JPH0722470B2 - Essential oil manufacturing method - Google Patents
Essential oil manufacturing methodInfo
- Publication number
- JPH0722470B2 JPH0722470B2 JP63053770A JP5377088A JPH0722470B2 JP H0722470 B2 JPH0722470 B2 JP H0722470B2 JP 63053770 A JP63053770 A JP 63053770A JP 5377088 A JP5377088 A JP 5377088A JP H0722470 B2 JPH0722470 B2 JP H0722470B2
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- Prior art keywords
- essential oil
- adventitious
- medium
- adventitious buds
- plant
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Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、植物の組織を培養し、分化誘導した不定芽か
ら精油成分を製造する方法に関する。TECHNICAL FIELD The present invention relates to a method for culturing plant tissue and producing an essential oil component from an adventitious shoot in which differentiation is induced.
精油は、植物体から圧搾、抽出等により得られる物質で
あって、化粧品、香料、食用フレーバー等の分野で広く
用いられている。そして、この精油は一般に栽培した植
物を原料として製造されている。Essential oils are substances obtained by squeezing, extracting, etc. from plants and are widely used in the fields of cosmetics, fragrances, edible flavors and the like. And this essential oil is manufactured using the cultivated plant as a raw material.
近年、植物の組織培養が比較的容易となり、この方法が
植物成分の製造手法として検討されている。そして、ペ
ラルゴニウム属、ゲラニウム属、ルタ属、ヴィオラ属、
メリッサ属、イリス属等の植物について、その不定芽か
ら精油を製造する方法が提案されている（特開昭59−21
3393号、特開昭60−71699号、特開昭60−248188号）。
しかし、アンテミス属に属する植物については、これま
で、全く報告がない。In recent years, tissue culture of plants has become relatively easy, and this method has been studied as a method for producing plant components. And the genus Pelargonium, the genus Geranium, the genus Ruta, the genus Viola,
For plants of the genus Melissa, genus Iris, etc., a method for producing an essential oil from adventitious buds thereof has been proposed (JP-A-59-21).
3393, JP-A-60-71699, JP-A-60-248188).
However, there have been no reports on plants belonging to the genus Antemis.
本発明者らは、従来の植物の栽培によるものに比べて気
象条件等の影響を受けず完全に制御された環境下で容易
かつ高収率で精油を製造する方法を開発すべく研究し、
本発明を完成するに至った。The present inventors have studied to develop a method for producing an essential oil easily and in high yield under a completely controlled environment that is not affected by weather conditions and the like as compared with the conventional plant cultivation,
The present invention has been completed.
〔課題を解決するための手段〕 本発明は、アンテミス（Anthemis）属に属する植物の組
織を培養し、培養物から不定芽を分化誘導せしめ、該不
定芽中に産生蓄積された精油成分を採取する精油の製造
方法である。そして、この不定芽としては増殖に最適な
植物ホルモンを含有する培地で継代培養し増殖したも
の、継代培養したものから選抜した精油高産生株が、加
えて該高産生株を液体培地で大量培養したものがそれぞ
れ使用される。さらに、本発明には、不定芽を継代培養
したものの中から、その形態による目視的選抜及びヘッ
ドスペースガスクロマトグラフィー法（以下HS−GC法と
いう）による選抜により精油高産生株を選抜する精油高
産生株の取得法が含まれる。[Means for Solving the Problems] The present invention cultivates the tissue of a plant belonging to the genus Anthemis, induces differentiation of adventitious buds from the culture, and collects essential oil components produced and accumulated in the adventitious buds. It is a method for producing essential oil. And, as this adventitious bud, those subcultured and propagated in a medium containing a plant hormone most suitable for growth, essential oil high-producing strains selected from the subcultured ones, in addition, the high-producing strains in a liquid medium A large amount of culture is used. Furthermore, in the present invention, an essential oil for selecting a strain highly producing essential oil by visual selection according to its morphology and selection by headspace gas chromatography method (hereinafter referred to as HS-GC method) from subcultured adventitious buds Includes methods for obtaining high-producing strains.
以下に、本発明を詳しく述べる。The present invention is described in detail below.
