JPH0347071A - Method for obtaining highly glycyrrhizin-producing strain of glycyrrhiza glabra l. var root stem cell - Google Patents
Method for obtaining highly glycyrrhizin-producing strain of glycyrrhiza glabra l. var root stem cellInfo
- Publication number
- JPH0347071A JPH0347071A JP1182180A JP18218089A JPH0347071A JP H0347071 A JPH0347071 A JP H0347071A JP 1182180 A JP1182180 A JP 1182180A JP 18218089 A JP18218089 A JP 18218089A JP H0347071 A JPH0347071 A JP H0347071A
- Authority
- JP
- Japan
- Prior art keywords
- medium
- glycyrrhizin
- cells
- concentration
- licorice
- 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.)
- Pending
Links
- 239000004378 Glycyrrhizin Substances 0.000 title claims abstract description 30
- 229960004949 glycyrrhizic acid Drugs 0.000 title claims abstract description 30
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 title claims abstract description 28
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 235000019410 glycyrrhizin Nutrition 0.000 title claims abstract description 28
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 title claims abstract description 28
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 11
- 244000303040 Glycyrrhiza glabra Species 0.000 title abstract description 17
- 210000000130 stem cell Anatomy 0.000 title abstract 3
- 239000002609 medium Substances 0.000 claims abstract description 42
- 239000000122 growth hormone Substances 0.000 claims abstract description 18
- 230000008635 plant growth Effects 0.000 claims abstract description 18
- 102000018997 Growth Hormone Human genes 0.000 claims abstract description 15
- 108010051696 Growth Hormone Proteins 0.000 claims abstract description 15
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000004161 plant tissue culture Methods 0.000 claims abstract description 3
- 239000003104 tissue culture media Substances 0.000 claims abstract description 3
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 claims description 22
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 claims description 21
- 229940010454 licorice Drugs 0.000 claims description 21
- 241000202807 Glycyrrhiza Species 0.000 claims description 11
- 230000003247 decreasing effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 17
- 239000005556 hormone Substances 0.000 abstract description 16
- 229940088597 hormone Drugs 0.000 abstract description 16
- 239000000126 substance Substances 0.000 abstract description 13
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 229930192334 Auxin Natural products 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 5
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 abstract description 5
- 239000002363 auxin Substances 0.000 abstract description 5
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 abstract description 5
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004062 cytokinin Substances 0.000 abstract description 4
- 239000006870 ms-medium Substances 0.000 abstract description 4
- 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 abstract description 3
- 229930006000 Sucrose Natural products 0.000 abstract description 3
- 229910017053 inorganic salt Inorganic materials 0.000 abstract description 3
- 235000015097 nutrients Nutrition 0.000 abstract description 3
- 239000005720 sucrose Substances 0.000 abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 2
- -1 benzyladenine Natural products 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 abstract description 2
- 230000002503 metabolic effect Effects 0.000 abstract description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 abstract 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 abstract 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 abstract 1
- 239000013630 prepared media Substances 0.000 abstract 1
- 235000013343 vitamin Nutrition 0.000 abstract 1
- 239000011782 vitamin Substances 0.000 abstract 1
- 229940088594 vitamin Drugs 0.000 abstract 1
- 229930003231 vitamin Natural products 0.000 abstract 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 27
