Ehime University Institutional Repository

岡部, 雅人

Japanese

温泉施設設計担当者の情報伝達義務 : 渋谷温泉施設爆発事故最高裁決定(最決平成28・5・25LEX/DB25447979)

中原, ゆかり

長野共進会の木曽節 : ふたつの木曽節をめぐって

河村, 泰之

斧, 純司

 
 
 
 
 
 
 
 
 
 
 
Junior High School Programing Education in Ehime Prefecture 
 Practical Report at the Junior High School attached to university  
 
Yasuyuki KAWAMURA 
Junji ONO 
 
 
 
 
 
 
213
愛媛大学教育学部紀要　第63巻　213〜217　2016
   
 
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C, C++  
 
OS 
Ubuntu 14.04 LTS  Remix 
(ubuntu-ja-14.04-desktop-amd64.iso)  
Windows 7 Professional (64 bit) 
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 gcc/g++, python, ruby 1.9, gdb, DDD 
 eclipse-cdt, Geany 
 Gnu Emacs, vim, vim-gnome (gvim), 
gedit, Kate, KWrite 
 C++ STL Document  ( )  
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愛媛県における中学校でのプログラミング教育 : 附属中学校での実践報告

千代田, 憲子

-4  
	 	 	 	 
	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 	 
	 
A Study on Elements which Contribute to Roadside Scape –４  
Educative Means for the Public Design	 
	 
Noriko CHIYODA 
 
１.	 研究の背景と目的	 
( )
 
	 
２.	 研究・調査の方法	 
２.１.ゲームの企画デザイン	 
２.１.１.	 ゲームの種類の選定	 	 	 
２.１.２.	 ゲームの内容とルール	 	 
２.１.３.	 ゲームの構成要素	 
	 
２.２.	 実施	 
	 
285
愛媛大学教育学部紀要　第63巻　285〜293　2016
２.３.	 実施後の検討	 
３.	 研究の結果	 
３.１.	 ゲームの企画デザイン	 
３.１.１.	 ゲームの種類の選定	 
３.１.２.	 ゲームの内容とルール	 
３.１.３.	 ゲームの構成要素	 
(１)タイトル	 
	 
	 
	 
	 
	 
	 
(２)ロゴマークとロゴカラー
	 
(３)キーワードのアイコン制作	 
(４)キャラクターの設定	 
286
-4  
(５)景観カード
図１	 アイコンと景観・効果カードの一覧
287
図２	 景観カード(一部)	 
(６)効果カード
(７)ゲームボード	 
	 (８)コマ	 
(９)ポイントチップ	 
(１０)ルールブック	 
	 
288
-4  
図３	 ボードゲーム	 K-冠グランプリの構成要素	 
	 (上：ゲームボード	 下左より：認定証、サイコロ、コマ、
チップ、ルールブック、景観カード、効果カード)	 
	 	 
(１１)パッケージ	 
３.２.	 実施	 
３.２.１.	 打ち合わせ	 
289
	 
図４	 パッケージ	 
３.２.２.広報と掲示	 
	 
３.２.３.	 認定証と参加賞	 
	 	 
	 
３.２.４.	 実施当日の状況	 
	 
図５	 ゲームの様子	 
	 
表１	 実施状況一覧
290
-4  
３.３.	 実施後の検討	 
３.３.１.	 ゲームの制作について：参加学生の声	 
３.３.２.	 インストラクターについて：参加学生の声	 
４.	 考察と提案	 
４.１.	 ゲームに関して	 
４.１.１.	 ゲームの内容とルール	 
４.１.２.	 ゲームの構成要素	 
291
４.１.３.	 「景観マスター」認定証と参加賞	 
	 
４.２.	 パブリックデザインの学習モデルとしての可能性	 
４.２.１.	 沿道景観に関する学びの導入として	 
 
 
４.２.２.	 インストラクターの介在  
４.３.	 コンセプト形成力の深化	 
	 
４.４.	 今後の課題と展開	 
４.４.１.	 制作環境	 
	 
４.４.２.	 ゲームのカスタマイズや展開
292
-4  
４.４.３.	 パブリックデザインの学習の充実	 
	 
	 
謝辞	 
	 
注	 
	 
参考文献・資料	 
293


沿道景観の貢献要素に関する考察 -4 : パブリックデザインの学習モデル

魁生, 由美子

愛媛県西条市における多様性の学び方 : <地域から学校へ/教室から地域へ>をつなぐ諸活動

不破, 茂

国際訴訟競合の規律再論(下)