本発明においては、アンテミス属に属する植物に組織を
寒天培地中で培養し、培養物から不定芽を分化誘導し、
この不定芽を最適な植物ホルモンの存在下に継代培養し
て増殖させ、得られる不定芽の中から精油高産生株を選
抜し、この不定芽から精油を製造する。更に原料の不定
芽としては上記精油高産生株を液体培地中で大量培養し
たものを用いることができる。In the present invention, a tissue belonging to a plant belonging to the genus Antemis is cultured in an agar medium to induce differentiation of adventitious shoots from the culture,
The adventitious buds are subcultured and grown in the presence of an optimum plant hormone, and a strain producing a high essential oil is selected from the adventitious buds obtained, and an essential oil is produced from the adventitious buds. Furthermore, as the adventitious buds of the raw material, those obtained by culturing the above-mentioned essential oil highly producing strain in a large amount in a liquid medium can be used.
本発明で使用されるアンテミス属に属する植物としては
アンテミス・ノビリス（Anthemis nobolis），アンテミ
ス・チンクトリア（Anthemis tinctoria），アンテミス
・アルベンシス（Anthemis arvensis），アンテミス・
コチュラ（Anthemis cotula）が挙げられる。そして、
植物体としては葉、茎、根の断片のいずれでもが使用で
きる。植物の組織から不定芽を分化誘導する培地として
は、植物組織培養において通常使用されるもの、例え
ば、リンスマイヤー・スクーグ（LS）培地、ムラシゲ・
スクーグ培地、B5培地、ニッチ＆ニッチ培地、ホワイト
培地等が使用されるが、好ましくはLS培地が用いられ
る。そして、実際に培養するに際しては、これら基本培
地に糖類、ビタミン類及び植物ホルモンを添加して用い
る。植物ホルモンとしては、例えばインドールー３−酢
酸（IAA）、インドールー３−酪酸（IBA）、α−ナフタ
レン酢酸（NAA）、2,4−ジクロロフェキシ酢酸（2,4−
Ｄ）等のオーキシン類、カイネチン（Kn）、６−ベンジ
ルアデニン（６−BA）等のサイトカイニン類等が挙げら
れ、これらオーキシンとサイトカイニンを組み合せて使
用される。特に好ましい組み合せはオーキシン：サイト
カイニンが10-6M:10-6Ｍである。この培地中で明下20〜
30℃、好ましくは25℃で２〜３週間培養することにより
カルス化し、不定芽及び不定根の誘導が起る。光の条件
は2000〜3000ルックスの光照射を用いる。The plants belonging to the genus Anthemis used in the present invention include Anthemis nobolis, Anthemis tinctoria, Anthemis arvensis, and Anthemis arvensis.
An example is Cotula (Anthemis cotula). And
As the plant body, any of leaf, stem and root fragments can be used. As a medium for inducing the differentiation of adventitious shoots from plant tissues, those usually used in plant tissue culture, for example, Rinsmeier-Skoog (LS) medium, Murashige
Skoo's medium, B5 medium, niche & niche medium, white medium and the like are used, but LS medium is preferably used. When actually culturing, sugars, vitamins and plant hormones are added to these basal media before use. Examples of plant hormones include indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthalene acetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-
D) and other auxins, kinetin (Kn), 6-benzyladenine (6-BA) and other cytokinins, and the like are used, and these auxins and cytokinins are used in combination. A particularly preferred combination is auxin: cytokinin 10 −6 M: 10 −6 M. 20 ~ under the light in this medium
By culturing at 30 ° C., preferably 25 ° C. for 2 to 3 weeks, callus is formed, and induction of adventitious buds and adventitious roots occurs. The light conditions are light irradiation of 2000 to 3000 lux.
この不定芽は新芽を含む葉２〜３枚の小さい株に分けて
継代培養して増殖を行うが、そのときの培地としては上
記基本培地が用いられる。そして、その際使用する植物
ホルモンの組成により異なった形態（葉の形、色等）の
不定芽が得られる。即ち、植物ホルモンとしてIBA10
-6M,BA10-6M;IBA10-5M,BA10-6M;NAA10-5M,BA10-6Ｍなど
を用いたところ、その不定芽の形態が３種類に分類され
た。This adventitious bud is divided into 2-3 small leaves containing shoots and subcultured for growth, and the above basic medium is used as the medium. Then, adventitious buds having different forms (leaf shape, color, etc.) are obtained depending on the composition of the plant hormone used at that time. That is, IBA10 as a plant hormone
-6 M, BA10 -6 M; IBA10 -5 M, BA10 -6 M; NAA10 -5 M, BA10 -6 was used like M, the form of the adventitious buds were classified into three types.
母植物に近い形態のタイプI,葉の分岐があまり無いタイ
プII,葉と茎の区別がつかないタイプIIIの３種類であ
る。その形態的分類では、第１表に示す通りタイプＩで
のみ良好な精油成分が産生した。したがって、タイプＩ
の不定芽の増殖に好適な植物ホルモンは、例えばIBA:10
-6ＭとBA:10-6〜10-7Ｍとを組み合わせたものである。There are three types: type I with a morphology similar to that of the mother plant, type II with few branching of leaves, and type III with indistinguishable leaves and stems. In its morphological classification, only Type I produced good essential oil components as shown in Table 1. Therefore, type I
A suitable plant hormone for the growth of adventitious buds is, for example, IBA: 10.
-6 M and BA: 10 -6 to 10 -7 M in combination.
不定芽を増殖して得られる株の中から精油の高産生株を
選抜するためには、不定芽の形態及び精油成分をHS−GC
法（恩田宣彦：ぶんせき、66,12（1987））にかけて行
った。従来、精油成分の分析には蒸留や溶媒抽出等の煩
雑な操作及び多量の試料が必要であるが、このHS−GC法
では少量の試料で簡単にかつ短時間で分析が可能であ
る。In order to select strains with high essential oil production from strains obtained by growing adventitious buds, the morphology of adventitious buds and essential oil components are determined by HS-GC.
I went to the law (Nobuhiko Onda: Bunseki, 66 , 12 (1987)). Conventionally, analysis of essential oil components requires complicated operations such as distillation and solvent extraction and a large amount of sample, but this HS-GC method enables simple and short-time analysis with a small amount of sample.
この精油高産生株は、液体培地においても大量培養する
ことができる。この液体培地は次の組成から成る。This essential oil high-producing strain can be mass-cultured in a liquid medium. This liquid medium has the following composition.
（液体培地組成） （１）基本培地 LS培地の濃度の１〜1/2培 （２）ショ糖 ３％ （３）ホルモン IBA 10-6Ｍ BA 10-7Ｍ （４）窒素源 アンモニア熊窒素／硝酸熊 窒素＝1/5〜1/11 培養は、25℃で７〜10日間明所（2000〜3000ルックス）
下で行う。(Liquid medium composition) (1) Basic medium 1-1 / 2 times the concentration of LS medium (2) Sucrose 3% (3) Hormone IBA 10 -6 M BA 10 -7 M (4) Nitrogen source Ammonia Bear nitrogen / Bear Nitrate Nitrogen = 1/5 to 1/11 Culture at 25 ° C for 7 to 10 days in the light (2000 to 3000 lux)
Do it below.
このようにして得られた不定芽はアンテミス属の植物と
同様の精油成分を含有しており、これら不定芽から、例
えば石油エーテル等の溶媒を用いて抽出する方法、水蒸
気蒸留法等、通常使用される公知の方法により精油成分
が容易に取得できる。The adventitious buds thus obtained contain the same essential oil components as plants of the genus Antemis, and from these adventitious buds, for example, a method of extracting using a solvent such as petroleum ether, a steam distillation method, etc. The essential oil component can be easily obtained by a known method.
本発明の方法は、従来の栽培によるものに比べて気象条
件等の影響を受けず、完全に制御された環境の下で短時
間に高収率かつ多量に精油を製造することができる。ま
た、原料として使用する精油高産生不定芽株が形態の目
視選抜及びHS−GC法により容易に選抜できる。The method of the present invention is not affected by weather conditions and the like as compared with the conventional cultivation, and can produce a large amount of essential oil in high yield in a short time under a completely controlled environment. In addition, the adventitious bud strain that produces a high amount of essential oil used as a raw material can be easily selected by visual selection of the morphology and the HS-GC method.
実施例１ （１）不定芽の分化誘導 水洗したアンテミス・ノビリスの葉を70％のエタノール
で１分間、予備滅菌し、次いで１％アンチホルミンで５
〜10分間滅菌した。次に滅菌蒸留水で薬剤を完全に除去
した葉を無菌的に約5mm角に細断し、外植片とした。こ
の細断葉を植物ホルモンとして、オーキシン（IBAまた
はNAA）の10-5Ｍ〜10-6とサイトカイニン（BAまたはカ
イネチン）の10-5Ｍ〜10-6Ｍとの組合わせで、それぞれ
添加した３％のショ糖１％の寒天を含むph5.6に調節し
たLS培地上に置床し、25℃で2000〜3000ルックスの光照
射下で培養を行ないカルスを形成せしめ、２〜３週間後
に不定芽及び、または不定根が分化誘導した。Example 1 (1) Induction of Adventitious Bud Differentiation The leaves of water-washed Antemis nobilis were pre-sterilized with 70% ethanol for 1 minute, and then 5% with 1% antiformin.
Sterilized for ~ 10 minutes. Next, the leaves from which the drug had been completely removed with sterile distilled water were aseptically cut into pieces of about 5 mm square, and used as explants. This shredded leaf was added as a plant hormone in a combination of 10 -5 M to 10 -6 of auxin (IBA or NAA) and 10 -5 M to 10 -6 M of cytokinin (BA or kinetin), respectively. After placing on LS medium adjusted to pH5.6 containing 3% sucrose 1% agar and culturing at 25 ° C under light irradiation of 2000-3000 lux, callus was formed, and after 2-3 weeks, indefinite Differentiation was induced by shoots and / or adventitious roots.
（２）不定芽の継代培養及び精油高産生株の選抜この不
定芽から葉２〜３枚を含む新芽のある小さな株を選抜し
同じ培地及び培養条件で３〜４週間継代培養した。各培
地で得られる不定芽の形態（葉の形、色等）及びHS−GC
の結果は第１表の通りである。この結果についてはHS−
GCによって得られたクロマトグラムのピーク高さから成
分量を求め、クトマトグラムに表われた成分全量に対す
る百分率で表示した。このことからタイプＩが精油の高
産生株であることが判った。この結果から判る通り、継
代培養した不定芽の中から精油の高産生株が目視的に容
易に選抜できる。(2) Subculture of adventitious buds and selection of highly oil-producing strains From these adventitious buds, small strains with shoots containing 2-3 leaves were selected and subcultured for 3 to 4 weeks under the same medium and culture conditions. Adventitious bud morphology (leaf shape, color, etc.) and HS-GC obtained with each medium
The results are shown in Table 1. HS-
The amount of the component was obtained from the peak height of the chromatogram obtained by GC and expressed as a percentage of the total amount of the component shown in the tomatogram. From this, it was found that type I is a high-producing strain of essential oil. As can be seen from these results, strains with high essential oil production can be easily visually selected from the subcultured adventitious buds.
この方法で得られる不定芽を培養し、精油を製造した結
果につきその培養条件、増殖度、精油の成分等を第２表
に示す。The adventitious buds obtained by this method are cultivated, and the essential oil production results are shown in Table 2 along with the culturing conditions, growth rate, essential oil components and the like.
なお、第１表および第２表において、不定芽の増殖度、
香気の官能試験及び精油成分の分析は次の方法により行
った。 In Tables 1 and 2, the degree of growth of adventitious shoots,
The sensory test of aroma and the analysis of essential oil components were carried out by the following methods.
（１）不定芽の増殖度（倍） （２）香気の官能試験 不定芽を手指で押しつぶし香気を10人の専門員によって
臭覚により判定した。対照は原植物とした。その平均値
を以って官能評価とした。(1) Growth rate of adventitious shoots (fold) (2) Sensory test of aroma The adventitious buds were crushed with fingers and the aroma was judged by 10 experts. The control was the original plant. The average value was used for sensory evaluation.
評価基準： 1.原植物の精製成分の臭いなし 2. 〃 かすかに有り 3. 〃 有り 4. 〃 強し 5. 〃 非常に強し （３）精油成分の分析 ヘッドスペースガスクロマトグラフィー（HS−GC）を用
い、クロマトグラムのピークの高さから成分量を求め、
クロマトグラムに表われた成分全量に対する百分率で表
示した。定性分析はガスクロマトグラフィー及び質量分
析計により標準物質との保持時間及びマススペクトルを
比較する事により行った。Evaluation criteria: 1. No odor of refined components of original plant 2. Slightly present 3. 〃 Present 4. 〃 Strong 5. 〃 Very strong (3) Analysis of essential oil components Headspace gas chromatography (HS-GC) Using, the amount of components is calculated from the peak height of the chromatogram,
It was expressed as a percentage of the total amount of the components shown in the chromatogram. The qualitative analysis was carried out by comparing the retention time and mass spectrum with the standard substance by gas chromatography and mass spectrometry.
（測定条件） １）ガスクロマトグラフィー 機種:HP5890Aガスクロマトグラフ カラム:DB−WAXヒューズド・シリカ・キャピラリーカラ
ム（径0.25mm×60m） 検出器:FID インジェクション温度:250℃ オープン温度:70℃〜180℃（４℃/minで昇温） キャリアガス流量（He）:0.78ml/min スプリット比:1/20 ２）ヘッドスペースサンプラー 機種:HP19395A 保温温度:75℃ 保温時間:90分 ３）質量分析 機種:HP59970C イオン化電圧:70eV イオン源温度:260℃ マスフィルタ：双曲線四重極 実施例２ 実施例１で得られた精油の高産生株の新芽を含み葉が２
〜３枚ついた株を、IBA10-6M,BA10-7M,を添加して、３
％ショ糖を加え、更にアンモニア態窒素と硝酸態窒素を
１対５から１対11の比率にし、基本培地の農度が１倍〜
1/2倍であるリンスマイヤー・スクーグ培地（pH5.6）に
投入し、25℃で2000〜3000ルッスクの光照射下で、100
r.p.m.の振とう培養を行った。(Measurement conditions) 1) Gas chromatography Model: HP5890A Gas chromatograph Column: DB-WAX Fused silica gel capillary column (diameter 0.25 mm x 60 m) Detector: FID Injection temperature: 250 ° C Open temperature: 70 ° C to 180 ° C (4 Carrier gas flow rate (He): 0.78 ml / min Split ratio: 1/20 2) Headspace sampler Model: HP19395A Thermal insulation temperature: 75 ° C Thermal insulation time: 90 minutes 3) Mass spectrometry model: HP59970C Ionization Voltage: 70 eV Ion source temperature: 260 ° C. Mass filter: Hyperbolic quadrupole Example 2 The leaves containing the shoots of the high-producing strain of essential oil obtained in Example 1 and 2 leaves
Add 3 to 3 strains by adding IBA10 -6 M, BA10 -7 M,
% Sucrose was added, and the ratio of ammonia nitrogen and nitrate nitrogen was adjusted to 1 to 5 to 1 to 11, and the basal medium had a farming ratio of 1 to 10%.
Add to 1/2 times Rinsemeier-Skoog medium (pH 5.6) and 100 at 100C under light irradiation of 2000-3000 rusks at 25 ℃.
Shaking culture was carried out at rpm.
培養後２日後から、不定芽の茎基部より、新たな不定芽
が出現し、以後その数が増加した。Two days after culture, new adventitious buds appeared from the stem base of adventitious buds, and the number increased thereafter.
10日後の培養結果を実施例２として、表２に示す。表２
より液体培養によって得られた不定芽（実施例２）は、
実施例１と比べて、増殖度が良い。又、栽培によって得
られる植物と同等な組成の精油が得られた事がわかる。The results of culturing after 10 days are shown in Table 2 as Example 2. Table 2
Adventitious buds obtained by more liquid culture (Example 2),
Compared with Example 1, the degree of proliferation is better. Further, it can be seen that an essential oil having the same composition as the plant obtained by cultivation was obtained.