- 239000007787 solid Substances 0.000 description 13
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000002513 implantation Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 4
- 229960001669 kinetin Drugs 0.000 description 4
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 4
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 4
- 229940023877 zeatin Drugs 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 244000000626 Daucus carota Species 0.000 description 2
- 235000002767 Daucus carota Nutrition 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 239000006286 aqueous extract Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 238000010187 selection method Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- KAWVXVCNEQRALX-DNVSUFBTSA-N (+)-Echinatine Natural products O=C(OCC=1[C@@H]2[C@@H](O)CC[N+]2CC=1)[C@@](O)([C@@H](O)C)C(C)C KAWVXVCNEQRALX-DNVSUFBTSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- KBLDMOWERKLSAQ-UHFFFAOYSA-N 5-benzylpurin-6-amine Chemical compound NC1=NC=NC2=NC=NC12CC1=CC=CC=C1 KBLDMOWERKLSAQ-UHFFFAOYSA-N 0.000 description 1
- 244000015329 Aeginetia indica Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920002148 Gellan gum Polymers 0.000 description 1
- 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 description 1
- 240000004670 Glycyrrhiza echinata Species 0.000 description 1
- 235000000554 Glycyrrhiza uralensis Nutrition 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 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 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- NMPPLGMVCCNRTJ-UHFFFAOYSA-N echinatine Natural products C1CC(O)C2C(COC(=O)C(O)(C(C)O)C(C)C)CCN21 NMPPLGMVCCNRTJ-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- LTINPJMVDKPJJI-UHFFFAOYSA-N iodinated glycerol Chemical compound CC(I)C1OCC(CO)O1 LTINPJMVDKPJJI-UHFFFAOYSA-N 0.000 description 1
- SFVVQRJOGUKCEG-UHFFFAOYSA-N isoechinatine Natural products C1CC(O)C2C(COC(=O)C(O)(C(C)O)C(C)C)=CCN21 SFVVQRJOGUKCEG-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- SFVVQRJOGUKCEG-OPQSFPLASA-N β-MSH Chemical compound C1C[C@@H](O)[C@H]2C(COC(=O)[C@@](O)([C@@H](C)O)C(C)C)=CCN21 SFVVQRJOGUKCEG-OPQSFPLASA-N 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はカンゾウ根茎細胞の選抜方法に係り、特にグリ
チルリチン高生産株の大量取得に好適な選抜方法に関す
る。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a selection method for licorice rhizome cells, and particularly to a selection method suitable for obtaining a large amount of glycyrrhizin-producing strains.
植物の細胞培養（カルス培養を含む）において幼苗の大
量生産や植物の有用な二次代謝物の生産を目的とした、
培地組成、培養環境の最適化に関する研究がなされてい
る０例えば、ミシマサイコの葉および茎のカルスを、オ
ーキシン濃度を変化させることにより再分化し、天然と
同一のサイコサポニンを生産する方法（生薬学雑誌：２
８く２＞、１５２〜１６０）、また、薬用にんじんの組
織培養物を糖類のバランスを調整し、硝酸アンモニウム
を除去したＭＳ培地で培養することにより、天然と同一
のサポニンを多量に含有するにんじんを生産する方法が
報告されている。（特公昭 第６３−２１４７０号公報
）
これらの場合、有効物質を高効率で生産する細胞群の選
別は、カルスやプロトプラストの視覚的選抜法や、細胞
の二次代謝物の分析によって行われていた。しかし、こ
れらの方法は非常に手間がかかる上、−度に大量のサン
プルを処理することはできない。For the purpose of mass production of seedlings in plant cell culture (including callus culture) and production of useful secondary metabolites of plants,
Research has been conducted on optimizing the medium composition and culture environment.For example, a method of redifferentiating callus from leaves and stems of A. japonica by changing the auxin concentration to produce the same saucosaponin as naturally occurring (Herbal Pharmacology) Magazine: 2
8, 2>, 152-160), and by culturing medicinal carrot tissue culture in MS medium in which the balance of sugars has been adjusted and ammonium nitrate has been removed, carrots containing a large amount of the same natural saponin can be produced. A method for producing it has been reported. (Special Publication No. 63-21470) In these cases, cell groups that produce effective substances with high efficiency are selected by visual selection of callus and protoplasts or by analysis of secondary metabolites of cells. Ta. However, these methods are very time-consuming and cannot process a large number of samples at once.
