins of gynostemma pentaphylla are further increased.
Disclosure of Invention
The invention provides a method for rapidly culturing gynostemma pentaphylla total saponins, which can rapidly culture gynostemma pentaphylla total saponins by a cell suspension culture method and further improve the content of gynostemma pentaphylla total saponins.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: 35-40 g/L of sucrose, 4.1-4.5 g/L of agar, 0.8-0.9 mg/L of naphthylacetic acid, 60-70 mg/L of inositol, 0.9-1.1. 1.1mg/L, TDZ 0.05.05-0.1 mg/L of 6-BA, pH range of 5.5-5.7, and culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coralline cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake culture, wherein the MS liquid culture medium comprises the following components: sucrose 40-45 g/L, naphthylacetic acid 0.8-0.9 mg/L, inositol 60-70 mg/L, 6-BA 0.9-1.1 mg/L, TDZ 0.05-0.1 mg/L, pH range of 5.5-5.7, and maintaining culture temperature at 22-24 ℃, wherein dark culture and light culture are alternately performed in one day, dark culture is performed for 6-10 h, light culture is performed in the rest time, and light intensity is 2000-10001 ux; adding 1-3 mg/L of citric acid and 30-50 mg/L of yeast extract when culturing for 4-5 d, adding 1-3 mg/L of malic acid and 0.2-0.6 mL/L of methyl jasmonate when culturing for 12-15 d, and harvesting gynostemma pentaphylla suspension cells on 20-26 days.
Preferably, in the step S1, the MS semi-solid medium consists of the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, and TDZ0.05mg/L.
Preferably, in the step S2, the MS liquid medium is composed of the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L and TDZ0.09mg/L.
Preferably, in the step S2, the inoculation amount is 20-25 mg/L.
Preferably, in the step S2, 2mg/L of citric acid and 45mg/L of yeast extract are added when culturing for 5d, and 2mg/L of malic acid and 0.3mL/L of methyl jasmonate are added when culturing for 13 d.
The invention has the following advantages:
(1) The invention adds TDZ into the semisolid culture medium of callus culture and the culture medium of gynostemma pentaphylla suspension cell culture, which can effectively improve the content of gynostemma pentaphylla total saponin in the callus and gynostemma pentaphylla suspension cell.
(2) The invention is also beneficial to improving the content of the total saponins of the gynostemma pentaphylla by adopting the combination of illumination culture and dark culture when culturing the gynostemma pentaphylla suspension cells.
(3) The invention also adds citric acid and yeast extract to induce the first total saponin, and also adds malic acid and methyl jasmonate to induce the second total saponin, so that the content of the gynostemma pentaphylla total saponin can reach 8.5wt% of the dry cell weight, which is beneficial to marketing popularization of gynostemma pentaphylla total saponin.
(4) The method of the invention is not affected by seasons, illumination and production places, and the synthetic period is smaller than the planting period of the gynostemma pentaphylla plant, thus providing a new idea for standardized cultivation of gynostemma pentaphylla saponin.
Drawings
FIG. 1 is a standard graph of ginsenoside.
Detailed Description
The present invention is further illustrated below with reference to specific examples, but the scope of the present invention is not limited to the following examples.
Example 1
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, TDZ0.05mg/L, pH range of 5.5-5.7, culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L of TDZ0.09mg/L, pH range of 5.5-5.7, and cultivation temperature of 22-24 ℃, wherein dark cultivation and light cultivation are alternately carried out in one day, dark cultivation is carried out for 8 hours, and light cultivation is carried out in the rest time, and the light intensity is 10001ux; adding 2mg/L of citric acid and 45mg/L of yeast extract when culturing for 5d, adding 2mg/L of malic acid and 0.3mL/L of methyl jasmonate when culturing for 13d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the total saponin content of gynostemma pentaphylla.
