CN102994443B - A kind of loquat cell suspending culture solution and suspension culture method - Google Patents
A kind of loquat cell suspending culture solution and suspension culture method Download PDFInfo
- Publication number
- CN102994443B CN102994443B CN201210457733.9A CN201210457733A CN102994443B CN 102994443 B CN102994443 B CN 102994443B CN 201210457733 A CN201210457733 A CN 201210457733A CN 102994443 B CN102994443 B CN 102994443B
- Authority
- CN
- China
- Prior art keywords
- loquat
- culture
- ursolic acid
- suspension culture
- acid content
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of loquat cell suspending culture solution and suspension culture method.Wherein, nutrient solution comprises: MS minimum medium, 2,4-D0.5-6.0mg/L; NAA0.5-6.0mg/L; IBA0.5-6.0mg/L; KT0-4.0mg/L; BA0-4.0mg/L; Sucrose 1-3.5%; PH4.0-6.5.Cultural method is that callus is cultivated in this nutrient solution.The present invention establishes loquat cellular liquid culture system, and adopt best mode of the present invention to cultivate, in loquat cell culture, the yield of ursolic acid is 36.49mg/gDW.
Description
Technical field
The present invention relates to biological technical field, relate to loquat cell suspension culture method particularly.
Background technology
Ursolic acid is most important secondary metabolites in Loquat Leaf, and be expected to become low toxicity, effective new type natural cancer therapy drug by tumour hospital of Chinese Medicine academy of sciences experiment proof, market is very in short supply.Current product is many carries out organic extraction from loquat blade, and cost is high.The visible CN200710008809.9 of extracting method, 200910154645.X, 201210057030.7 etc.
Mainly concentrate in prior art: to the callus culture of loquat and the foundation of Clonal regeneration system; To extraction and the pharmacological research thereof of Loquat Leaf active ingredient.Plant cell suspension culture approach production secondary metabolite is the important technology route during current Chinese medicine is produced, but at present in loquat research, there is no and set up suspension culture system to extract the report of the triterpene compounds such as ursolic acid.
Summary of the invention
The object of the present invention is to provide a kind of loquat cell suspension culture method, produce natural ursolic acid, to solve the above-mentioned problems in the prior art.
Technical scheme provided by the invention is as follows:
Technical scheme one:
For a nutrient solution for loquat cell suspension culture, comprising:
MS minimum medium
2,4-D(2,4-dichlorophenoxyacetic acid) 0.5-6.0mg/L;
NAA(a-naphthylacetic acid) 0.5-6.0mg/L;
IBA(indolebutyric acid) 0.5-6.0mg/L;
6-BA(6-benzyladenine) 0-4.0mg/L;
KT(kinetin, kinetin) 0-4.0mg/L;
Sugarcane 1-3.5%(weight ratio);
pH 4.0-6.5。
Nutrient solution scheme best in the present invention is: MS minimum medium+0.5mg/L NAA+3% sucrose+PH 6.0
Technical scheme two:
A kind of loquat cell suspension culture method, comprises the steps:
(1) acquisition loquat callus is cultivated;
(2) aforesaid loquat cell suspending culture solution is provided;
(3) callus carries out suspension culture in suspending nutrient solution;
(4) harvested cell, extracts ursolic acid.
In the preferred embodiment, the callus for suspension culture is faint yellow, that growth conditions is good, the induction of open-textured loquat rataria comes callus.Obtain callus and can adopt prior art.After subculture 5 times, proceed to suspension culture.
A kind of loquat cell suspension culture method, cell suspension culture based formulas is: MS minimum medium+0.5mg/L NAA+3% sucrose, and pH value is 6.0; Extraneous control condition is: illumination condition is 400Lux(12h/d), adopt the bottled liquid 100ml of triangle of 250ml, inoculum size is 40-60g/LFW, and shaking speed is 130r/min, adopts air-permeable envelope sealing, cultivates 18 days results.