一方、カンデラは根茎に含有するグリチルリチンが有効
物質として広く使用されるが、国内生産はほとんどなさ
れていない。カンデラの細胞培養は、シナカンゾウ（Ｇ
ｌｙｃｙｒｒｈｉｚａ ｅｃｈｉｎａｔａ）の細胞をア
ルギン酸カルシウムビーズに固定化することにより、エ
キナチンを細胞レベルで長期間生産させる方法（発酵と
工業： Ｖｏｆｆｉ４５ Ｎα１２）が報告されて
いる。しかし、グリチルリチンを含有するスペインカン
ゾウ（Ｇｌｙｃｙｒｒｈｉｚａ ｇｌａｂｒａ）やウラ
ルカンゾウ（Ｇｌｙｃｙｒｒｈｉｚａ ｕｒａｌｅｎｓ
ｉｓ）の細胞培養、またグリチルリチンを高効率で作る
細胞の培養法は見られない。On the other hand, glycyrrhizin contained in the rhizome of candela is widely used as an active substance, but it is hardly produced domestically. Candela cell culture is based on Chinese licorice (G.
A method for producing echinatine at the cellular level for a long period of time (fermentation and industry: Voffi45 Nα12) has been reported by immobilizing cells of lycyrrhiza echinata on calcium alginate beads. However, Spanish licorice (Glycyrrhiza glabra) and Ural licorice (Glycyrrhiza uralens), which contain glycyrrhizin,
is), and no method for culturing cells that produces glycyrrhizin with high efficiency has been found.
上記のように、従来グリチルリチンを含むカンゾウ根茎
細胞の培養や、グリチルリチン高生産株取得については
明らかにされていない。このため培養細胞のグリチルリ
チン生産能を明らかにし、グリチルリチン生産細胞を効
率的に選抜できる方法が要望されていた。As mentioned above, the culture of licorice rhizome cells containing glycyrrhizin and the acquisition of strains with high glycyrrhizin production have not been clarified so far. Therefore, there has been a need for a method that can clarify the glycyrrhizin-producing ability of cultured cells and efficiently select glycyrrhizin-producing cells.
本発明の目的は、カンゾウ根茎細胞に効率よくグリチル
リチンを生産させ、グリチルリチン高生産株を取得する
方法を提供することにある。An object of the present invention is to provide a method for efficiently producing glycyrrhizin in licorice rhizome cells and obtaining a highly glycyrrhizin-producing strain.
上記した目的は、カンゾウ根茎細胞を植物組織培養培地
における植物成長ホルモンの濃度が変化する培地に移植
し、化学的ストレスを与えることによって達成される。The above objectives are achieved by transplanting licorice rhizome cells into a plant tissue culture medium with varying concentrations of plant growth hormone and by applying chemical stress.
カンゾウ細胞は、ＨＮＯ，、ＮＨ４ＮＯ３。Licorice cells are HNO, NH4NO3.
ＣａＣ１ｘ 、Ｍｇ５Ｏａ 、ＫＨｚ ＰＯａ等の多量
無機塩１１１００〜３０００ｐｐｍ、Ｆｅ５Ｏａ。11100-3000 ppm of large amounts of inorganic salts such as CaC1x, Mg5Oa, KHz POa, Fe5Oa.
Ｍｎ５Ｏａ 、Ｈ３ＢＯｉ 、Ｋ Ｉ、Ｃｕ５Ｏｎ 。Mn5Oa, H3BOi, KI, Cu5On.
Ｃｏ Ｃｌ ！等の微量無機塩Ｗ（０、Ｏ１〜５０ ｐ
ｐ ｍ、またビタミンＢ群、ビタミンＣ，グリシン、
アデニン等の有機栄養素０．１〜５０ｐｐｍ含む任意の
固体培地又は液体培地で培養することができ、その基礎
培地にはＢ５．ＭＳ、ＳＨ，ｗｈｉｔｅ等がある。本発
明において特に好ましい培地は、シェルライト０．５〜
２．０％を含む２に希釈したＭＳ培地である。CoCl! Trace inorganic salt W (0, O1~50 p
PM, also vitamin B group, vitamin C, glycine,
It can be cultured in any solid or liquid medium containing 0.1 to 50 ppm of organic nutrients such as adenine, and the basal medium contains B5. There are MS, SH, white, etc. Particularly preferred medium in the present invention is Shelllite 0.5~
MS medium diluted to 2 containing 2.0%.
