JPH0365180A - Plant tissue culture of callus from eucommia ulmoldes - Google Patents
Plant tissue culture of callus from eucommia ulmoldesInfo
- Publication number
- JPH0365180A JPH0365180A JP1201846A JP20184689A JPH0365180A JP H0365180 A JPH0365180 A JP H0365180A JP 1201846 A JP1201846 A JP 1201846A JP 20184689 A JP20184689 A JP 20184689A JP H0365180 A JPH0365180 A JP H0365180A
- Authority
- JP
- Japan
- Prior art keywords
- callus
- plant tissue
- ulmoldes
- eucommia
- culture
- 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.)
- Granted
Links
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 36
- 241000208688 Eucommia Species 0.000 title abstract 4
- 238000004161 plant tissue culture Methods 0.000 title description 9
- 241000196324 Embryophyta Species 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 239000004480 active ingredient Substances 0.000 claims description 12
- 241000208367 Euonymus Species 0.000 claims description 4
- 101800004637 Communis Proteins 0.000 claims 2
- 230000006698 induction Effects 0.000 abstract description 6
- 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 3
- 239000004062 cytokinin Substances 0.000 abstract description 3
- FFDULTAFAQRACT-NYYYOYJKSA-N (-)-syringaresinol O,O'-bis(beta-D-glucoside) Chemical compound COC1=CC([C@H]2[C@H]3[C@H]([C@@H](OC3)C=3C=C(OC)C(O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)=C(OC)C=3)CO2)=CC(OC)=C1O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FFDULTAFAQRACT-NYYYOYJKSA-N 0.000 abstract description 2
- USOSNFWVEWONDB-UHFFFAOYSA-N liriodendrin Natural products COc1cc(cc(OC)c1OC2OC(CO)C(O)C(O)C2O)C3OC(C)(C)C4C(OC(C)(C)C34)c5cc(OC)c(OC6OC(CO)C(O)C(O)C6O)c(OC)c5 USOSNFWVEWONDB-UHFFFAOYSA-N 0.000 abstract description 2
- HGXBRUKMWQGOIE-AFHBHXEDSA-N (+)-pinoresinol Chemical compound C1=C(O)C(OC)=CC([C@@H]2[C@@H]3[C@@H]([C@H](OC3)C=3C=C(OC)C(O)=CC=3)CO2)=C1 HGXBRUKMWQGOIE-AFHBHXEDSA-N 0.000 abstract 1
- 230000003276 anti-hypertensive effect Effects 0.000 abstract 1
- RJWJHRPNHPHBRN-FKVJWERZSA-N aucubin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@@H]2C(CO)=C[C@@H](O)[C@@H]2C=CO1 RJWJHRPNHPHBRN-FKVJWERZSA-N 0.000 abstract 1
- UTDFQMAXCUGNJR-UHFFFAOYSA-N aucubin Natural products OCC1OC(Oc2ccoc2C3C(O)CCC3O)C(O)C(O)C1O UTDFQMAXCUGNJR-UHFFFAOYSA-N 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 239000002609 medium Substances 0.000 abstract 1
- 230000003285 pharmacodynamic effect Effects 0.000 abstract 1
- OHOPKHNWLCMLSW-UHFFFAOYSA-N pinoresinol Natural products C1=C(O)C(OC)=CC(C2C3C(C(OC3)C=3C=C(CO)C(O)=CC=3)CO2)=C1 OHOPKHNWLCMLSW-UHFFFAOYSA-N 0.000 abstract 1
- 235000007221 pinoresinol Nutrition 0.000 abstract 1
- QJVXKWHHAMZTBY-GCPOEHJPSA-N syringin Chemical compound COC1=CC(\C=C\CO)=CC(OC)=C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 QJVXKWHHAMZTBY-GCPOEHJPSA-N 0.000 abstract 1
- QJVXKWHHAMZTBY-KSXIZUIISA-N syringin Natural products COc1cc(C=CCO)cc(OC)c1O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O QJVXKWHHAMZTBY-KSXIZUIISA-N 0.000 abstract 1
- BURBOJZOZGMMQF-UHFFFAOYSA-N xanthoxylol Natural products C1=C(O)C(OC)=CC=C1C1C(COC2C=3C=C4OCOC4=CC=3)C2CO1 BURBOJZOZGMMQF-UHFFFAOYSA-N 0.000 abstract 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 8
- 239000007787 solid Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 6
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000000644 propagated effect Effects 0.000 description 5
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000003375 plant hormone Substances 0.000 description 3
- SODPIMGUZLOIPE-UHFFFAOYSA-N (4-chlorophenoxy)acetic acid Chemical compound OC(=O)COC1=CC=C(Cl)C=C1 SODPIMGUZLOIPE-UHFFFAOYSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 2
- HXKWSTRRCHTUEC-UHFFFAOYSA-N 2,4-Dichlorophenoxyaceticacid Chemical compound OC(=O)C(Cl)OC1=CC=C(Cl)C=C1 HXKWSTRRCHTUEC-UHFFFAOYSA-N 0.000 description 2
- 229930192334 Auxin Natural products 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002363 auxin Substances 0.000 description 2
- 239000003617 indole-3-acetic acid Substances 0.000 description 2
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229930189508 virginol Natural products 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 240000006570 Euonymus japonicus Species 0.000 description 1
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000013138 pruning Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- UZKQTCBAMSWPJD-UQCOIBPSSA-N trans-Zeatin Natural products OCC(/C)=C\CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-UQCOIBPSSA-N 0.000 description 1
- UZKQTCBAMSWPJD-FARCUNLSSA-N trans-zeatin Chemical compound OCC(/C)=C/CNC1=NC=NC2=C1N=CN2 UZKQTCBAMSWPJD-FARCUNLSSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229940023877 zeatin Drugs 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野コ
この発明は、杜仲（Ｅｕｃｏｓｍｌａ ｕｌｓｏｌｄｅ
ｓ）の植物組織片から誘導される新規植物組織培養カル
スに関する。[Detailed description of the invention] [Industrial field of application] This invention is directed to Eucosmla ulsold.
The present invention relates to a novel plant tissue culture callus derived from the plant tissue piece of s).
［従来技術およびその問題点］
現在、生薬として用いられている杜仲は、はとんど中国
大陸その他で自生ないしは栽培しているものであって、
樹齢２０年程度の成木を伐採し、その樹皮を剥離し、得
られた皮部分を薬用原料として利用している。[Prior art and its problems] Most of the Du Zhong that is currently used as herbal medicine grows naturally or is cultivated in mainland China and other countries.
Mature trees that are about 20 years old are cut down, their bark is peeled off, and the resulting bark is used as a medicinal raw material.
しかし、自生の杜仲はなかなか見つけ難く、また樹齢２
０年もの成木を栽培するには広大な面積と長い時間が必
要であり、その上樹木の伐採および樹皮の剥離にもそれ
ぞれ多大な労力を費やす。However, wild trees are difficult to find, and trees that are only 2 years old
Cultivating a 0-year-old mature tree requires a vast area and a long time, and on top of that, a great deal of labor is required to cut down the tree and peel off the bark.
杜仲の植物組織片から誘導された植物組織培養カルスが
、それ自体、杜仲の樹皮中に存在する薬用有効成分を含
有しているのであれば、この誘導カルスを増殖させ、得
られたカルスから杜仲の薬用有効成分を分離採取するこ
とによって、上記のように広大な面積、多大な労力およ
び長い時間を費やすことなく、１ｎ−ｖｌｔｒｏで杜仲
の有効成分を取得することができる。If the plant tissue culture callus derived from the plant tissue pieces of Morifolia itself contains the medicinal active ingredients present in the bark of Morifolia, then this induced callus can be propagated and the resulting callus can be used to extract the By separating and collecting the medicinal active ingredients of Du Zhong, it is possible to obtain the active ingredients of Du Zhong in 1n-vltro without spending a large area, much labor, and long time as described above.
この発明は、上記のような観点からなされたもので、杜
仲の薬用有効成分を含有する植物組織培養カルスを提供
することを目的とする。The present invention was made from the above-mentioned viewpoint, and an object of the present invention is to provide a plant tissue culture callus containing a medicinal active ingredient of Mori mori.
ｃ問題点の解決手段］
この発明による杜仲の植物組織培養カルスは、上記目的
の達成のために、杜仲の植物組織片の培養によって形成
され、かつ杜仲の薬用有効成分を含有することを特徴と
する。Solution to Problem c] In order to achieve the above-mentioned object, the tissue-cultured callus of Euonymus japonica according to the present invention is characterized in that it is formed by culturing pieces of tissue of the Euonymus annuus and contains a medicinal active ingredient of Ori annuus. do.
ここで、杜仲の植物組織片の培養すべき部位としでは、
大量の材料（外植体）を容易に確保することができる器
官を使用する必要があることから、当年技（徒長枝）と
りわけその茎節部や上胚軸がよく使用される。ただし、
培養すべき部位はこれらに限定されない。Here, the parts to be cultured of plant tissue pieces of Mori Zhong are:
Since it is necessary to use organs from which a large amount of material (explants) can be easily obtained, the current crop (elongated branches), especially their stem nodes and epicotyls, are often used. however,
The sites to be cultured are not limited to these.
杜仲の植物組織片の培養は、植物組織片からカルスを誘
導させるいわゆる誘導培養と、誘導されたカルスを増殖
させるいわゆる増殖培養とより成る。カルスの誘導培養
は好ましくは寒天培地のような固形培地で行われ、また
カルスの増殖培養は好ましくは液体培地における継代培
養の繰り返しにより行われる。Cultivation of mori plant tissue pieces consists of so-called induction culture in which callus is induced from the plant tissue pieces, and so-called multiplication culture in which the induced callus is propagated. Induction culture of callus is preferably carried out in a solid medium such as an agar medium, and callus growth culture is preferably carried out by repeated subculturing in a liquid medium.
固形培地としてはカルス誘導培養用の通常の培地が使用
でき、たとえば、表１に示すＢ−５（Ｇａｍｂｏｒｇ）
１９８６または表２に示すＷＰＭ（Ｗｏｏｄｙ ｐｌａ
ｎｔｍｅｄ１ｕａ＋）１９８２をベースとした組成に、
植物ホルモン（生長調整物質）としてオーキシンおよび
サイトカイニンを添加したものがよく用いられる。オー
キシンとしては、ＮＡＡ （ナフタレン酢酸）、ＩＡＡ
（インドール酢酸）、２．４−Ｄ （２，４−ジクロロ
フェノキシ酢酸）、ＣＰＡ （４−クロロフェノキシ酢
酸）、ＩＢＡ（インドール酪酸）などがよく用いられる
。また、サイトカイニンとしては、６−ＢＡ （Ｎ６−
ベンジルアデニン）、カイネチン（Ｎ ６−フルフリル
アミノプリン） 、２ｉｐ（Ｎ６ ｒ 、ｒ−ジメチル
アリルアミノプリン）、ゼアチンなどがよく用いられる
。また、液体培地としてはカルス増殖培養用の通常の培
地が使用でき、たとえば、上記固形培地の組成から寒天
を省いた組成のものがよく用いられる。As the solid medium, a normal medium for callus induction culture can be used, such as B-5 (Gamborg) shown in Table 1.
1986 or WPM (Woody pla
The composition is based on ntmed1ua+) 1982,
Those containing auxin and cytokinin as plant hormones (growth regulators) are often used. Auxins include NAA (naphthalene acetic acid), IAA
(indoleacetic acid), 2.4-D (2,4-dichlorophenoxyacetic acid), CPA (4-chlorophenoxyacetic acid), IBA (indolebutyric acid), etc. are often used. In addition, as cytokinin, 6-BA (N6-
benzyladenine), kinetin (N6-furfurylaminopurine), 2ip (N6r, r-dimethylallylaminopurine), zeatin, and the like are often used. Further, as the liquid medium, a usual medium for callus growth culture can be used, and for example, a solid medium having a composition in which agar is omitted from the composition of the above-mentioned solid medium is often used.
カルスの誘導培養およびカルスのクローン増殖培養にお
いて、通常は、培養温度は好ましくは２５℃±２℃であ
り、増殖培養においては、液体培地のベース組成に植物
ホルモンを複数の濃度段階で添加した培地を用いて、約
１カ月単位で数回〜十数回継代培養を行なう。In callus induction culture and callus clonal propagation culture, the culture temperature is usually preferably 25°C ± 2°C, and in propagation culture, a medium in which plant hormones are added at multiple concentration levels to the base composition of the liquid medium is used. The subculture is carried out several times to more than 10 times every about one month.
カルスの誘導培養およびカルスのクローン増殖培養のう
ち、少なくとも後者の培養は、好ましくは明所で行われ
る。この明所培養は、一定期間たとえば１０〜２４時間
、１００〜２００００ルツクス、好ましくは１０００〜
１００００ルツクスの光照射下に培養物を置くことによ
る培養方法である。Of callus induction culture and callus clonal propagation culture, at least the latter culture is preferably carried out in the light. This light cultivation is carried out for a certain period of time, for example 10 to 24 hours, at 100 to 20,000 lux, preferably at 1,000 to 20,000 lux.
This is a culturing method in which the culture is placed under light irradiation of 10,000 lux.
ただし、上記培養条件および培地の組成はいずれも限定
的なものではない。However, neither the above culture conditions nor the composition of the medium are limited.
かくして杜仲の植物組織片から誘導・増殖された植物組
織培養カルスは、杜仲の樹皮中に存在する薬用有効成分
、とりわけ顕著な血圧降下作用を有するビルジノール・
ジー〇−β−Ｄ−グルコシド（Ｐｌｎｏｒｅｓｉｎｏｌ
−ｄｉ−０−β−１）−ｇｌｕｃ。In this way, the plant tissue culture callus induced and propagated from the plant tissue pieces of Morifolia can contain the medicinal active ingredients present in the bark of Morifolia, especially virginol, which has a remarkable hypotensive effect.
D-β-D-glucoside (Plnoresinol)
-di-0-β-1)-gluc.
５ｉｄｅ）を含有する。この成分は、たとえば、杜仲の
植物組織培養カルス（１５０，６ｇ）中に約０．４５ｍ
ｇ含まれている（なお、この成分は杜仲の樹皮中には約
０．０４％含まれていることが知られている。）。その
他に、このカルス中にはアウクビン（Ａｕｃｕｂｌｎ）
、シリンシン（Ｓｙｒｔｎｇｔｎ）、リリオデンドリン
（Ｌｌｒｌｏｄｅｎｄｒｉｎ）などが含まれている。カ
ルスの上記含有成分の単離、同定などの分析は、溶媒抽
出、薄層クロマトグラフィ（ＴＬＣ）、核磁気共鳴スペ
クトル（ＮＭＲ） 、紫外線吸収スペクトル（Ｕ Ｖ）
、赤外線吸収スペクトル（ＩＲ）、マススペクトル（Ｍ
Ｓ）などを用いた手法によって行われる。5ide). For example, approximately 0.45 m of this component is contained in Du Zhong plant tissue culture callus (150.6 g).
g (It is known that this component is contained in the bark of Mori annua at about 0.04%). In addition, this callus contains Aucubln.
, Syrtngtn, Llrlodendrin, and the like. Analysis such as isolation and identification of the above-mentioned components of callus can be performed using solvent extraction, thin layer chromatography (TLC), nuclear magnetic resonance spectroscopy (NMR), and ultraviolet absorption spectroscopy (UV).
, infrared absorption spectrum (IR), mass spectrum (M
This is done using a method such as S).
［発明の効果］
この発明によれば、杜仲の植物組織片から誘導された植
物組織培養カルスは、それ自体、杜仲の樹皮中に存在す
る薬用有効成分を含有している。したがって、この誘導
カルスを増殖させ、得られたカルスから杜仲の薬用有効
成分を分離採取することによって、杜仲の成木から上記
薬用有効成分を採取する場合のように広大な面積、多大
な労力および長い時間を☆やすことなく、Ｉｎ−ｖｌｔ
ｒｏで杜仲の有効成分を取得することができる。そのた
め、この発明によるカルスは生薬の材料として極めて有
用な物質である。[Effects of the Invention] According to the present invention, the plant tissue culture callus derived from the plant tissue pieces of Morifolia itself contains the medicinal active ingredient present in the bark of Morifolia. Therefore, by propagating this induced callus and separating and collecting the medicinal active ingredients of Morifolia from the resulting callus, it takes a vast area, a great deal of labor, and a large amount of effort, as compared to the case where the medicinal active ingredients are collected from adult Mori trees. In-vlt without spending a long time☆
The active ingredients of Mori Zhong can be obtained through RO. Therefore, the callus according to the present invention is an extremely useful substance as a raw material for crude drugs.
［実施例］
つぎに、この発明をその実施例によって具体的に説明す
る。[Examples] Next, the present invention will be specifically explained with reference to Examples.
ａ）カルスの形成
樹齢３年の実生苗を温室内に搬入し、伸長してきた当年
技を約３０ｃｍの長さに切り取り、この切り取り片を２
％のアンチフォルミン（次亜塩素酸ナトリウム溶液）と
共に２０分間回転させることによって殺菌処理を行なっ
た。この殺菌処理品を剪定鋏で長さ３ｃｍ程度の多数の
小片に切り、これら小片を試験管内の固形培地に置床（
捕し木）した。この固形培地は、族１に示すＢ−５（Ｇ
ａｍｂｏｒｇ） １９６Ｂの寒天培地に、０．５ｇｇ／
ｌのＮＡＡ （ナフタレン酢酸）と０．６厘ｇ／ ／の
６−ＢＡ （Ｎ６−ベンジルアデニン）をそれぞれ添加
して調整したものである。培養温度は２５℃±２℃に維
持した。こうして、試験管内において杜仲のカルスを誘
導させた。a) Formation of callus A 3-year-old seedling was brought into the greenhouse, and the elongated plant of the current year was cut into a length of about 30 cm, and this cut piece was divided into 2 pieces.
Sterilization was performed by rolling with % antiformin (sodium hypochlorite solution) for 20 minutes. Cut this sterilized product into many small pieces with a length of about 3 cm using pruning shears, and place these small pieces on a solid medium in a test tube (
I did it. This solid medium is B-5 (G
amborg) 196B agar medium, 0.5 gg/
It was prepared by adding 1 ml of NAA (naphthalene acetic acid) and 0.6 ml of 6-BA (N6-benzyladenine). The culture temperature was maintained at 25°C±2°C. In this way, Mori callus was induced in the test tube.
つぎに、この誘導カルスを液体培養によってクローン増
殖させた。すなわち、３００ｍ／のコニカルフラスコを
用いて、液体培地として、上記固形培地の組成から寒天
を省いた組成に上記植物ホルモンを１５段階の濃度で添
加した１５区の培地を用いて、約１カ月単位で連続５回
線代培養を行なった。この培養は、温度２５℃±２℃で
、１６峙間、３０００ルツクスの光照射下に培養物を置
く明所培養によって行なった。Next, this induced callus was clonally propagated by liquid culture. That is, using a 300 m conical flask as a liquid medium, a 15-section medium containing the above-mentioned solid medium with agar omitted and the above-mentioned plant hormones added at 15 levels of concentration was used as a liquid medium for about 1 month. Five consecutive subcultures were performed. This cultivation was carried out by light cultivation in which the culture was placed under light irradiation of 3000 lux for 16 hours at a temperature of 25°C ± 2°C.
こうして、液体培養によって杜仲のカルスをクローン増
殖させた。In this way, calli of Mori zhong were clonally propagated by liquid culture.
ｂ）、カルス含有成分の分析
こうして得られた杜仲の植物組織培養カルス（１５０，
６ｇ）を、第１図に示すフローシートにしたがって、メ
タノール抽出、その抽出物の水中懸濁、その水可溶物の
ポリスチレン系樹脂ＭＣＩゲルカラムクロマトグラフィ
、およびそのフラクションＦｒ、２とフラクションＦｒ
。b) Analysis of callus-containing components The plant tissue culture callus (150,
6g) was subjected to methanol extraction, suspension of the extract in water, polystyrene resin MCI gel column chromatography of the water-soluble product, and fractions Fr, 2 and Fr.
.
３のシルカゲルカラムクロマトグラフィに順次付して、
フラクションＦｒ、３”２ｃ （４，５ｍｇ）を得た。3. Sequentially subjected to silica gel column chromatography,
A fraction Fr, 3"2c (4.5 mg) was obtained.
また、ＣＨＣ／ ３ ： Ｍｅ ＯＨ：Ｈ２Ｏ−７：
２．５：０．２の展開溶媒を用いて、上記フラクション
Ｆｒ、３をシルカゲル薄層クロマトグラフィに付した。Also, CHC/3:MeOH:H2O-7:
The above fraction Fr, 3 was subjected to silica gel thin layer chromatography using a developing solvent of 2.5:0.2.
その展開状態を第２図に示す。The unfolded state is shown in FIG.
第２図の展開状態において、フラクションＦｒ、１−１
およびＦｒ、２−１については、それらのシルカゲル薄
層クロマトグラフィのＲｆ値および２０％硫酸水溶液中
での加熱による呈色反応を、標品のものと比較すること
によって、フラクションＦｒ、１−１はアラキュビンで
あると同定され、またフラクションＦｒ、２−１はシリ
ンシンであると同定された。In the developed state shown in Fig. 2, the fraction Fr, 1-1
By comparing the Rf value of silica gel thin layer chromatography and the color reaction by heating in a 20% sulfuric acid aqueous solution with that of the standard product, the fraction Fr, 1-1 was determined. It was identified as aracubine, and fraction Fr, 2-1 was identified as cilinsin.
フラクションＦｒ、３−２ｃについては、そのシルカゲ
ル薄層クロマトグラフィのＲｆ値、第３図に示すＩＨ−
ＮＭＲスペクトル、および第４図に示す”Ｃ−ＮＭＲス
ペクトルを、標品のものと比較することによって、この
フラクションＦｒ、３−２ｃはリリオデンドリンである
と同定された。Regarding the fraction Fr, 3-2c, its Rf value of silica gel thin layer chromatography, IH-
By comparing the NMR spectrum and the "C-NMR spectrum shown in FIG. 4 with that of the standard product, this fraction Fr, 3-2c was identified as liriodendrin.
フラクションＦｒ、３−２ｃには、第４図の”Ｃ−ＮＭ
Ｒスペクトルに示すように、１００．５ 。Fraction Fr, 3-2c contains "C-NM" in Figure 4.
100.5 as shown in the R spectrum.
１１１．０ 、 １１５．８ 、１１８．３ 、１３５
．０ 、１４Ｂ、０ 、 １４９．１などに弱いシグナ
ルを示す物質が含まれている。これらシグナルの位置す
なわちケミカルシフト値を標品のものと比較することに
よって、上記含有物質はビルジノール・ジー〇−β−Ｄ
−グルコシドであると同定された。フラクション ンＦ
ｒ、 ３−２ ｃ （４，５ｍｇ）の結晶中のりリ
オデンドリンとビルジノール・ジーＯ−β−Ｄ−グルコ
シドとの含有比は約１０：１であるので、杜仲の薬用有
効成分であるビルジノール・ジー０−β−Ｄ−グルコシ
ドは、杜仲の植物組織培養カルス（１５０，６ｍｇ）中
に約０．４５ｍｇ含まれていることになる。111.0, 115.8, 118.3, 135
．． It contains substances that show weak signals at 0, 14B, 0, 149.1, etc. By comparing the positions of these signals, that is, the chemical shift values, with those of the standard, it was determined that the above-mentioned contained substance was Virginol G-β-D.
-Identified as a glucoside. Fraction F
r, 3-2c (4.5 mg), the content ratio of Nori-Ryodendrin and Virginol-di O-β-D-glucoside is about 10:1, so Virginol-Di, the medicinal active ingredient of Duzhong, Approximately 0.45 mg of 0-β-D-glucoside is contained in the plant tissue culture callus (150.6 mg) of Duchuan.
（以下余白）
族１ （Ｇａｍｂｏｒｇ、ＭＩＩＩｅｒ ａｎｄ Ｏｊ
１ｍａ培地（Ｂ−５） １９８８の組成）族２ （Ｗｏ
ｏｄｙ ｐｌａｎｔ培地（ＷＰＭ）１９８０のＭ５゜
６に調整した。(Left below) Family 1 (Gamborg, MIIIer and Oj
Composition of 1ma medium (B-5) 1988) Group 2 (Wo
ody plant medium (WPM) 1980 was adjusted to M5°6.
５゜ ６に調整した。5゜ Adjusted to 6.
図面はこの発明の実施例を示すものであって、第１図は
カルスの分析方法を示すフローシート、第２図はフラク
ションＦｒ、３のシルカゲル薄層クロマトグラフィの展
開状態を示す図、第３図は’Ｈ−ＮＭＲスペクトルを示
す図、第４図は”Ｃ−ＮＭＲスペクトルを示す図である
。
以 上The drawings show examples of the present invention, in which Fig. 1 is a flow sheet showing a callus analysis method, Fig. 2 is a drawing showing the development state of silica gel thin layer chromatography of fraction Fr, 3, and Fig. 3 is a flow sheet showing a callus analysis method. Figure 4 shows the 'H-NMR spectrum, and Figure 4 shows the 'C-NMR spectrum.
Claims (1)
薬用有効成分を含有することを特徴とする杜仲の植物組
織培養カルス。1. A tissue culture callus of Euonymus communis, which is formed by culturing a piece of tissue of the Euonymus elegans plant and contains a medicinal active ingredient of Euonymus communis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1201846A JPH07108217B2 (en) | 1989-08-03 | 1989-08-03 | Tochu's plant tissue culture callus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1201846A JPH07108217B2 (en) | 1989-08-03 | 1989-08-03 | Tochu's plant tissue culture callus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0365180A true JPH0365180A (en) | 1991-03-20 |
| JPH07108217B2 JPH07108217B2 (en) | 1995-11-22 |
Family
ID=16447861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1201846A Expired - Fee Related JPH07108217B2 (en) | 1989-08-03 | 1989-08-03 | Tochu's plant tissue culture callus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07108217B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0630768A (en) * | 1992-07-20 | 1994-02-08 | Hitachi Zosen Corp | Method for proliferating cultured cell of euconymus japonica thumb |
| CN106770695A (en) * | 2016-05-21 | 2017-05-31 | 广州今典精方药业有限公司 | The quality standard and manufacturing process of the bark of eucommia qualitative, quantitative prepared slices of Chinese crude drugs |
-
1989
- 1989-08-03 JP JP1201846A patent/JPH07108217B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| BIOLOGICAL ABSTRACTS * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0630768A (en) * | 1992-07-20 | 1994-02-08 | Hitachi Zosen Corp | Method for proliferating cultured cell of euconymus japonica thumb |
| CN106770695A (en) * | 2016-05-21 | 2017-05-31 | 广州今典精方药业有限公司 | The quality standard and manufacturing process of the bark of eucommia qualitative, quantitative prepared slices of Chinese crude drugs |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07108217B2 (en) | 1995-11-22 |
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