Because traditional Loquat Leaf ursolic acid extracting method exists multiple limitation, the invention provides the nutrient solution for loquat cell suspension culture and cultural method.The present invention with loquat rataria for material, carry out the induction of excellent callus, excellent callus proceeds to liquid nutrient medium through repeatedly succeeding transfer culture, then the screening carrying out condition of suspension culture detects the content of target product ursolic acid simultaneously, thus establish loquat callus suspension culture system, produce for the industrialization of being extracted ursolic acid by loquat cell suspension culture and lay the foundation.Wherein, adopt best mode to cultivate, in loquat cell culture, the yield of ursolic acid is 36.49mg/gDW.About 160.46% is improve than the content (14.01mg/gDW) of the highest ursolic acid in Loquat Leaf (South Asia loquat) in current big vast swallow duckweed bibliographical information.
Accompanying drawing explanation
Fig. 1 is loquat cell suspension culture growth curve and ursolic acid content figure in the embodiment of the present invention.
Embodiment
Test materials: loquat young fruit, picks up from Putian City fruit tree research institute of Fujian Province.
The extracting method of loquat cell effective constituent ursolic acid is prior art ultrasonic extraction, is specially: solvent is 95% ethanol, solid-liquid ratio 1:30(g/ml), ultrasonic extraction, extraction temperature 40 DEG C, extraction frequency is 25KHZ, extraction power 260W, extraction time 1 time, ultrasonic time 40min.Measure by liquid chromatography (HPLC).
The acquisition of embodiment 1 loquat callus
Adopt prior art, obtain from the induction of loquat young fruit, inducing culture is MS minimum medium+1mg/L2,4-D+0.5mg/L KT+ sucrose 2%+6.8g/L agar powder, PH 5.8,25 DEG C, dark culturing, after same substratum and culture condition succeeding transfer culture 5 times, proceeds to suspension culture.
The foundation of loquat cell suspension culture system and cultural method
Material: choose faint yellow on solid medium, that growth conditions is good, quality is loosened, loquat rataria is induced and come embryo callus.With original solid culture based formulas and culture condition (in embodiment 1, inducing culture is MS minimum medium+1mg/L 2,4-D+0.5mg/L KT+ sucrose 2%, PH 5.8,25 DEG C, dark culturing, shaking speed 110r/min) more than continuous subculture 5 generation, obtain homogeneous suspension, to carry out follow-up study on regulation (embodiment 2-9).The nutrient solution formula adopted in study on regulation and culture condition are: MS minimum medium+1mg/L 2,4-D+0.5mg/L KT+ sucrose 2%, PH 5.8,25 DEG C, and dark culturing, shaking speed 110r/min, when investigating certain factor, fixing other factors is studied.
Paper examines index: suspension culture fresh weight specific growth rate, ursolic acid content.
Ursolic acid is with ultrasonic extraction, and content liquid chromatography (HPLC) measures
Embodiment 2 hormone is on suspended culture cell growth and the impact of ursolic acid content
Table 12,4D is on suspended culture cell growth and the impact of ursolic acid content
| Process number | 2,4-D（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0.5 | 162.11 | 11.68 |
| 2 | 1.0 | 147.58 | 8.37 |
| 3 | 2.0 | 148.75 | 6.84 |
| 4 | 4.0 | 111.08 | 5.34 |
| 5 | 6.0 | 92.40 | 7.83 |
The comparatively suitable loquat cell suspension culture of conclusion: 2,4-D 0.5
Table 2NAA is on suspended culture cell growth and the impact of ursolic acid content
| Process number | NAA（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0.5 | 257.61 | 36.49 |
| 2 | 1.0 | 272.80 | 30.52 |
| 3 | 2.0 | 286.21 | 28.00 |
| 4 | 4.0 | 280.96 | 29.02 |
| 5 | 6.0 | 271.04 | 28.63 |
Conclusion: NAA0.5 is suitable for loquat cell suspension culture
Table 3IBA is on suspended culture cell growth and the impact of ursolic acid content
| Process number | IBA（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0.5 | 29.06 | 10.53 |
| 2 | 1.0 | 24.28 | 10.84 |
| 3 | 2.0 | 44.26 | 10.39 |
| 4 | 4.0 | 56.85 | 9.75 |
| 5 | 6.0 | 59.13 | 10.56 |
Conclusion: each scope of growth hormone IBA is all not suitable for loquat cell suspension culture, in trial stretch, IBA6.0 is relatively suitable.