培地にはシュークロース、グルコース、ソルビトール等
の炭素源１〜１０％が添加されるが、２〜３％のシェー
クロースを添加したものが好ましい。The medium contains 1 to 10% of a carbon source such as sucrose, glucose, or sorbitol, preferably 2 to 3% of sucrose.
培地に添加される植物成長ホルモンとしては、オーキシ
ン種として２．４−Ｄ、ＮＡＡ、ＩＡＡ、サイトカイニ
ン種として、ヘンシルアデニン、カイネチン、ゼアチン
等があり、継代維持培養においてはそれぞれ２種のホル
モンよりひとつを選択し、各０．０１ｐｐｍ〜２０ｐｐ
ｍ添加する。特に２．４−Ｄとベンジルアデニン、又は
ＮＡＡとカイネチンの組み合わせで各０．１〜２．０ｐ
ｐ ｍが好ましい。Plant growth hormones added to the culture medium include auxin species such as 2.4-D, NAA, and IAA, and cytokinin species such as hensyl adenine, kinetin, and zeatin, and two types of hormones are added to each culture in subculture maintenance. Select one from 0.01ppm to 20pp each
Add m. In particular, the combination of 2.4-D and benzyladenine, or NAA and kinetin, each 0.1 to 2.0 p.
pm is preferred.
カンゾウ細胞に与える化学的ストレスは、培地中にホル
モンを含有する培地からホルモンを含有しない培地へ、
又はホルモン濃度が０．５ｐ ｐ ｍ〜２０ＰＰｍの培
地から０．０１〜０．５ｐ ｐ ｍの培地へ細胞を移す
ことによって与えられる。特に、０゜２．０ｐ ｐ ｍ
のホルモン含有培地より、０〜０．５ｐｐｍのホルモン
含有培地に移すことが望ましい。The chemical stress applied to licorice cells changes from a medium containing hormones to a medium containing no hormones.
or by transferring cells from a medium with a hormone concentration of 0.5 ppm to 20 ppm to a medium with a hormone concentration of 0.01 to 0.5 ppm. Especially, 0゜2.0p p m
It is preferable to transfer to a hormone-containing medium of 0 to 0.5 ppm rather than a medium containing hormones of 0 to 0.5 ppm.
この時、ホルモン濃度の差は、０．５ｐ ｐ ｍ以上で
あるとよい。At this time, the difference in hormone concentration is preferably 0.5 ppm or more.
化学゛的ストレスは培地にオーキシン、サイトカイニン
の両者を含有するものから、どちらか一方を除去するこ
と、又は濃度を下げることによっても与えられる。さら
に、あらかじめオーキシン。Chemical stress can also be applied by removing or lowering the concentration of either auxin or cytokinin in a medium that contains both. Additionally, auxin in advance.
サイトカイニンの一方のみ含有するものにおいて、上記
のようにストレスを与えても効果がある。特にベンジル
アデニン又はゼアチンを０．１〜２０ｐｐｍ含有する培
地より、ホルモンを含有しない培地に移すことが好まし
く、ベンジルアデニン０．５〜２．０ｐ ｐ ｍ含有す
る培地からのホルモンを含有しない培地に移植する場合
、最も効果が大きい。In those containing only one of the cytokinins, even if stress is applied as described above, it is effective. In particular, it is preferable to transfer to a hormone-free medium from a medium containing 0.1 to 20 ppm benzyladenine or zeatin, and transfer from a medium containing 0.5 to 2.0 ppm benzyladenine to a hormone-free medium. The effect is greatest when
化学的ストレスを与えると同時に、低温ストレス等の物
理的ストレスを与えても効果がある。It is effective to apply physical stress such as low temperature stress in addition to chemical stress.
ホルモン濃度差の変化による化学的ストレスを与える際
、培地中の他の成分、例えば無機栄養素などの濃度を変
えてもよいが、変えない方が望ましい。When applying chemical stress due to a change in hormone concentration, the concentration of other components in the medium, such as inorganic nutrients, may be changed, but it is preferable not to do so.
