CN102505031B - Method for synthesizing arbutin by utilizing hairy roots of scutellaria baicalensis to convert hydroquinone - Google Patents
Method for synthesizing arbutin by utilizing hairy roots of scutellaria baicalensis to convert hydroquinone Download PDFInfo
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- CN102505031B CN102505031B CN201110300403.4A CN201110300403A CN102505031B CN 102505031 B CN102505031 B CN 102505031B CN 201110300403 A CN201110300403 A CN 201110300403A CN 102505031 B CN102505031 B CN 102505031B
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- flowered skullcap
- resorcinol
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- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 title claims abstract description 68
- 229960000271 arbutin Drugs 0.000 title claims abstract description 34
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 240000004534 Scutellaria baicalensis Species 0.000 title claims abstract description 22
- 235000017089 Scutellaria baicalensis Nutrition 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 22
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 title abstract 8
- 230000002194 synthesizing effect Effects 0.000 title abstract 2
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 claims description 78
- 241000207929 Scutellaria Species 0.000 claims description 53
- 239000006870 ms-medium Substances 0.000 claims description 19
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 13
- 229930006000 Sucrose Natural products 0.000 claims description 13
- 239000005720 sucrose Substances 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 10
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 239000011573 trace mineral Substances 0.000 claims description 10
- 235000013619 trace mineral Nutrition 0.000 claims description 10
- 230000009514 concussion Effects 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000004471 Glycine Substances 0.000 claims description 5
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 5
- 239000000413 hydrolysate Substances 0.000 claims description 5
- 229960000367 inositol Drugs 0.000 claims description 5
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 5
- 229960003512 nicotinic acid Drugs 0.000 claims description 5
- 235000001968 nicotinic acid Nutrition 0.000 claims description 5
- 239000011664 nicotinic acid Substances 0.000 claims description 5
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 5
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 5
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 5
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 5
- 229940088594 vitamin Drugs 0.000 claims description 5
- 235000013343 vitamin Nutrition 0.000 claims description 5
- 239000011782 vitamin Substances 0.000 claims description 5
- 229930003231 vitamin Natural products 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 239000006227 byproduct Substances 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 229960005190 phenylalanine Drugs 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000011169 microbiological contamination Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 244000139693 Arctostaphylos uva ursi Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 206010040829 Skin discolouration Diseases 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cosmetics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for synthesizing arbutin by utilizing hairy roots of scutellaria baicalensis to convert hydroquinone. In the method, the arbutin is synthesized by cultivating the hairy roots of scutellaria baicalensis and converting hydroquinone by cells of the hairy roots of scutellaria baicalensis. The content of the arbutin in the converted hairy roots of scutellaria baicalensis can reach 4 to 6 percent. The conversion rate of the hydroquinone can reach 50 to 60 percent. The method has the characteristics of low pollution, few by-products and the like. A novel technology and a novel approach are provided for the production of the arbutin.
Description
Technical field
The invention belongs to conversion technology field, be specifically related to a kind of method of utilizing the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol.
Background technology
Arbutin is a kind of composition that comes from Ericaceae manzanita uva ursi.Research shows that arbutin has restraint of tyrosinase activity, antibechic, diuresis, the biological activity such as antibacterial.Arbutin can be combined with tyrosine oxidase effectively, makes melanic synthetic minimizing, thereby reduces pigment deposition in skin, is to be usually used in the main raw material in skin-lightening cosmetic both at home and abroad at present, and domestic and international market demand is very huge.The production of arbutin is mainly by chemosynthesis at present, but chemosynthesis needs 4～6 steps to react, and synthetic cost is high, pollution is large, by product is many.
Bio-transformation refers to by culturing micro-organisms, vegetable cell, plant tissue, utilizes enzyme in its body to carry out structural modification to external source substrate and obtains a kind of technology of valuable product.This technology, compared with chemosynthesis, has the advantages such as nontoxic, pollution-free, less energy-consumption, high-level efficiency, highly selective.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol that cost is low, pollution-free and transformation efficiency is high.
In order to achieve the above object, the present invention has adopted following technical scheme:
Cultivate root of large-flowered skullcap hairly root, utilize the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol after cultivating.