Table 4BA is on suspended culture cell growth and the impact of ursolic acid content
| Process number | BA（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0 | -4.59 | 22.10 |
| 2 | 0.25 | -5.73 | 22.14 |
| 3 | 0.5 | 3.32 | 20.93 |
| 4 | 1.0 | 13.06 | 24.75 |
| 5 | 2.0 | 25.32 | 23.21 |
| 6 | 4.0 | 51.47 | 24.88 |
Conclusion: BA2.0 can be used for loquat cell suspension culture
Table 5KT is on suspended culture cell growth and the impact of ursolic acid content
| Process number | KT（mg/L） | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0 | 28.70 | 17.00 |
| 2 | 0.25 | 28.47 | 21.03 |
| 3 | 0.5 | 44.21 | 20.50 |
| 4 | 1.0 | 51.55 | 21.05 |
| 5 | 2.0 | 71.16 | 22.95 |
| 6 | 4.0 | 82.96 | 24.98 |
Conclusion: KT4.0 is suitable for loquat cell suspension culture
Brief summary: the comprehensive specific growth rate of single-factor hormone regulating and controlling and ursolic acid content, is advisable with NAA0.5mg/L.
Embodiment 3PH value is on suspended culture cell growth and the impact of ursolic acid content
After selected minimum medium and optimum hormone proportioning, pH value different in observation liquid nutrient medium is on the impact of suspended culture cell growth and ursolic acid content.
Table 6pH value grows suspended culture cell and the impact of ursolic acid content
| Process number | PH | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 4 | 218.78 | 12.24 |
| 2 | 4.5 | 192.43 | 11.83 |
| 3 | 5 | 239.88 | 11.57 |
| 4 | 5.5 | 228.82 | 12.09 |
| 5 | 6 | 261.72 | 13.23 |
| 6 | 6.5 | 217.47 | 11.67 |
Conclusion: loquat cell suspension culture all can grow within the scope of PH4.0-6.5, but with PH6.0 for best pH value.Embodiment 4 illumination is on suspended culture cell growth and the impact of d-Bomeol content
Light on cells suspension culture may have larger impact, therefore studies the illumination condition of suspension culture system.
Choose 0,400,1000,2000 and 4000Lux light intensity to culture 12h/d irradiate (simulating nature), to filter out favourable optimum illumination condition.
Table 7 illumination condition is on suspended culture cell growth and the impact of ursolic acid content
| Process number | Intensity of illumination (lux) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 0 | 301.42 | 11.85 |
| 2 | 400 | 336.11 | 17.18 |
| 3 | 1000 | 265.28 | 12.14 |
| 4 | 2000 | 203.64 | 13.33 |
| 5 | 4000 | 180.31 | 14.03 |
Conclusion: 400lux illumination condition is comparatively suitable.
Embodiment 5 inoculum size is on suspended culture cell growth and the impact of ursolic acid content
To the clone of any suspension culture, there is the density that its growth is applicable to.In 250ml triangular flask, often bottled enter 100ml nutrient solution, observation different vaccination amount is on the impact of suspended culture cell growth and ursolic acid content.
Table 8 inoculum size is on suspended culture cell growth and the impact of ursolic acid content
| Process number | Inoculum size (g/100ml) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 2 | 108.13 | 8.38 |
| 2 | 4 | 243.25 | 11.85 |
| 3 | 6 | 242.67 | 15.62 |
| 4 | 8 | 239.00 | 16.90 |
| 5 | 10 | 182.80 | 15.91 |
| 6 | 12 | 147.92 | 16.94 |
Conclusion: inoculum size 8g/L optimum loquat cell suspension culture
Embodiment 6 shaking speed is on suspended culture cell growth and the impact of ursolic acid content
Culture is placed in respectively 50,70,90,110,130,150, the rotary shaker of 170r/min carries out suspension culture, observation shaking speed is on the impact of suspended culture cell growth and ursolic acid content.
Table 9 shaking speed is on suspended culture cell growth and the impact of ursolic acid content
The most applicable loquat cell suspension culture of conclusion: shaking speed 130rpm/min.