（発明の効果〕
以上のように本発明によれば、植物成長ホルモンの濃度
差による化学的ストレスをカンゾウ根茎細胞に与えるこ
とによってグリチルリチン生産が促進される。(Effects of the Invention) As described above, according to the present invention, glycyrrhizin production is promoted by applying chemical stress to licorice rhizome cells due to a difference in concentration of plant growth hormone.
これは、植物体の防御機構とも考えられる。すなわち、
二次代謝系が化学的ストレスによって刺激され、グリチ
ルリチン生産が促進されるためと考えられる。This is also considered to be a plant defense mechanism. That is,
This is thought to be because the secondary metabolic system is stimulated by chemical stress, promoting glycyrrhizin production.
以下、本発明を実施例により更に詳細に説明する。 Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例１
％に希釈したＭＳ培地で、シｇｔＪ！２０．ＯＯＯｐｐ
ｍ、塩酸チアミン０．１ｐ ｐ ｍ、塩酸ピリドキシン
０．１ｐ ｐ ｍ、ニコチン酸０．５ｐ ｐｍ、 ｍｙ
ｏ −イノシトール１１００ｐｐ、グリシン３．０ｐ
ｐ ｍ、ジェルライト０．６％を含むものを基本固体
培地とし、これに植物成長ホルモンとして、ナフタレン
酢酸（ＮＡＡ）と、カイネチンを各ｌｐｐｍずつ添加し
た固体培地で、温度１０°Ｃで継代培養したカンゾウ根
茎カルス約０．１ｇを、植物成長ホルモンを添加しない
上記の基本固体培地に着床して温度１０℃で培養した。Example 1 In MS medium diluted to 1%, SigtJ! 20. OOOpp
m, thiamine hydrochloride 0.1p pm, pyridoxine hydrochloride 0.1p pm, nicotinic acid 0.5p pm, my
o-inositol 1100pp, glycine 3.0p
The basic solid medium was one containing 0.6% Gelrite, and the plant growth hormones naphthalene acetic acid (NAA) and kinetin were added to this solid medium at a temperature of 10°C. Approximately 0.1 g of cultured licorice rhizome callus was implanted on the above-mentioned basic solid medium to which no plant growth hormone was added and cultured at a temperature of 10°C.
４２日後、カルス重量を測定すると、重量は着床時の約
２倍であった。また、カルスを超音波破砕し、水で抽出
した抽出液を液体クロマトグラフィーで分析すると、グ
リチルリチンを２．４Ｘ１０−”％（カルス乾燥重量）
含有していた。When the weight of the callus was measured after 42 days, the weight was approximately twice that of implantation. In addition, when callus was crushed ultrasonically and the extract extracted with water was analyzed by liquid chromatography, glycyrrhizin was found to be 2.4 x 10% (callus dry weight).
It contained.
比較例１
実施例１と同様にして得たカルスを、同様の基本固体培
地に、植物成長ホルモンとして、ＮＡＡとカイネチンを
各ｌｐｐｍずつ添加した固体培地に着床し、温度１０°
Ｃで培養した。４２日後、カルス重量は着床時の約３．
５倍であった。また、実施例１と同様にして得たカルス
の水抽出液はグリチルリチンを１．８Ｘ１０−”％（ｔ
Ｊルス乾燥重量）を含有していた。Comparative Example 1 Calli obtained in the same manner as in Example 1 were implanted on the same basic solid medium to which lppm each of NAA and kinetin were added as plant growth hormones, and the temperature was 10°C.
Cultured in C. After 42 days, the callus weight was about 3.5 mm at implantation.
It was 5 times more. In addition, the water extract of callus obtained in the same manner as in Example 1 contained glycyrrhizin at 1.8×10-”% (t
J. russ dry weight).
実施例１．比較例１より、最初からグリチルリチンを生
産するカルスにホルモン除去による化学的ストレスを与
えると、グリチルリチン生産性は向上することが判明し
た。Example 1. From Comparative Example 1, it was found that glycyrrhizin productivity was improved when chemical stress was applied by hormone removal to callus that produced glycyrrhizin from the beginning.