The substratum of described cultivation root of large-flowered skullcap hairly root is modified MS medium, and this modified MS medium comprises macroelement, trace element, molysite and organic composition, and pH is 5.4～6.0;
The KNO that described macroelement comprises 950mg/L
3, 42.5mg/L KH
2pO
4, 185mg/L MgSO
47H
2the CaCl of O, 220mg/L
22H
2the lactoalbumin hydrolysate of O and 500mg/L;
The KI that described trace element comprises 0.83mg/L, the H of 6.2mg/L
3bO
3, 22.3mg/L MnSO
44H
2the ZnSO of O, 8.6mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CuSO of O, 0.025mg/L
45H
2the CoCl of O and 0.025mg/L
2;
The Na that described molysite comprises 18.65mg/L
2the FeSO of EDTA and 13.9mg/L
47H
2o;
The nicotinic acid of vitamin, 0.5mg/L pyridoxine hydrochloride and the 0.5mg/L of the inositol that described organic composition comprises 100mg/L, the glycine of 2mg/L, 0.1mg/L.
Above-mentioned modified MS medium by change existing MS substratum (1962 by Murashige, T. and Skoo-ge, F. bis-people's designs, therefore be called for short MS substratum.) composition, be more suitable for the growth of root of large-flowered skullcap hairly root.
The method of described cultivation root of large-flowered skullcap hairly root is:
1) root of large-flowered skullcap hairly root is inoculated in root of large-flowered skullcap hairly root seed culture medium to lucifuge concussion under 100～150r/min with 4-5g/L and cultivates 18～20 days to obtain root of large-flowered skullcap hairly root seed, 25～28 DEG C of the temperature of cultivating, root of large-flowered skullcap hairly root seed culture medium is the modified MS medium of adding 40-50g/L sucrose;
2) root of large-flowered skullcap hairly root seed being inoculated in substratum to lucifuge concussion under 100～150r/min with 4-5g/L cultivates 20～30 days, the temperature of cultivating is 25～28 DEG C, and substratum is the modified MS medium of adding 30-60g/L sucrose, glucose or its mixture and 0.4mmol/L phenylalanine.Add the effect of a certain amount of sucrose, glucose and 0.4mmol/L phenylalanine to be to promote root of large-flowered skullcap hairly root Fast Growth, improved the ability of the synthetic arbutin of hairy roots of scutellaria baicalensis, be conducive to save production cost, increase economic efficiency.
The time of described conversion is 30～40h, and temperature when conversion is 20～28 DEG C.
Described Resorcinol dissolves with the aqueous ethanolic solution of volume fraction 70% before adding, and dissolving ratio is that every 1-5mL aqueous ethanolic solution dissolves 20mg Resorcinol.The effect of aqueous ethanolic solution is to reduce the probability of root of large-flowered skullcap hairly root microbiological contamination in the process that adds Resorcinol, avoids microbiological contamination in hairy roots of scutellaria baicalensis Resorcinol process.
The add-on of described Resorcinol is to add 15～25mg in the nutrient solution of every 100mL root of large-flowered skullcap hairly root.In the time that the add-on of every 100mL nutrient solution exceedes 25mg, Resorcinol suppresses the growth of root of large-flowered skullcap hairly root.
Compared with existing arbutin production technology, the present invention has following useful technique effect:
1. compared with chemical synthesis, production technique of the present invention is easy, and the transformation efficiency of substrate reaches 50～60%, has the advantages such as low pollution, by product are few; 2. with compared with plant extraction process, arbutin is with short production cycle, and condition is controlled, is not subject to seasonal restrictions; 3. the present invention utilizes the glucosides transformation function of root of large-flowered skullcap hairly root, can be used as the bio-reactor of new product, for arbutin synthetic provides a kind of new way, also for development of new medicinal material provides new way and new approaches.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and described embodiment is explanation of the invention instead of restriction.
The invention discloses a kind of method of utilizing the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol, by preculture root of large-flowered skullcap hairly root, then join a certain amount of Resorcinol as substrate in root of large-flowered skullcap hairly root nutrient solution, utilize the Glycosylase in scutellaria baicalensis hairy shape root cells to transform Resorcinol generation arbutin.