青木, 亮人

文学や漫画から見る近現代の遍路

Ehime University (愛媛大学)博士(農学)thesi

三井, 一寿

MITSUI, Kazuhisa

（様式５）(Style5) 
学位論文全文に代わる要約 
Extended Summary in Lieu of Dissertation 
 
 
氏名： 
Name 
三井 一寿 
 
学位論文題目： 
Title of Dissertation 
The in-depth study of aroma compounds formed during tobacco combustion via 
Maillard reaction and/or caramelization 
（シガレット燃焼過程でのメイラード反応およびカラメル化反応による
加熱香気成分生成に関する研究） 
 
学位論文要約： 
Dissertation Summary 
 
The aroma of foods is the most important sensory attribute affecting the acceptance and preference of 
consumers. It is widely accepted that the olfactory perceptions were caused by volatile or semi-volatile 
substances and depends in part on the aroma compounds present in the raw material and in part on how the food 
was prepared (thermally treated). Thermal treatment of foods results in desired and undesired effects which all 
derive from the chemical reactions, namely Maillard reaction, caramelization and to a minor extent, lipid 
oxidation. Through the Maillard reaction, together with caramelization, a large number and various quantities of 
compounds are generated. Several of these formed products play an important role in the aroma and taste of 
foods and consumer products. However, it’s a complicated process which consists of many reactions and still it 
has not been clarified completely. Therefore the efforts for its understanding and control are of utmost importance 
to support the development of food industries. Beside the relevance in the applicative fields of food science, a 
deep understanding of the reaction mechanisms are essential for fundamental biological issue such as those 
related to the ingestion of these products. 
This dissertation demonstrated some of the investigation designed to lead to an increased understanding 
of the chemistry of the Maillard reaction and caramelization for real-life samples, based on recent analytical 
developments. The novel analytical techniques was developed and applied to precursor screening, intermediate 
monitoring and trace level of aroma compounds detection, formed during tobacco combustion as one of the 
highly complex real-life sample, to clarify the influence on cigarette smoke aroma properties. 
In chapter 1, the Maillard reaction and caramelization mechanism for the aroma compounds formed by 
thermal treatment of raw food and the practical case in daily food processing were summarized. In view of the 
large numbers of stages and the sensitivity of each stage in the reaction to changes in the conditions, it is very 
complicated to investigate and control the whole Maillard reaction and caramelization. The extent of, and the 
pathway for, change are controlled by many factors. These factors could be distinguished as those relating to the 
reactant composition, e.g., sugar type, type of amino acid, sugar and amino acid ratio and external factors such as 
moisture level, temperature, pH and so on. Additionally, traditional gas chromatography–mass spectrometry 
(GC–MS) and related novel analytical techniques, multi-dimensional GC, analytical pyrolysis and modern omics 
approach, of volatile and semi-volatile aroma compounds were reviewed. 
In chapter 2, analytical method for the aroma precursor compounds affecting the cigarette smoke aroma 
formed during tobacco combustion was discussed. Analytical pyrolysis combined with GC–MS (Py-GC–MS) is 
an effective technique for studying thermally treated natural products and processes like cigarette smoking. Direct 
pyrolysis of tobacco leaf results in a very complex mixture of organic compounds containing volatile, 
semi-volatile and non-volatile compounds. Consequently, detecting and identifying the main cigarette smoke 
aroma compounds and their formation pathways are very difficult. 
（様式５）(Style5) 
In this study, an innovative workflow that can elucidate the aroma precursor compounds in tobacco leaf 
was presented using a recently developed resistive heating pyrolysis device, namely a modified thermal 
desorption system (TDU-Pyro, Gerstel GmbH, Mülheim an der Ruhr, Germany). The workflow consisted of LC 
fractionation of a tobacco leaf extract, recombination of fractions in a fraction omission approach, Py-GC–MS 
and multivariate chemometric data analyses. Based on previous studies, an aqueous tobacco extraction was 
selected to demonstrate the importance of polar constituents such as carbohydrates and/or amino acids on the 
formation of Maillard reaction and caramelization aroma compounds. In the first step, the aqueous tobacco leaf 
extract was fractionated using semi-preparative liquid chromatography (prep-LC). In the next step, the LC 