実施例２
実施例１と同様の基本固体培地に、植物成長ホルモンと
して、ゼアチンを０．５ｐ ｐ ｍ添加した固体培地で
、ｍＫ２ｏ°Ｃで継代培養したカンゾウ根茎カルス約０
．１ｇを植物成長ホルモンを添加しない上記の基本固体
培地に着床し、温度２０°Ｃで培養した。４２日後、カ
ルス重量は着床時の約３．５倍であった。また、実施例
１と同様にして得たカルスの水抽出液はグリチルリチン
を１．３Ｘ１０−”％（カルス乾燥重量）含有していた
。Example 2 Approximately 0 licorice rhizome callus was subcultured at mK2o°C on the same basic solid medium as in Example 1 to which 0.5 ppm of zeatin was added as a plant growth hormone.
．． 1 g was implanted on the above-mentioned basic solid medium to which no plant growth hormone was added, and cultured at a temperature of 20°C. After 42 days, the callus weight was about 3.5 times that of implantation. Further, the water extract of callus obtained in the same manner as in Example 1 contained 1.3 x 10-''% (callus dry weight) of glycyrrhizin.
比較例２
実施例２と同様にして得たカルスを、同様の基本固体培
地に植物成長ホルモンとして、ゼアチン０．５ｐ ｐ
ｍ添加した固体培地に着床し、温度２０°Ｃで培養した
。４２日後、カルス重量は着床時の約７．０倍であった
。しかし、実施例１と同様にして得たカルスの水抽出液
からグリチルリチンは検出されなかった。Comparative Example 2 Callus obtained in the same manner as in Example 2 was placed on the same basic solid medium as a plant growth hormone, and zeatin 0.5 p p
The cells were implanted on a solid medium supplemented with m and cultured at a temperature of 20°C. After 42 days, the callus weight was about 7.0 times that of implantation. However, no glycyrrhizin was detected in the aqueous extract of callus obtained in the same manner as in Example 1.
実施例３
実施例１と同様の基本固体培地に植物成長ホルモンとし
て、ベンジルアデニンを０．５ｐ ｐ ｍ添加した固体
培地を用い、温度２０°Ｃで継代したカンヅウ根茎カル
ス約０．１ｇを、植物成長ホルモンを添加しない上記の
基本固体培地に着床し、温度２０゛Ｃで培養した。４２
日後、カルス重量は着床時の約５．０倍だった。また、
実施列ｌと同様にして得たカルスの水抽出液は、グリチ
ルリチンを１．５Ｘ １０−”％（カルス乾燥重量）含
有していた。Example 3 Using a basic solid medium similar to that of Example 1 to which 0.5 ppm of benzyladenine was added as a plant growth hormone, approximately 0.1 g of C. rhizome callus was subcultured at a temperature of 20°C. The plants were implanted on the above-mentioned basic solid medium to which no plant growth hormone was added and cultured at a temperature of 20°C. 42
Days later, the callus weight was approximately 5.0 times that of implantation. Also,
The aqueous callus extract obtained in the same manner as in Example 1 contained 1.5 x 10-''% glycyrrhizin (callus dry weight).
比較例３
実施例３と同様にして得たカルスを、同様の基本固体培
地に植物成長ホルモンとして、ベンジルアデニン０．５
ｐ ｐ ｍ添加した固体培地に着床し、温度２０°Ｃで
培養した。４２日後、カルス重量は約７．０倍であった
。しかし、実施例１と同様にして得たカルスの水抽出液
からグリチルリチンは検出されなかった。Comparative Example 3 Callus obtained in the same manner as in Example 3 was placed on the same basic solid medium as a plant growth hormone, and 0.5 benzyladenine was added.
The cells were implanted on a solid medium supplemented with p p m and cultured at a temperature of 20°C. After 42 days, the callus weight was about 7.0 times larger. However, no glycyrrhizin was detected in the aqueous extract of callus obtained in the same manner as in Example 1.