実施例３ 実施例２と同様な高産生株を、実施例２と同様の培地に
投入した。ただし、本実施例においては、振とう培養の
代わりに、細かい泡（ガラスフィルターG2程度）で通気
培養を行った。Example 3 The same high-producing strain as in Example 2 was added to the same medium as in Example 2. However, in this example, aeration culture was performed with fine bubbles (about glass filter G2) instead of shaking culture.
10日培養後の結果を実施例３として第２表に示す。実施
例２と比べて、良好な増殖が得られ、かつ栽培によって
得られる植物と同等な組成の精油が得られたことがわか
る。The results after 10 days of culture are shown in Table 2 as Example 3. It can be seen that, compared with Example 2, good growth was obtained, and an essential oil having a composition similar to that of a plant obtained by cultivation was obtained.
Claims (1)
キシンとしてIBAを10-6M,サイトカイニンとしてBA10-6
Ｍを添加したLS寒天培地にて培養し、培養物から母植物
に近い形態を有する不定芽を分化誘導せしめ、該不定芽
中に産生蓄積された精油成分を採取することを特徴とす
る精油の製造方法。The method according to claim 1] Antemisu genus plant tissue belonging to the IBA as auxin 10 -6 M, a cytokinin BA10 -6
Culturing in an LS agar medium supplemented with M to induce differentiation of adventitious buds having a morphology similar to a mother plant from the culture, and collecting essential oil components produced and accumulated in the adventitious buds. Production method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63053770A JPH0722470B2 (en) | 1988-03-09 | 1988-03-09 | Essential oil manufacturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63053770A JPH0722470B2 (en) | 1988-03-09 | 1988-03-09 | Essential oil manufacturing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01228413A JPH01228413A (en) | 1989-09-12 |
| JPH0722470B2 true JPH0722470B2 (en) | 1995-03-15 |
Family
ID=12952050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63053770A Expired - Lifetime JPH0722470B2 (en) | 1988-03-09 | 1988-03-09 | Essential oil manufacturing method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0722470B2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0637A (en) * | 1992-06-17 | 1994-01-11 | P C C Technol:Kk | Method for collecting highly productive strain by plant tissue culture and method for producing secondary metabolite using the same |
| CN103230352B (en) * | 2011-09-27 | 2014-08-20 | 沈志荣 | Method for preparing plant extract and hydrolyzed pearl-containing toner composition |
| CN103340811B (en) * | 2011-09-27 | 2015-03-25 | 浙江欧诗漫生物股份有限公司 | Lotion composition containing sambucus canadensis extracts, pansy extracts and hydrolyzed pearls |
| CN103239371B (en) * | 2011-09-27 | 2014-07-30 | 沈志荣 | Preparation method of toning lotion composition containing chamomile extract and panax ginseng extract |
| CN103222946B (en) * | 2011-09-27 | 2015-04-15 | 沈志荣 | Preparation method for instantaneous penetration skin regeneration water composition containing American elderberry extract, guava extract and hydrolyzed pearl |
| CN103340810B (en) * | 2011-09-27 | 2015-03-25 | 浙江欧诗漫生物股份有限公司 | Lotion composition containing coconut/seaweed composite extracts, ginseng root extracts and hydrolyzed pearls |
| CN103222943B (en) * | 2011-09-27 | 2015-03-25 | 沈志荣 | Preparation method for instantaneous penetration skin regeneration water composition containing pansy flower extract, guava extract and hydrolyzed pearl |
-
1988
- 1988-03-09 JP JP63053770A patent/JPH0722470B2/en not_active Expired - Lifetime
Non-Patent Citations (2)
| Title |
|---|
| 山田康之編集「植物培養細胞の変異と選抜」１９８５，Ｐ．５７−５９，Ｐ．７４・７７ |
| 萩森学，ＢＩＯＩｎｄｕｇｔｈｙ，ｖｏｌ．２，Ｎｏ．１２，１９８５，Ｐ．５４−６０ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01228413A (en) | 1989-09-12 |
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