fractions were recombined in an omission approach. In this approach, a series of “pooled” extracts was obtained, 
and in each extract an LC fraction was omitted. In that way, certain tobacco leaf constituents are eliminated from 
the “pooled” extracts. All extracts were then concentrated and analyzed by Py-GC–MS using a resistive heating 
pyrolysis system installed on a thermal desorption unit. The obtained pyrograms were statistically evaluated and 
compared with a whole extract (all LC-fractions present) using a multivariate chemometric approach and the key 
LC-fraction was identified that contribute mostly to the formation of typical cigarette smoke aroma. Chemical 
information from the samples was obtained by analysis of both volatile and non-volatile compounds, followed by 
processing of chromatograms and reduction in the number of variables using specialized software. Chemometric 
analysis can rapidly classify and differentiate significant fractions for further analysis. In addition, GC–MS 
analysis of derivatized LC-fraction was performed to identify the precursors of these aroma compounds and the 
main constituents were identified as aspartic acid, proline, malic acid and sugars (fructose, glucose and sucrose). 
These are natural constituents in tobacco leaves and their degradations through Maillard-type reactions contribute 
significantly to the aroma character of cigarette smoke. The analytical approach was suitable to correlate the 
effect of different classes of compounds in tobacco leaf to the typical aroma compounds in cigarette smoke. The 
LC-fraction has the greatest effect on the composition of the volatile fraction of the pyrolysate of an aqueous 
extract of tobacco leaf. This evidence confirms that the sugars and amino acids detected in that fraction play a 
major role in the aroma of cigarette smoke. A huge number of amino acids and sugars through Maillard reactions 
contribute to tobacco leaf and cigarette smoke aroma. Therefore, the described analytical pyrolysis systems will 
provide an advanced technique for understanding these Maillard reaction pathways. [1] 
For more in-depth study, then, analytical method was modified to analyze intermediate compounds in 
chapter 3. Typically, pyrolysis products are analyzed using Py-GC–MS. However, some products, and especially 
intermediate compounds, are too polar and thermally unstable to be analyzed directly using GC–MS. Therefore, 
various derivatization methods are applied in combination with Py-GC–MS. On the other hand, LC–MS can be 
also applied to analyze polar, non-volatile fractions of pyrolysates, but the combination of pyrolysis with LC–MS 
is much less documented compared with Py-GC–MS. 
In this study, a recently developed resistive heating pyrolysis devices, namely a generic pyrolysis unit 
(PyroVial, Research Institute for Chromatography, Kortrijk, Belgium) was applied. The PyroVial system is an 
off-line pyrolysis system based on a modified 2 mL autosampler vial. In this system, the sample is placed in a 
bottom-tapered quartz insert vial that is inserted in a heating filament, mounted inside the 2 mL vial. After 
pyrolysis, several options are possible: the headspace gas can be injected in GC, solvent can be added and the 
dissolved fraction can be analysed by GC or LC and/or derivatization reagent can be added for post-pyrolysis 
derivatization of the pyrolysate. Firstly, a mixture of glucose and proline was pyrolysed as model. These two 
compounds were selected because they represent, respectively, an abundant sugar and amino acid content present 
in tobacco leaf. The volatile fractions of the pyrolysates were analyzed using headspace-GC–MS, semi-volatile 
using liquid injection GC–MS and non-volatiles using LC–MS. In addition, a tobacco leaf extract was analyzed 
by Py-LC–MS, whereby the micro-vial pyrolysis simulates the tobacco leaf combustion process. Using MS 
deconvolution, molecular feature extraction and differential analysis it was possible to identify Amadori 
（様式５）(Style5) 
intermediates of the Maillard reaction in the tobacco leaf extract. The intermediate disappeared was the case for 
1-deoxy-1-prolino-β-D-fructose or the concentration decreased in the pyrolysate compared to the original extract 
such as for the 1-deoxy-1-[2-(3-pyridyl)-1-pyrrolidinyl]-β-D-fructose isomers, indicating that Amadori 
intermediates are important precursors for aroma compound formation. 
1-deoxy-1-[2-(3-pyridyl)-1-pyrrolidinyl]-β-D-fructose isomers were identified by LC–Q-TOF-MS analysis in 
MS/MS mode to determine the possible chemical structures, and using Molecular Structure Correlator (MSC) 