Example 2
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: 35. 35 g/L of sucrose, 4.5g/L of agar, 0.8mg/L of naphthylacetic acid, 60mg/L of inositol, 0.9mg/L of 6-BA, 0.05mg/L of TDZ, and a pH range of 5.5-5.7, wherein the culture temperature is 22-24 ℃, and calli which are continuously subcultured for 4 times and are vigorous in growth, loose in texture and coral-shaped cell clusters are selected;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 20mg/L, and the MS liquid culture medium consists of the following components: 45g/L of sucrose, 0.8mg/L of naphthylacetic acid, 60mg/L of inositol, 6-BA0.9mg/L of TDZ0.06mg/L, pH range of 5.5-5.7, and cultivation temperature of 22-24 ℃, wherein dark cultivation and light cultivation are alternately performed in one day, dark cultivation is performed for 7 hours, and light cultivation is performed in other times, wherein light intensity is 8001ux; adding 1mg/L of citric acid and 50mg/L of yeast extract when culturing for 4d, adding 1mg/L of malic acid and 0.6mL/L of methyl jasmonate when culturing for 12d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the content of gynostemma pentaphylla total saponins.
Example 3
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 40g/L, agar 4.1g/L, naphthylacetic acid 0.9mg/L, inositol 70mg/L, 6-BA1.0mg/L, TDZ0.09mg/L, pH range 5.5-5.7, culturing at 22-24 deg.C, selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: sucrose 40g/L, naphthylacetic acid 0.9mg/L, inositol 70mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range of 5.5-5.7, and culture temperature of 22-24deg.C, dark culture and light culture alternate in one day, dark culture is carried out for 7h, and light culture is carried out for the rest time, and light intensity is 10001ux; adding 3mg/L of citric acid and 42mg/L of yeast extract when culturing for 5d, adding 3mg/L of malic acid and 0.6mL/L of methyl jasmonate when culturing for 12d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the total saponin content of gynostemma pentaphylla.
Example 4
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 40g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 62mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range 5.5-5.7, culturing at 22-24 deg.C, selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: sucrose 40g/L, naphthylacetic acid 0.8mg/L, inositol 62mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range of 5.5-5.7, and culture temperature of 22-24deg.C, dark culture and light culture alternate in one day, dark culture is carried out for 7h, light culture is carried out in other times, and light intensity is 9001ux; adding 2mg/L of citric acid and 45mg/L of yeast extract when culturing for 5d, adding 2mg/L of malic acid and 0.3mL/L of methyl jasmonate when culturing for 12d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the total saponin content of gynostemma pentaphylla.
Comparative example 1
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, pH range of 5.5-5.7, culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coralloid cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L, pH of 5.5-5.7, maintaining the culture temperature at 22-24 ℃, alternately performing dark culture and light culture in one day, performing dark culture for 8 hours, performing light culture in the rest time, and controlling the light intensity to 10001ux; adding 2mg/L of citric acid and 45mg/L of yeast extract when culturing for 5d, adding 2mg/L of malic acid and 0.3mL/L of methyl jasmonate when culturing for 13d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the total saponin content of gynostemma pentaphylla.
Comparative example 2
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, TDZ0.05mg/L, pH range of 5.5-5.7, culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L of TDZ0.09mg/L, pH range of 5.5-5.7, culture temperature of 22-24 ℃ and light irradiation culture, and light intensity of 10001ux; adding 2mg/L of citric acid and 45mg/L of yeast extract when culturing for 5d, adding 2mg/L of malic acid and 0.3mL/L of methyl jasmonate when culturing for 13d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the total saponin content of gynostemma pentaphylla.
Comparative example 3
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 40g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 62mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range 5.5-5.7, culturing at 22-24 deg.C, selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: sucrose 40g/L, naphthylacetic acid 0.8mg/L, inositol 62mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range of 5.5-5.7, and culture temperature of 22-24deg.C, dark culture and light culture alternate in one day, dark culture is carried out for 7h, and light culture is carried out for the rest time, and light intensity is 10001ux; adding 45mg/L of yeast extract when culturing for 4d, adding 0.3mL/L of methyl jasmonate when culturing for 12d, extracting the gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the content of gynostemma pentaphylla total saponins.