Embodiment 1
1) root of large-flowered skullcap hairly root is inoculated in root of large-flowered skullcap hairly root seed culture medium with 4g/L, 28 DEG C, 120r/min, lucifuge is cultivated 18 days; Described root of large-flowered skullcap hairly root seed culture medium is the modified MS medium of adding 50g/L sucrose;
2) will be through step 1) cultivate the root of large-flowered skullcap hairly root of 18 days, be inoculated in substratum with 5g/L, 28 DEG C, 150r/min, lucifuge is cultivated 28 days; Described substratum is the modified MS medium of adding 50g/L sucrose.
3) Resorcinol is dissolved in the aqueous ethanolic solution of volume fraction 70%, dissolving ratio is that every 1mL aqueous ethanolic solution dissolves 20mg, under aseptic condition, join through step 2) cultivate after root of large-flowered skullcap hairly root nutrient solution in, utilize hairy roots of scutellaria baicalensis 40h, temperature when conversion is 25 DEG C, transforms and carries out under lucifuge condition; The add-on of described Resorcinol is 20mg/100mL nutrient solution;
Described modified MS medium adopts following formula:
Comprise macroelement, trace element, molysite and organic composition, pH is 5.4～6.0;
Described macroelement is KNO
3: 950mg/L, KH
2pO
4: 42.5mg/L, MgSO
47H
2o:185mg/L and CaCl
22H
2o 220mg/L; Lactoalbumin hydrolysate: 500mg/L;
Trace element is KI:0.83mg/L, H
3bO
3: 6.2mg/L, MnSO
44H
2o:22.3mg/L, ZnSO
47H
2o:8.6mg/L, Na
2moO
42H
2o:0.25mg/L, CuSO
45H
2o:0.025mg/L and CoCl
2: 0.025mg/L;
Molysite is Na
2eDTA:18.65mg/L and FeSO
47H
2o13.9mg/L;
Organic composition is: inositol: 100mg/L, glycine: 2mg/L, vitamin: 0.1mg/L, pyridoxine hydrochloride: 0.5mg/L and nicotinic acid: 0.5mg/L;
4) in HPLC mensuration hairly root, the content of arbutin is 4.42%, and the transformation efficiency of Resorcinol is 51.6%.
Embodiment 2
1) root of large-flowered skullcap hairly root is inoculated in root of large-flowered skullcap hairly root seed culture medium with 5g/L, 28 DEG C, 100r/min, lucifuge is cultivated 20 days; Described root of large-flowered skullcap hairly root seed culture medium is the MS substratum that adds the improvement of 40g/L sucrose;
2) by through step 1) the fresh and tender root of large-flowered skullcap hairly root seed cultivated 20 days is inoculated in substratum with 5g/L, and 28 DEG C, 100r/min, lucifuge is cultivated 20 days; Described substratum is the modified MS medium of adding 50g/L sucrose and 10g/L glucose and 0.4mmol/L phenylalanine; Described modified MS medium adopts as the formula of embodiment 1;
3) Resorcinol is dissolved in the aqueous ethanolic solution of volume fraction 70%, dissolving ratio is that every 2mL aqueous ethanolic solution dissolves 20mg, under aseptic condition, join through step 2) cultivate after root of large-flowered skullcap hairly root nutrient solution in, utilize hairy roots of scutellaria baicalensis 40h, temperature when conversion is 28 DEG C; Transform and carry out under lucifuge condition, the add-on of described Resorcinol is 20mg/100mL nutrient solution;
4) in HPLC mensuration hairly root, the content of arbutin is 4.71%, and the transformation efficiency of Resorcinol is 56.13%.Compared with embodiment 1, by hairly root cultivation stage substratum in add glucose and phenylalanine, improved the transformation efficiency of Resorcinol.
Embodiment 3
Cultivate root of large-flowered skullcap hairly root, utilize the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol after cultivating, the time of described conversion is 30h, temperature when conversion is 20 DEG C, transform and carry out under lucifuge condition, described Resorcinol dissolves with the aqueous ethanolic solution of volume fraction 70% before adding, and dissolving ratio is that every 1mL aqueous ethanolic solution dissolves 20mg Resorcinol, and the add-on of Resorcinol is to add 15mg in the nutrient solution of every 100mL root of large-flowered skullcap hairly root.
The method of described cultivation root of large-flowered skullcap hairly root is:
1) root of large-flowered skullcap hairly root is inoculated in root of large-flowered skullcap hairly root seed culture medium to lucifuge concussion under 150r/min with 5g/L and cultivates 19 days to obtain root of large-flowered skullcap hairly root seed, 25 DEG C of the temperature of cultivating, root of large-flowered skullcap hairly root seed culture medium is the modified MS medium of adding 40g/L sucrose;
2) root of large-flowered skullcap hairly root seed is inoculated in substratum to lucifuge concussion under 120r/min with 4g/L and cultivates 30 days, the temperature of cultivation is 25 DEG C, and substratum is the modified MS medium of adding 30g/L glucose and 0.4mmol/L phenylalanine.
Above-mentioned modified MS medium, comprises macroelement, trace element, molysite and organic composition, and pH is 5.4～6.0;
The KNO that described macroelement comprises 950mg/L
3, 42.5mg/L KH
2pO
4, 185mg/L MgSO
47H
2the CaCl of O, 220mg/L
22H
2the lactoalbumin hydrolysate of O and 500mg/L;
The KI that described trace element comprises 0.83mg/L, the H of 6.2mg/L
3bO
3, 22.3mg/L MnSO
44H
2the ZnSO of O, 8.6mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CuSO of O, 0.025mg/L
45H
2the CoCl of O and 0.025mg/L
2;
The Na that described molysite comprises 18.65mg/L
2the FeSO of EDTA and 13.9mg/L
47H
2o;
The nicotinic acid of vitamin, 0.5mg/L pyridoxine hydrochloride and the 0.5mg/L of the inositol that described organic composition comprises 100mg/L, the glycine of 2mg/L, 0.1mg/L.
The content that HPLC measures arbutin in hairly root can reach 4～6%, and the transformation efficiency of Resorcinol can reach 50～60%.
Embodiment 4
Cultivate root of large-flowered skullcap hairly root, utilize the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol after cultivating, the time of described conversion is 35h, temperature when conversion is 22 DEG C, transform and carry out under lucifuge condition, described Resorcinol dissolves with the aqueous ethanolic solution of volume fraction 70% before adding, and dissolving ratio is that every 5mL aqueous ethanolic solution dissolves 20mg Resorcinol, and the add-on of Resorcinol is to add 25mg in the nutrient solution of every 100mL root of large-flowered skullcap hairly root.
The method of described cultivation root of large-flowered skullcap hairly root is:
1) root of large-flowered skullcap hairly root is inoculated in root of large-flowered skullcap hairly root seed culture medium to lucifuge concussion under 130r/min with 4g/L and cultivates 19 days to obtain root of large-flowered skullcap hairly root seed, 26 DEG C of the temperature of cultivating, root of large-flowered skullcap hairly root seed culture medium is the modified MS medium of adding 50g/L sucrose;
2) root of large-flowered skullcap hairly root seed is inoculated in substratum to lucifuge concussion under 140r/min with 4g/L and cultivates 24 days, the temperature of cultivation is 27 DEG C, and substratum is the modified MS medium of adding 60g/L sucrose and 0.4mmol/L phenylalanine.
Above-mentioned modified MS medium, comprises macroelement, trace element, molysite and organic composition, and pH is 5.4～6.0;
The KNO that described macroelement comprises 950mg/L
3, 42.5mg/L KH
2pO
4, 185mg/L MgSO
47H
2the CaCl of O, 220mg/L
22H
2the lactoalbumin hydrolysate of O and 500mg/L;
The KI that described trace element comprises 0.83mg/L, the H of 6.2mg/L
3bO
3, 22.3mg/L MnSO
44H
2the ZnSO of O, 8.6mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CuSO of O, 0.025mg/L
45H
2the CoCl of O and 0.025mg/L
2;
The Na that described molysite comprises 18.65mg/L
2the FeSO of EDTA and 13.9mg/L
47H
2o;
The nicotinic acid of vitamin, 0.5mg/L pyridoxine hydrochloride and the 0.5mg/L of the inositol that described organic composition comprises 100mg/L, the glycine of 2mg/L, 0.1mg/L.
The content that HPLC measures arbutin in hairly root can reach 4～6%, and the transformation efficiency of Resorcinol can reach 50～60%.
HPLC measures the method for arbutin in root of large-flowered skullcap hairly root: the hairly root after results transform, and 80 DEG C of oven dry, 95% ethanol, with 1: 100 solid-liquid ratio, refluxing extraction 5h; HPLC method is measured the condition determination of arbutin content in hairly root: waters-2487, Symmetry-C
18(250mm × 4.6mm, 5 μ m), flow velocity 0.8mL/min, moving phase: V
methyl alcohol: V
water=15: 85, detect wavelength: 287nm.
The method of utilizing the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol of the present invention, the method is by cultivating root of large-flowered skullcap hairly root, utilizing the Glycosylase in scutellaria baicalensis hairy shape root cells, transforms Resorcinol, generates arbutin.In root of large-flowered skullcap hairly root after conversion, the content of arbutin can reach 4～6%, and the transformation efficiency of Resorcinol can reach 50～60%.The features such as the low pollution of tool of the present invention, by product lack, produce a kind of new technology, new way are provided for arbutin.
Claims (2)
1. a method of utilizing the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol, is characterized in that comprising the following steps: cultivate root of large-flowered skullcap hairly root, utilize the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol after cultivating;
The method of described cultivation root of large-flowered skullcap hairly root is:
1) root of large-flowered skullcap hairly root is inoculated in root of large-flowered skullcap hairly root seed culture medium to lucifuge concussion under 100～150r/min with 4-5g/L and cultivates 18～20 days to obtain root of large-flowered skullcap hairly root seed, 25～28 DEG C of the temperature of cultivating, root of large-flowered skullcap hairly root seed culture medium is the modified MS medium of adding 40-50g/L sucrose;
One of 2) root of large-flowered skullcap hairly root seed is inoculated in substratum to lucifuge concussion under 100～150r/min with 4-5g/L and cultivates 20～30 days, the temperature of cultivation is 25～28 DEG C, and described substratum is on the basis of modified MS medium, add (a)-(c):
(a) phenylalanine of 0.4mmol/L and 30-60g/L sucrose;
(b) phenylalanine of 0.4mmol/L and 30-60g/L glucose;
(c) mixture of the phenylalanine of 0.4mmol/L and 30-60g/L sucrose and glucose;
The time of described conversion is 30～40h, and temperature when conversion is 20～28 DEG C;
The add-on of described Resorcinol is to add 15～25mg in the nutrient solution of every 100mL root of large-flowered skullcap hairly root;
Described modified MS medium comprises macroelement, trace element, molysite and organic composition, and pH is 5.4～6.0;
Described macroelement is the KNO of 950mg/L
3, 42.5mg/L KH
2pO
4, 185mg/L MgSO
47H
2the CaCl of O, 220mg/L
22H
2the lactoalbumin hydrolysate of O and 500mg/L;
Described trace element is the KI of 0.83mg/L, the H of 6.2mg/L
3bO
3, 22.3mg/L MnSO
44H
2the ZnSO of O, 8.6mg/L
47H
2the Na of O, 0.25mg/L
2moO
42H
2the CuSO of O, 0.025mg/L
45H
2the CoCl of O and 0.025mg/L
2;
Described molysite is the Na of 18.65mg/L
2the FeSO of EDTA and 13.9mg/L
47H
2o;
Described organic composition is the nicotinic acid of vitamin, 0.5mg/L pyridoxine hydrochloride and the 0.5mg/L of glycine, the 0.1mg/L of inositol, the 2mg/L of 100mg/L.
2. a kind of method of utilizing the synthetic arbutin of hairy roots of scutellaria baicalensis Resorcinol according to claim 1, it is characterized in that: described Resorcinol dissolves with the aqueous ethanolic solution of volume fraction 70% before adding, dissolving ratio is that every 1-5mL aqueous ethanolic solution dissolves 20mg Resorcinol.
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