software. The identified compounds correspond to Amadori rearrangement intermediates, previously reported 
and naturally present in tobacco leaf and the two isomers were the fructopyranose (major) and fructofuranose 
(minor) isomers, respectively. Pyrolysis at 500°C led to the formation of pleasant and sweet aromas. 
1-deoxy-1-[2-(3-pyridyl)-1-pyrrolidinyl]-β-D-fructose is the Amadori product from glucose and nornicotine, a 
secondary alkaloid present in Nicotiana tobacum L. The novel vial-based resistive heating pyrolysis device, 
PyroVial, was successfully applied to study Maillard reactions and to identify Amadori intermediates. The 
obtained information is very useful to further elucidate the formation of aroma compounds during thermal 
treatment or pyrolysis of natural products. [2] 
Finally, advanced comprehensive analytical systems applied to detect a trace level of sulfuric aroma 
compounds in cigarette smoke in chapter 4. Volatile sulfur compounds in food and beverage have received 
special attentions due to their extremely low odor threshold levels and high sensory impact, but these compounds 
are mostly often present at very low levels in complex matrices. Sulfur compounds which are derived from 
Maillard reaction of sugar and amino acids degradation products also play an important role in the aroma of 
cigarette smoke. However, the number of identified sulfur compounds in cigarette smoke is still limited due to 
one of the most complex sample matrices. To analyze volatile sulfur compounds in cigarette smoke, it is therefore 
essential to have advanced GC separation technique and instrumentation. An effective way to improve the 
chromatographic resolution and identification capability is through multi-dimensional GC coupled to a hybrid 
quadrupole time-of-flight mass spectrometer (MDGC–Q-TOF-MS) with element-specific detection. 
In this study, a large volume injection (LVI), 10 μL of cigarette smoke extract, followed by selectable 
1
D/
2
D GC–Q-TOF-MS that is possible to select one-dimensional (
1
D) GC separation or heart-cutting 
two-dimensional (
2
D) GC separation without any additional instrumental set-up, with parallel sulfur 
chemiluminescence detection (SCD) was applied for identification of trace sulfur compounds in cigarette smoke. 
The selectable 
1
D/
2
D GC–SCD/Q-TOF-MS system has also the capability to integrate parallel olfactometry in 
both 
1
D and 
2
D separations. To unravel the complexity and identify important sulfur compounds, 28 sulfur 
fractions selected from 
1
D GC–SCD on a non-polar pre-column are sequentially transferred onto a polar 
main-column and then further separated, detected and identified using 
2
D GC–SCD/Q-TOF-MS. In order to 
identify each individual sulfur compound, sequential heart-cuts of 28 sulfur fractions from 
1
D GC to 
2
D GC were 
performed with the three MS detection modes (SCD/EI-TOF-MS, SCD/PCI-TOF-MS, and 
SCD/PCI-Q-TOF-MS). Thirty sulfur compounds were positively identified by MS library search, 
1
D/
2
D linear 
retention indices (LRI), elemental information (sulfur), molecular mass determination using positive chemical 
ionization (PCI) accurate mass spectra, formula calculation using electron ionization (EI) and PCI accurate mass 
spectra, and structure elucidation using collision activated dissociation (CAD) of the protonated molecule. 
Additionally, the best candidate molecular formulas could be obtained for 11 unknown sulfur compounds. Also, 
sulfur compounds are quantified with the use of the linear and equimolar response of the 
2
D GC–SCD to sulfur 
compounds. The determined values of the identified and unknown sulfur compounds were in the range of 
10–740 ng mg total particulate matter (TPM)
-1
 (RSD: 1.2–12%, n = 3). Forty-one sulfur compounds could thus 
be determined at ng mg TPM
−1
 levels. Of these sulfur compounds, 13 compounds have not previously been 
reported in cigarette smoke. [3] 
（様式５）(Style5) 
Through the efforts of this dissertation, innovative analytical work flows designed to lead to an increased 
understanding of the chemistry of the Maillard reaction and caramelization using real-life samples could be 
demonstrated. In tobacco science, but also in other fields such as food technology and biological application, 
sophisticated GC–MS and LC–MS techniques combined with computation analyses for aroma precursors 
screening, intermediates monitoring, trace level of aroma compounds detection generated by thermal processing 
and elucidating their formation pathways that has been developed in this dissertation are widely applicable, 
important and required for advanced research. 
 