Comparative example 4
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, TDZ0.05mg/L, pH range of 5.5-5.7, culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L of TDZ0.09mg/L, pH range of 5.5-5.7, and cultivation temperature of 22-24 ℃, wherein dark cultivation and light cultivation are alternately carried out in one day, dark cultivation is carried out for 8 hours, and light cultivation is carried out in the rest time, and the light intensity is 10001ux; adding 45mg/L of yeast extract when culturing for 5d, adding 2mg/L of malic acid and 0.3mL/L of methyl jasmonate when culturing for 13d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the content of gynostemma pentaphylla total saponins.
Comparative example 5
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, TDZ0.05mg/L, pH range of 5.5-5.7, culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L of TDZ0.09mg/L, pH range of 5.5-5.7, and cultivation temperature of 22-24 ℃, wherein dark cultivation and light cultivation are alternately carried out in one day, dark cultivation is carried out for 8 hours, and light cultivation is carried out in the rest time, and the light intensity is 10001ux; adding 2mg/L of citric acid and 45mg/L of yeast extract when culturing for 5d, adding 0.3mL/L of methyl jasmonate when culturing for 13d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the content of gynostemma pentaphylla total saponins.
Comparative example 6
A method for rapidly culturing gynostemma pentaphylla total saponin comprises the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium comprises the following components: sucrose 40g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 62mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range 5.5-5.7, culturing at 22-24 deg.C, selecting callus which is continuously subcultured for 4 times and has vigorous growth, loose texture and coral cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake cultivation, wherein the inoculum size is 25mg/L, and the MS liquid culture medium consists of the following components: sucrose 40g/L, naphthylacetic acid 0.8mg/L, inositol 62mg/L, 6-BA1.1mg/L, TDZ0.1mg/L, pH range of 5.5-5.7, and culture temperature of 22-24deg.C, dark culture and light culture alternate in one day, dark culture is carried out for 7h, and light culture is carried out for the rest time, and light intensity is 10001ux; adding 45mg/L of yeast extract and 0.3mL/L of methyl jasmonate when culturing for 4d, adding 45mg/L of yeast extract and 0.3mL/L of methyl jasmonate when culturing for 12d, extracting gynostemma pentaphylla suspension cells every other day after culturing for 6-30 days, and measuring the content of gynostemma pentaphylla total saponins.
Comparing the total saponin content of gynostemma pentaphylla:
1. test method
1.1 preparation of Standard solution
Precisely weighing 10mg of ginsenoside Re standard, placing into a 10ml volumetric flask, dissolving with methanol, diluting to scale, and shaking to obtain ginsenoside Re standard solution (containing ginsenoside Re 1mg per ml).
1.2 drawing of Standard Curve
1.3 preparation of sample solutions
Cell preparation, namely taking extracted gynostemma pentaphylla suspension cells, freeze-drying, grinding into powder, sieving with a 40-mesh sieve, and drying at 55 ℃ to constant weight.
Extracting total saponins by weighing 3g of stem cells, adding 100ml of methanol, reflux-extracting with Soxhlet extractor for 6h, recovering methanol extract, evaporating to dryness in water bath, adding 30ml of water to dissolve residues, extracting with water saturated n-butanol solution for 3 times, sequentially mixing n-butanol solutions with volumes of 30ml, 30ml and 20m1, and evaporating to dryness under reduced pressure with a rotary evaporator.
The preparation of total saponin sample solution comprises dissolving the residue with small amount of methanol, transferring to 50ml volumetric flask, diluting with methanol to scale, and shaking to obtain total saponin sample solution.
1.4 determination of total saponins content of sample
And (3) precisely weighing 50ul of the sample solution, placing the sample solution in a test tube, operating according to a drawing method of a standard curve, measuring the absorbance, and calculating the total saponin content of the gynostemma pentaphylla according to the standard curve. The total saponin content of gynostemma pentaphylla is expressed as a percentage of the weight of stem cells.