Embodiment 7 harvest time is on suspended culture cell growth and the impact of ursolic acid content
Respectively at the 18th day results after cultivation, observation harvest time is on the impact of suspension culture callus, Growth of Cells and ursolic acid content.The results are shown in Figure 1.
As seen from Figure 1, the growth curve of culture presents S type substantially, and 7-15d is growth logarithmic phase, and enter the growth platform phase after 15d, in growth cycle, the content of ursolic acid first declines and rises afterwards, and the content of ursolic acid is also accumulate in a large number after 15d.
Embodiment 8 liquid amount is on suspended culture cell growth and the impact of ursolic acid content
Material is cultivated with the culturing bottle of 250ml, and the ratio of new and old substratum is fixed as 1:1.
Table 10 liquid amount is on suspended culture cell growth and the impact of ursolic acid content
| Process number | Liquid amount (ml) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 40 | 255.42 | 11.21 |
| 2 | 70 | 287.39 | 9.22 |
| 3 | 100 | 308.99 | 8.26 |
| 4 | 130 | 227.90 | 9.62 |
| 5 | 160 | 78.84 | 9.13 |
The culturing bottle of conclusion: 250ml is good with the liquid amount of 100ml.
Embodiment 9 sucrose content is on suspended culture cell growth and the impact of ursolic acid content
Table 11 sucrose content is on suspended culture cell growth and the impact of ursolic acid content
| Process number | Sucrose content (%) | Fresh weight specific growth rate (%) | Ursolic acid content (mg/g) |
| 1 | 1.0 | 191.61 | 6.34 |
| 2 | 1.5 | 167.51 | 6.88 |
| 3 | 2.0 | 210.92 | 6.90 |
| 4 | 2.5 | 200.80 | 6.63 |
| 5 | 3.0 | 230.89 | 7.57 |
| 6 | 3.5 | 190.41 | 7.22 |
Conclusion: sucrose content is the most applicable with 3%.
Claims (1)
1. for extracting a loquat cell suspension culture method for ursolic acid, cell suspension culture based formulas is: MS minimum medium+0.5mg/L NAA+3% sucrose, and pH value is 6.0; Extraneous control condition is: illumination condition is 400Lux, 12h/d, and adopt the bottled liquid 100ml of triangle of 250ml, inoculum size is 40-60g/LFW, and shaking speed is 130r/min, adopts air-permeable envelope sealing, cultivates 18 days results.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210457733.9A CN102994443B (en) | 2012-11-14 | 2012-11-14 | A kind of loquat cell suspending culture solution and suspension culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210457733.9A CN102994443B (en) | 2012-11-14 | 2012-11-14 | A kind of loquat cell suspending culture solution and suspension culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102994443A CN102994443A (en) | 2013-03-27 |
| CN102994443B true CN102994443B (en) | 2015-08-26 |
Family
ID=47923556
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210457733.9A Active CN102994443B (en) | 2012-11-14 | 2012-11-14 | A kind of loquat cell suspending culture solution and suspension culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102994443B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105325300B (en) * | 2015-11-30 | 2017-10-20 | 华南农业大学 | The methods and applications of Eriobotrya distant hybrid progeny material are quickly bred by embryo culture |
| CN105816368B (en) * | 2016-03-29 | 2019-02-19 | 福建省亚热带植物研究所 | It is a kind of prepared from loquat cell natural beauty is black, antibacterial anti-inflammatory scalp massage cream |
| CN110710452A (en) * | 2019-11-05 | 2020-01-21 | 南京农业大学 | Tissue culture and rapid propagation method of eriobotrya japonica |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007004827A1 (en) * | 2005-06-30 | 2007-01-11 | Samyang Genex Corporation | The method for production of corosolic acid in suspension culture of plant cells |
| TW201114433A (en) * | 2009-10-28 | 2011-05-01 | Jen Li Biotech Co Ltd | Loquat leaf cell extracts useful for hepatoprotection and/or reducing fat and preparation methods and uses of the same |
| CN102487819A (en) * | 2011-11-22 | 2012-06-13 | 嘉兴浙华农业技术开发有限公司 | Cyclocarya paliurus callus and suspension culture method for callus particles |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100338998C (en) * | 2005-03-07 | 2007-09-26 | 四川农业大学 | Method for culturing embryoid of woody plant anther |