 
 
 
 
[1] Kazuhisa Mitsui, Frank David, Emmie Dumont, Nobuo Ochiai, Hirotoshi Tamura, Pat Sandra. 
LC fractionation followed by pyrolysis GC–MS for the in-depth study of aroma compounds formed 
during tobacco combustion. Journal of Analytical and Applied Pyrolysis, 116:68–74. (2015) 
[2] Kazuhisa Mitsui, Frank David, Bart Tienpont, Koen Sandra, Nobuo Ochiai, Hirotoshi Tamura, 
Pat Sandra. Analysis of the reaction products from micro-vial pyrolysis of the mixture 
glucose/proline and of a tobacco leaf extract: Search for Amadori intermediates. Journal of 
Chromatography A, 1422:27–33. (2015) 
[3] Nobuo Ochiai, Kazuhisa Mitsui, Kikuo Sasamoto, Yuta Yoshimura, Frank David, Pat Sandra. 
Multidimensional gas chromatography in combination with accurate mass, tandem mass 
spectrometry, and element-specific detection for identification of sulfur compounds in tobacco 
smoke. Journal of Chromatography A, 1358:240–251. (2014) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
(注) 要約の文量は，学位論文の文量の約１０分の１として下さい。図表や写真を含めても構いません。
(Note)  The Summary should be about 10％ of the entire dissertation and may include illustrations 


English

The in-depth study of aroma compounds formed during tobacco combustion via Maillard reaction and/or caramelization

Ehime University (愛媛大学)博士(学術)thesi

Wilma, Joanna Carolina Moka

 
 
（第５号様式）  
                        学位論文審査の結果の要旨 
 
    氏    名 
Wilma Joanna Carolina Moka 
審 査 委 員  
         論  文  名 
Genetic study of hatchery cultured small type rotifer, Brachionus plicatilis species complex 
（種苗施設で生産された S型シオミズツボワムシの遺伝的研究） 
 
審査結果の要旨 
 輪形動物門のシオミズツボワムシは海水魚類養殖における種苗の初期餌料生物として重要な動物プラ
ンクトンであるが、隠蔽種を含むとともに多くの株が存在し、増殖特性はそれぞれ異なるものと考えら
れる。しかし、これらの株は配布業者や種苗生産場間で譲渡がなされており、それぞれの種苗生産場で
どのような株が使われているのか明らかではない。また、シオミズツボワムシの培養中に増殖率が急激
に減少し、種苗の餌料が不足することが問題となっており、それぞれの種苗生産場のシオミズツボワム
シ株を明らかにすることが求められる。 
 本論文では、国内において多く用いられる S 型シオミズツボワムシ Brachionus plicatilis sp. 
complex 株の遺伝的識別を行うため、ミトコンドリア DNA の COI 領域を用いた株判別技術の最適化を行
い、国内の種苗生産場で用いられている株について明らかにした。また、マイクロサテライト DNA マー
カーを開発し、COI 領域とマイクロサテライトマーカーにより、より詳細に株判別を行い、大量培養に
おけるクローンの多様性と増殖率との関連を明らかにした。さらに、種苗生産場から流出したワムシが
当該環境へ定着する可能性があることを示した。 
 
以下、論文を構成するテーマごとに、その内容と評価できる点を整理し、審査結果を記述する。 
 
１．ミトコンドリア COI 領域を用いた S型シオミズツボワムシ株判別技術の最適化 
S 型シオミズツボワムシの効率的な DNA 抽出法とミトコンドリア DNA の COI 領域を用いた株判別技術
を開発した。DNA 抽出は S 型ワムシを直接、凍結または粉砕してからキレート樹脂に浸漬する方法につ
いて比較したところ、粉砕および凍結法により抽出された DNA の増幅効率が高かった。また、S 型ワム
シに適合するミトコンドリア DNA の COI 領域増幅用プライマーを設計し、PCR における増幅効率をユニ
バーサルプライマーと比較したところ、S 型ワムシに適合するプライマーによる増幅効率が顕著に高か
った。種苗生産で用いられている S 型ワムシ株の 1 ロットについて PCR-RFLP 法とシークエンスを行っ
たところ、Cayman 株であることが判明し、本法が今後の S 型ワムシ株判別に有効であることが示された。
また、本法は他の S型ワムシ株や L型ワムシにも応用可能であることが示された。 
 
 
 
２．わが国の種苗生産場で用いられている S 型ワムシ（Brachionus plicatilis sp. complex）の mtDNA 
COI 領域を用いた株型解析と遺伝的集団構造解析 
わが国の種苗生産場で使用されているシオミズツボワムシ Brachionus plicatilis sp. complex（以
下、S 型ワムシ）は DNA 系統解析を元にした株型判別が行われていないため、どのような S 型ワムシ系
統が各種苗生産場で用いられているのかは明らかとなっていない。本研究では、ミトコンドリア DNA の
COI 領域を用いた株型解析を行い、わが国で流通している S 型ワムシの株型を判別するとともに、ハプ
ロタイプの出現頻度から各株の遺伝的集団構造を明らかにした。解析に用いた S 型ワムシ 11 株からは 5
つのハプロタイプが得られ、系統解析の結果、2 つの“Cayman”系統と 1 つの名称不詳の系統に識別さ
れた。ハプロタイプの出現頻度から各株の遺伝的集団構造を調べたところ、4 株については単一のハプ
ロタイプから構成されていたが、残りの 7 株については複数のハプロタイプが混在した状態であった。
また、複数のハプロタイプが混在していた場合では、特定の系統が優占した状態となっていた。 
 
３．S型シオミズツボワムシの大量培養におけるクローンの多様性と増殖率との関連 
 本研究では S 型シオミズツボワムシ用のマイクロサテライトマーカーを開発し、ミトコンドリア DNA
の COI 領域と開発したマイクロサテライト DNA マーカーを用いて S 型ワムシのクローンを識別し、クロ
ーンの多様性の程度が大量培養における増殖率に与える影響について調べた。実験に用いた大量培養株
は３種の COI ハプロタイプと５種のマイクロサテライトジェノタイプから８種の遺伝型に識別された。
S 型シオミズツボワムシの大量培養においてバッチごとの遺伝的多様度と増殖率において相関がみられ
た。また、いくつかのクローンは、ハプロタイプとジェノタイプを共有しており、S 型シオミズツボワ
ムシの大量培養において有性生殖が生じていることが示唆された。 
 
４．天然海域における種苗生産場から流出した S型シオミズツボワムシの探索 
海産魚の種苗生産が始まって以来、初期餌料として種苗生産場で培養されてきたシオミズツボワムシ
（Brachionus plicatilis sp. complex）は飼育水と共に大量に海へ流出していると考えられるが、流
出したワムシが天然海域のワムシに与える遺伝的影響については考慮されてこなかった。本研究では、
種苗生産場の周辺海域で 1 年間にわたりシオミズツボワムシを採集し、ミトコンドリア DNA COI 領域の
部分配列を解析することで種苗生産場に由来するものかを調べた。種苗生産場の排水溝周辺では多数の
シオミズツボワムシが見られ、種苗生産場から流出したものだと考えられた。また、他の採集地点では
4 回のサンプリングで数個体のシオミズツボワムシが採集された。水温と塩分濃度を測定したところ、
採集地点の 1 つは種苗生産場の環境に類似しており、シオミズツボワムシの増殖並びに再生産が可能で
あることが示唆された。以上の結果から、種苗生産場から流出したワムシが生育に適した時期に、適し
た環境へと漂着した場合、当該環境へ定着する可能性があると考えられた。 
 
以上のように本論文はわが国で流通・培養されている S 型シオミズツボワムシの株型を判別し、複数
のハプロタイプが混在した状態であることを明らかにするとともに大量培養におけるクローンの多様性
と増殖率との関係について検討した。また、種苗生産場から流出したワムシが生育に適した時期に、適
した環境へと漂着した場合、当該環境へ定着する可能性を示した。このような実証的研究は、種苗生産
の現場との協力がなければ成り立たず、得られたデータは貴重である。また、本論文が提示したクロー
ンの多様性と増殖率に関する考察は、今後の当該分野の研究と生産に寄与すると考えられる。 
 
本論文に関する公開審査会は、平成２８年８月６日に香川大学農学部において開催され、論文発表と
質疑応答が行われた。引き続いて開催された学位審査委員会で本論文を慎重に審査し、審査委員全員一
致して博士（学術）の学位を授与するに値するものと判定した。 
 
 


Genetic study of hatchery cultured small type rotifer, Brachionus plicatilis species complex

Md., Sirajul Islam Khan

（第３号様式）(Form No. 3) 
 1 
学  位  論  文  要  旨  
Dissertation Abstract 
 
 
氏名：  
Name 
 
Md. Sirajul Islam Khan 
 
学位論文題目：  
Title of Dissertation 
 
Allelopathic Potential and Allelopathic Substances from Leaves 
of Three Tree Species   
(3 種の樹木の葉のアレロパシー活性及びアレロパシー物質 ) 
 
 
学位論文要旨：  
Dissertation Abstract 
 
 
 
 
Litchi (Litchi chinensis Sonn.), cannon-ball (Couroupita guianensis Aubl.) and mango 
(Mangifera indica L.) have been drawn the attention to many researchers because of 
their myriad of pharmacological activities. However, a few reports on the allelopathic 
properties of these species have been studied. Therefore, this research aimed to explore 
the allelopathic potential and allelopathic active substances in leaves of these species.  
    Seed germination of all test plants were significantly inhibited by the extracts 
obtained from litchi, cannon-ball and mango leaves at concentrations ≥0.01 g dry weight 
(DW) equivalent extract mL-1. The extracts of litchi and cannon-ball leaves completely 
inhibited germination of lettuce and significantly delayed germination of barnyard grass 
at the concentration of 0.1 g DW equivalent extract mL-1. At the same concentration, 
extracts obtained from three mango cultivars (Khirshapat, Himsagor and Sinduri) 
showed a significant delayed or/and complete inhibition of germination of all test plants 
except for Sinduri extracts on barnyard grass.  
     The hypocotyl/coleoptile and root growth of all test plants were significantly 
inhibited by extracts obtained from three species at concentrations ≥0.01 g DW 
equivalent extract mL-1. The inhibitory activities were dependent on test plants and 
extracts concentration. The extracts obtained from litchi leaves completely inhibited 
hypocotyl growth of cress, lettuce and alfalfa, and root growth of all test plants, while 
87, 90 and 62% coleoptile growth inhibition was found on timothy, Italian ryegrass and 
barnyard grass, respectively at the concentration of 0.1 g DW equivalent extract mL-1. 
At the same concentration, the hypocotyl/coleoptile and root growth of all test plants 
were inhibited by leaf extracts of cannon-ball to more than 90%, except for 
hypocotyl/coleoptile growth of alfalfa and barnyard grass. On the other hand, three 
mango extracts showed more than 70% hypocotyl/coleoptile and root growth inhibition 
of all test plants except barnyard grass at the concentration of 0.1 g DW equivalent 
（第３号様式）(Form No. 3) 
 2 
extract mL-1. The concentration required for 50% growth inhibition (I50) for 
hypocotyl/coleoptile and root growth of all test plants ranged from 0.002-0.079, 
0.001-0.010 and 0.003-0.103 g DW equivalent extract mL-1 for litchi, cannon-ball and 
mango, respectively.  
         As no significant differences among three mango cultivars were found for 
their inhibitory activities on germination and growth of hypocotyl/coleoptile as well as 
root of all test plants, all of them could be candidates for isolation and identification 
of allelopathic substances. Therefore, further isolation and identification of these 
substances from the extracts of Khirshapat leaves was first proceeded. The extracts of 
Khirshapat leaves was purified by several chromatographic separations through 
bioassay-directed fractionation, and an allelopathically active substance was identified 
by spectral data as methyl-3,4,5-trihydroxybenzoate (methyl gallate). The inhibitory 
effects on seedling growth of garden cress and foxtail fescue were observed at 
concentrations ≥1 mM of methyl gallate. The I50 values of methyl gallate on garden 
cress root and hypocotyl were 4.9 and 3.4 mM, respectively, whereas those on foxtail 
fescue root and coleoptile were 1.4 and 10.1 mM, respectively. Comparing I50 values, 
effectiveness of methyl gallate on hypocotyl was 1.4-fold greater than that of root of 
garden cress and on root was 7.2-fold greater than that of coleoptile of foxtail fescue, 
respectively. These results indicate that methyl gallate may contribute an important role 
in allelopathic activities of mango.  
         Leaves of litchi, cannon-ball and mango inhibited seed germination and 
seedling growth of different test plants including weeds. These results suggest that these 
three tree species have allelopathic potential and may possess allelopathic active 
substances. Therefore, these three species could be candidates for isolation and 
identification of those active substances to serve as weed inhibiting agents for 
sustainable crop production. An allelopathic substance was found as methyl gallate in 
mango leaves and the substance showed inhibitory activities on test plants at 
concentration dependent manner. However, further isolation and identification of 
allelopathic active substances from leaves of litchi and cannon-ball species should be 
investigated for assessing their effects on weeds.  
 
 
 
 
 
 
 


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