実施例２及び実施例３．比較例２及び比較例３より、最
初グリチルリチンを生産しないカルスにホルモン除去に
よる化学的ストレスを与えると、グリチルリチンを生産
しはじめることが判明した。Example 2 and Example 3. From Comparative Examples 2 and 3, it was found that when callus, which does not initially produce glycyrrhizin, is subjected to chemical stress by hormone removal, it begins to produce glycyrrhizin.
以上のように、カンゾウ根茎カルスを培養する際、あら
かじめホルモン含有培地で培養し、その後、培地中の植
物成長ホルモンを除去することによってカルスにグリチ
ルリチンを生産させ、高生産株を得ることができる。As described above, when culturing licorice rhizome callus, it is possible to culture in a hormone-containing medium in advance, and then remove the plant growth hormone in the medium to cause the callus to produce glycyrrhizin, thereby obtaining a high-producing strain.
Claims (3)
養するに際し、前記カンゾウ根茎細胞を培養する培地か
ら、この培地に対し植物成長ホルモン濃度が減少する培
地に移植して、カンゾウ根茎細胞に化学的ストレスを与
えることを特徴とするカンゾウ根茎細胞のグリチルリチ
ン高生産株取得方法。(1) When culturing licorice rhizome cells using a plant tissue culture medium, the licorice rhizome cells are transferred from the medium for culturing the licorice rhizome cells to a medium in which the concentration of plant growth hormone is decreased relative to this medium, and the licorice rhizome cells are A method for obtaining a high-glycyrrhizin-producing strain of licorice rhizome cells characterized by applying physical stress.
する培地から、植物成長ホルモンを含有しない培地に移
植することを特徴とする請求項（１）記載のカンゾウ根
茎細胞のグリチルリチン高生産株取得方法。(2) The method for obtaining a high-glycyrrhizin-producing strain of licorice rhizome cells according to claim (1), which comprises transplanting the licorice rhizome cells from a medium containing plant growth hormone to a medium not containing plant growth hormone.
長ホルモンが０．５ｐｐｍ以上の濃度差で減少する培地
に移植することを特徴とする請求項（１）記載のカンゾ
ウ根茎細胞のグリチルリチン高生産株取得方法。(3) The glycyrrhizin-rich glycyrrhizin-producing strain of licorice rhizome cells according to claim (1), characterized in that the licorice rhizome cells are transplanted from the medium in which the licorice rhizome cells are cultured to a medium in which the concentration of plant growth hormone is reduced by a concentration difference of 0.5 ppm or more. Acquisition method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1182180A JPH0347071A (en) | 1989-07-14 | 1989-07-14 | Method for obtaining highly glycyrrhizin-producing strain of glycyrrhiza glabra l. var root stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1182180A JPH0347071A (en) | 1989-07-14 | 1989-07-14 | Method for obtaining highly glycyrrhizin-producing strain of glycyrrhiza glabra l. var root stem cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0347071A true JPH0347071A (en) | 1991-02-28 |
Family
ID=16113742
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1182180A Pending JPH0347071A (en) | 1989-07-14 | 1989-07-14 | Method for obtaining highly glycyrrhizin-producing strain of glycyrrhiza glabra l. var root stem cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0347071A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0646840A (en) * | 1992-08-04 | 1994-02-22 | Nippon Zenyaku Kogyo Kk | Solution for freezing and storing cell |
| CN117448254A (en) * | 2023-10-23 | 2024-01-26 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
-
1989
- 1989-07-14 JP JP1182180A patent/JPH0347071A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0646840A (en) * | 1992-08-04 | 1994-02-22 | Nippon Zenyaku Kogyo Kk | Solution for freezing and storing cell |
| CN117448254A (en) * | 2023-10-23 | 2024-01-26 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
| CN117448254B (en) * | 2023-10-23 | 2024-04-09 | 广州梵之容化妆品有限公司 | Preparation method and application of Glycyrrhiza glabra stem cells |
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