2. Test results
TABLE 1 Total saponins content in Stem cells of examples 1-4
TABLE 2 Total saponins content in Stem cells of comparative examples 1-6
3. Conclusion(s)
The results of examples 1-4 and comparative examples 1-5 show that the addition of TDZ to the semisolid culture medium for callus culture and the culture medium for gynostemma pentaphylla suspension cell culture can effectively improve the content of gynostemma pentaphylla total saponins in the callus and gynostemma pentaphylla suspension cells.
When the gynostemma pentaphylla suspension cells are cultured, the combination of light culture and dark culture is adopted, so that the content of the gynostemma pentaphylla total saponins is improved.
In addition, citric acid and yeast extract are added to induce the first total saponins, and malic acid and methyl jasmonate are also added to induce the second total saponins, so that the content of the gynostemma pentaphylla total saponins can reach 8.5wt% of the dry cell weight, and the marketing popularization of the gynostemma pentaphylla total saponins is facilitated.
The method of the invention is not affected by seasons, illumination and production places, and the synthetic period is smaller than the planting period of the gynostemma pentaphylla plant, thus providing a new idea for standardized cultivation of gynostemma pentaphylla saponin.
Claims (5)
1. A method for rapidly culturing gynostemma pentaphylla total saponins is characterized by comprising the following steps:
s1, taking gynostemma pentaphylla young stems, carrying out light-shielding subculture on an MS semi-solid culture medium, wherein the MS semi-solid culture medium also comprises the following components: 35-40 g/L of sucrose, 4.1-4.5 g/L of agar, 0.8-0.9 mg/L of naphthylacetic acid, 60-70 mg/L of inositol, 0.9-1.1 mg/L, TDZ 0.05.05-0.1 mg/L of 6-BA, pH range of 5.5-5.7, and culture temperature of 22-24 ℃, and selecting callus which is continuously subcultured for 4 times and grows vigorously, has loose texture and coralline cell mass;
s2, inoculating the callus obtained in the step S1 into an MS liquid culture medium for shake culture, wherein the MS liquid culture medium also contains the following components: sucrose 40-45 g/L, naphthylacetic acid 0.8-0.9 mg/L, inositol 60-70 mg/L, 6-BA 0.9-1.1 mg/L, TDZ 0.05-0.1 mg/L, pH range of 5.5-5.7, and maintaining culture temperature at 22-24 ℃, wherein dark culture and light culture are alternately performed in one day, dark culture is performed for 6-10 h, light culture is performed in the rest time, and light intensity is 1000-20001 ux; adding 1-3 mg/L of citric acid and 30-50 mg/L of yeast extract when culturing for 4-5 d, adding 1-3 mg/L of malic acid and 0.2-0.6 mL/L of methyl jasmonate when culturing for 12-15 d, and harvesting gynostemma pentaphylla suspension cells on 20-26 days.
2. The method for rapid cultivation of gynostemma pentaphylla total saponin according to claim 1, wherein:
in the step S1, the MS semi-solid medium further contains the following components: sucrose 38 g/L, agar 4.2g/L, naphthylacetic acid 0.8mg/L, inositol 65mg/L, 6-BA1.0mg/L, and TDZ0.05mg/L.
3. The method for rapid cultivation of gynostemma pentaphylla total saponin according to claim 1, wherein:
in the step S2, the MS liquid medium further contains the following components: 42g/L of sucrose, 0.9mg/L of naphthylacetic acid, 68mg/L of inositol, 6-BA1.0mg/L and TDZ0.09mg/L.
4. The method for rapid cultivation of gynostemma pentaphylla total saponin according to claim 1, wherein:
in the step S2, the inoculation amount is 20-25 mg/L.
5. The method for rapid cultivation of gynostemma pentaphylla total saponin according to claim 1, wherein:
in the step S2, 2mg/L of citric acid and 45mg/L of yeast extract are added when the culture is carried out for 5 days, and 2mg/L of malic acid and 0.3mL/L of methyl jasmonate are added when the culture is carried out for 13 days.
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