| CN102499080B (en) * | 2011-10-23 | 2013-07-03 | 成都大学 | Plant fast propagating method using fagopyrum tataricum leaf stalks as explants |
-
2012
- 2012-11-14 CN CN201210457733.9A patent/CN102994443B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007004827A1 (en) * | 2005-06-30 | 2007-01-11 | Samyang Genex Corporation | The method for production of corosolic acid in suspension culture of plant cells |
| TW201114433A (en) * | 2009-10-28 | 2011-05-01 | Jen Li Biotech Co Ltd | Loquat leaf cell extracts useful for hepatoprotection and/or reducing fat and preparation methods and uses of the same |
| CN102487819A (en) * | 2011-11-22 | 2012-06-13 | 嘉兴浙华农业技术开发有限公司 | Cyclocarya paliurus callus and suspension culture method for callus particles |
Non-Patent Citations (3)
| Title |
|---|
| Regulation and improvement of triterpene formation in plant cultured cells of Eriobotrya japonica Lindl;Hui-ya Ho et al.;《Journal of Bioscience and Bioengineering》;20100724;第110卷(第5期);588-592 * |
| 青钱柳组织培养及次生代谢物调控的初步研究;王纪;《中国优秀硕士学位论文全文数据库 农业科技辑》;20100215(第2期);D049-96，摘要 * |
| 青钱柳细胞悬浮培养及三萜化合物积累;尹忠平 等;《深圳大学学报理工版》;20110930;第28卷(第5期);430-435 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102994443A (en) | 2013-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wan et al. | Sequential heterotrophy–dilution–photoinduction cultivation of Haematococcus pluvialis for efficient production of astaxanthin | |
| CN102119655B (en) | Natural light rapid breeding method for dendrobium officinale | |
| EP3441457B1 (en) | Method for increasing content of mogroside v in siraitia grosvenorii suspended cells | |
| CN110301353A (en) | Method masson pine embryo callus subculture proliferation and maintain culture | |
| CN102174463A (en) | Method for culturing siraitia grosvenorii callus cell suspension system | |
| CN105441340A (en) | High-producing strain of GA (gibberellin) 4+7 and application of high-producing strain | |
| CN102994443B (en) | A kind of loquat cell suspending culture solution and suspension culture method | |
| CN105199957A (en) | Optimized culture method of Dunaliella salina | |
| CN103651139A (en) | Induced redifferentiation method for improving paclitaxel content in calluses of Taxus wallichiana var. mairei | |
| CN102994444B (en) | Pseudolarix amabilis cell suspension culture method | |
| CN102532337A (en) | Method for producing glycyrrhizia polysaccharide by utilizing tissue culture method | |
| CN109430054A (en) | A method of induction blueberry bud differentiation and result | |
| US20230287336A1 (en) | Method for improving flavonoid phenylpropanoid compounds in saussurea involucrata cell culture | |
| CN101475929B (en) | Method for producing oleanolic acid by white birch suspension culture | |
| CN105850729A (en) | Verbena bonariensis cultivation method | |
| CN108277197A (en) | A method of improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell | |
| WO2012065545A1 (en) | Microalgae culturing method for oil and lutein rapid accumulation | |
| Murthy et al. | Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L. | |
| CN105349592B (en) | The method of the complementary plant cell miscegenation culture production secondary metabolite of metabolism | |
| CN108034681A (en) | Utilize the method for the stem of Radix Codonopsis lanceolatae forming layer stem cell production Catalpol for the culture that suspends | |
| CN104429961B (en) | A kind of Fast-propagation tissue culture method of Herba Dendrobii | |
| Krishnan et al. | Establishment of cell suspension culture in Marchantia linearis Lehm & Lindenb. for the optimum production of flavonoids | |
| CN102499095A (en) | Method for direct induction of bulb culture medium by isolated culture of garlic (Allium sativum L.) rachis | |
| CN102640709B (en) | Cultural method of aralia elates seem high-yield saponin callus | |
| CN112154919A (en) | Culture medium and method for inducing calli of paris polyphylla to directly grow seedlings |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |