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熊本大学学術リポジトリ

Kumamoto University Repository System

Title Study on Tomato-Saponin and Onion Sulfide :

Biotechnological Production of Natural Products

Author(s) El-Aasr, Mona Abdel-Hamid Mohamed

Citation

Issue date 2010-09-25

Type Thesis or Dissertation

URL http://hdl.handle.net/2298/22162

Right

2010

Study on Tomato-Saponin and Onion Sulfide

(Biotechnological Production of Natural Products)

Kumamoto University

Graduate School of Pharmaceutical Sciences

Doctoral course, Drug Development, Medicinal Chemistry,

Department of Natural Medicines

Mona Abdel-Hamid Mohamed El-Aasr

Study on Tomato-Saponin and Onion Sulfide (Biotechnological Production of Natural Products)

Graduate School of Pharmaceutical Sciences, Doctoral course, Drug Development,

Medicinal Chemistry, Department of Natural Medicines


Belief in the medicinal power of foods is not a recent event but has been a widely

accepted philosophy for generations. Hippocrates, the father of medicine stated almost

2,500 years ago, ‘‘Let food be thy medicine and medicine be thy food’’. Today, the

belief in the health benefits of selected foods and their components appears to

increase. The consumers are aware of the fact that a healthy diet is important and

necessary for improving human health. An increasing number of scientific studies is

supporting the knowledge that food is an important factor for preventing many of the

chronic disorders and diseases. I have been interested in vegetables which are

expected to have many biological activities. Among them, I have focused on the

sciences of tomato (Solanum lycopersicum L.) and onion (Allium cepa L.).

Regarding to tomato, I have clarified a chemical interrelation between esculeoside A

and esculeogenin B, which is a rare naturally occurring substance. A chemical

conversion of spirosolane skeleton-type, esculeoside A, into solanocapsine-type

skeleton, esculeogenin B, has successfully been attained by acid hydrolysis using 2 N

HCl in a solution of dioxane and water (1:1) to yield two kinds of esculeogenin B. Its

mechanism of conversion has been deduced. Now it has become possible to prepare

esculeogenin B for animal experiments.

O

O

N

GalGlcGlc

Xyl

3

2 4

OGlc

OAc

16

H

H

2 N HCl in 50% dioxane

O

HN

O

HN

CH2OH

OH

H

CH2OH

HOHO H H

H

OH

+

Esculeogenin B-2Esculeogenin B-1

3

Acid Treatment of Esculeoside A (1) with 2 N HCl in Dioxane and Water (1:1)

Next, I have determined the content variations of tomato-saponin, esculeoside A in the

fresh tomato, tomato boiled in water, tomato heated using a microwave oven, freeze-

dried tomato, and commercially available tomato products contained in plastic bottles

and cans in order to develop a health food. The yields of the tomato-saponin,

esculeoside A, in the mini and middy tomatoes were approximately four times that of

lycopene. The yields of mini and middy tomatoes were thus three times that of

Momotaro tomato. The tomato-saponin was not decomposed or changed upon heating

or upon heating under far-infrared light or using a microwave oven. In commercial

juices and cans, tomato-saponin could not be found.

We have developed a tomato-health food with co-operation of a company (N.D.R.

Co., Ltd.) and are trying to apply for persons suffering from high blood pressure, high

blood sugar level, and atopic dermatitis to accumulate the data for future use.

Meanwhile, onion is the most widely used Allium. It is a rich source of organosulfur

compounds, which are mainly responsible for many biological activities. I have

isolated a compound onionin A from the acetone extracts of bulbs of onion. It is a

novel, stable, sulfur-containing compound, and its structure has been characterized as

3,4-dimethyl-5-(1Z-propenyl)-tetrahydrothiophen-2-sulfoxide-S-oxide. The

biosynthetic pathway for production of onionin A could be estimated.

S+

H3C

H

H3C

H

S+

H O-

H

C C CH3

HH

O-

H

Onionin A

Then, we have examined the inhibitory effect of onionin A on CD163 expression by

cell-ELISA. We have found that onionin A significantly inhibited CD163 expression;

this finding suggests that onionin A has a potential to suppress tumor cell proliferation

by inhibition of M2 macrophage polarization.

Data are presented as the mean ± SD. * p < 0.001 vs. control

Effect of Onionin A on CD163 Expression

For exhibiting maximum biological activities from tomato, tomato should be used as

a fresh juice homogenized with water or freeze-dried. On the other hand, onion should

be taken in raw (uncooked) since onion contains beneficial sulfur compounds which

are destroyed by cooking.

* *

0

0.5

1

1.5

2

2.5

Non load Control 0.3 µM

Onionin A

1 µM

Onionin A

3 µM

Onionin A

10 µM

Onionin A

30 µM

Onionin A

Ab

sorb

ance

at

45

0 n

m

トマトサポニンならびにタマネギのトマトサポニンならびにタマネギのトマトサポニンならびにタマネギのトマトサポニンならびにタマネギの硫化物硫化物硫化物硫化物にににに関関関関するするするする研究研究研究研究

（（（（天然物天然物天然物天然物のののの生物工学的生産生物工学的生産生物工学的生産生物工学的生産））））

分子機能薬学専攻

創薬化学講座（天然薬物学分野）


古代のエーベルス・パピルス、デ・マテリア・メデイカ、アユルベーダ、漢方薬等の根源

的底流には「医食同源」という考えがあり、最近、日本の薬学分野における「食品薬学シン

ポジウム」の学会の設立も、食と医薬との関連性に強い関心が寄せられていることを示して

いる。そこで私は野菜機能性成分が見出されたなす科植物由来のトマト、並びにタマネギの

成分に関する研究を行い以下の結果を得た。

I.トマトサポニントマトサポニントマトサポニントマトサポニン

(1) Esculeoside A からからからから Esculeogenin B へのへのへのへの化学的変換化学的変換化学的変換化学的変換：：：：2003 年、野原らは本邦産、ならびに

イタリア産の成熟トマト(Solanum lycopersicum L.)より spirosolane-type ならび solanocapsine-

type のトマトサポニンを初めて得、それらを esculeoside A および B と命名した。 Esculeoside

A の sapogenol である esculeogenin A は apo 欠損マウスにて、抗動脈硬化作用を有することが

明らかにされた。一方、solanocapsine-type は天然では希少であることから、動物実験等を進

めるには天然に豊富に存在する入手豊富な spirosolane から solanocapsine-type への化学的変換

が必要である。そこで私は酸加水分解反応条件を種々検討し、2 N HCl-dioxane で変換に成功

した。

O

O

N

GalGlcGlc

Xyl

3

2 4

OGlc

OAc

H

H

Esculeoside A

O

HN

O

HN

CH2OH

OH

H

CH2OH

HOHO H H

H

OH

+


(2) 生食用生食用生食用生食用トマトのトマトおよびトマトのトマトおよびトマトのトマトおよびトマトのトマトおよび加工食品中加工食品中加工食品中加工食品中のトマトサポニンのトマトサポニンのトマトサポニンのトマトサポニン（（（（Esculeoside A））））のののの含量含量含量含量：：：：これ

までの野原らのなす科植物成分研究より、トマトサポニンは種々の生理作用を持つことが明

らかとなった。そこでトマト粉末の健康食品を開発する為に、桃太郎トマト、ミニトマト、

ミデイトマト、ならびに、多くの加工食品について esculeoside A の含量を測定した。その結

果、esculeoside A は成熟トマトの圧倒的主成分 (0.043%)で、熱に対しても安定である。しか

しながら、市販のジュースや缶詰には殆ど含まれないことが判明した。

* *

II. タマネギのタマネギのタマネギのタマネギの硫化物硫化物硫化物硫化物

(1) 新規新規新規新規 Sulfide のののの単離単離単離単離とととと化学構造化学構造化学構造化学構造：一般にタマネギ (Allium cepa L.) は、抗腫瘍、抗炎症、抗

免疫、抗酸化、抗糖尿病、抗微生物、強心作用等を有すると言われる。ニンニクと同様、こ

れまでにタマネギ全般については数多くの報告があるが、しかしながら化合物レベルでは核

心に至った論文は少ない。これまでに報告された sulfide は殆ど二次的生成物であり不安定と

見られる。私は健康食品にも応用出来るような、生物活性を有し、かつ安定な sulfide の単離

に着手し、onionin A と命名する新規 sulfide を得た。本化学構造は NMR (FGCOSY, HMQC,

HMBC, NOESY) 等により、3,4-dimethyl-5-(1Z-propenyl)-tetrahydrothiophen-2-sulfoxide-S-oxide

と決定した。Tetrahydrothiophen 骨格を有する特異な構造で非常に注目される。(+)-S-propenyl-

L-cysteine-S-oxide から propenylsulfenic acid を経由して生合成すると推定している。

S+

H3C

H

H3C

H

S+

H O-

H

C C CH3

HH

O-

H

Onionin A

Effect of Onionin A on CD163 Expression

(2) マクロファージマクロファージマクロファージマクロファージ活性化制御作用活性化制御作用活性化制御作用活性化制御作用：：：：タマネギより単離した新規化合物 onionin A の癌免疫賦

活作用を検討した。方法としては、onionin A のマクロファージ活性化に対する効果を M2 マ

クロファージマーカーである CD163 を指標に Cell- ELISA 法を用いて評価した。その結果、

onionin A は、M2 マクロファージマーカーである CD163 の発現を抑制したことから、マクロ

ファージの活性化を制御することで腫瘍の増殖を抑制する可能性が示唆された。

0

0.5

1

1.5

2

2.5


Onionin A

1 µM

Onionin A

3 µM

Onionin A

10 µM

Onionin A

30 µM

Onionin A

Abso

rbance a

t 450 n

m

Preface

This thesis is the outcome of my three years research as a doctoral course student at

the Graduate School of Pharmaceutical Sciences, Kumamoto University, Drug

Development, Medicinal Chemistry, Department of Natural Medicines.

It is based on the following publications:

1. Conversion of Esculeoside A into Esculeogenin B.

Mona El-Aasr,Yukino Oshiro,Yukio Fujiwara, Hiroyuki Miyashita, Tsuyoshi

Ikeda, Masateru Ono, Hitoshi Yoshimitsu, and Toshihiro Nohara

Chem. Pharm. Bull., 56 (7), 926–929 (2008).

2. Content Variations of Tomato-Saponin, Esculeoside A, in Various Processed

Tomatoes.

Hideyuki Manabe, Yoshihiro Murakami, Mona El-Aasr, Tsuyoshi Ikeda, Yukio

Fujiwara, Masateru Ono, and

Toshihiro Nohara

J. Nat. Med. (MS No. 10066, in press).

3. Onionin A from Allium cepa Inhibits Macrophage Activation.

Mona El-Aasr, Yukio Fujiwara, Motohiro Takeya, Tsuyoshi Ikeda, Sachiko

Tsukamoto, Masateru Ono, Daisuke Nakano, Masafumi Okawa,

Junei Kinjo, Hitoshi Yoshimitsu, and Toshihiro Nohara

J. Nat. Prod., 73, 1306–1308 (2010).

Abbreviations

TLC thin layer chromatography

HR high resolution

FAB-MS

EI-MS

fast atom bombardment mass spectroscopy

electron ionization mass spectroscopy

IR infra-red 1H-NMR

13C-NMR

proton-1 nuclear magnetic resonance

carbon-13 nuclear magnetic resonance

COSY proton proton correlation spectroscopy

HMQC Heteronuclear multiple quantum correlation

HMBC

NOESY

ApoE

MФ

heteronuclear multiple bond correlation

nuclear Overhauser effect spectroscopy

Apolipoprotein E

macrophage

CD163

IL-3

cluster of differentiation 163

interleukin-3

IL-10

IL-13

PFA

AM3K

HRP-conjugated antibodies

IgG

GM-CSF

LPS

interleukin-10

interleukin-13

fixing cells with paraformaldehyde

anti-macrophage surface antigen antibody

horseradish peroxidase conjugated antibodies

immunoglobulin G

granulocyte macrophage colony-stimulating

factor receptor

lipopolysaccharides

IFNγ

M-CSF

interferon-gamma

macrophage colony stimulating factor

PGE

PBS

Tween 80

prostaglandin E

phosphate-buffered saline

polyoxyethylene (20) sorbitan monooleate

CONTENTS

Introduction ----------------------------------------------------------------------------- 1

Main Issue-------------------------------------------------------------------------------- 11

I. Tomato-Saponin---------------------------------------------------------------------- 11

1. Chemical Conversion of Esculeoside A into Esculeogenin B---------------- 11

(1) Acid Treatment of Esculeoside A (1) with 2 N HCl ------------------------- 12

(2) Acid Treatment of Esculeoside A (1) with 2 N HCl-MeOH ---------------- 15

(3) Acid Treatment of Esculeoside A (1) with 3 N H2SO4-MeOH ------------ 18

(4) Acid Treatment of Esculeoside A (1) with 2 N HCl in Dioxane and

Water (1:1) ------------------------------------------------------------------------ 19

2. Content Variations of Tomato-Saponin, Esculeoside A, in Various

Processed Tomatoes ----------------------------------------------------------------- 24

3. Development of Tomato Health Food ------------------------------------------- 36

4.Treatment Trials of Tomato Product for Some Volunteers ----------------- 37

5. Consideration of The Efficacy of Steroidal Glycosides --------------------- 43

II. Onion-Sulfide ------------------------------------------------------------------------- 46

1. Extraction and Isolation ----------------------------------------------------------- 47

2. Chemical Structure ----------------------------------------------------------------- 49

3. Configuration of Sulfoxide -------------------------------------------------------- 60

4. Plausible Biosynthetic Pathway for Production of Onionin A ------------ 63

5. Effect of Onionin A on Macrophage Activation ------------------------------ 65

Conclusion ------------------------------------------------------------------------------- 68

Experimental --------------------------------------------------------------------------- 74

References and Notes ------------------------------------------------------------- 87

Acknowledgements ----------------------------------------------------------------- 91

1

Introduction

The use of medicinal herbs had been mentioned in the ancient Egyptian medical papyri

that were discovered by Ebers in 1862 and translated into English in 1937 by Ebbell.

More than 800 prescriptions in Ebers Papyrus emphasized that medicines were

originally foods. Thus, humans from different civilizations discovered medicinal

substances from food in the same way.1)

China is the home of traditional Chinese medicine. There is an ancient saying that food

and medicine are from the same source (synonym), which is also the foundation of

functional foods today. Presently, China is one of the world’s most important and

developed markets for functional foods, which are based on traditional dietary culture

and the rapid economic development among individuals and communities.2)

With the rapid increase in the senior population in Japan and the development of a

senior society, chronic diseases of aging such as diabetes, cardiovascular diseases,

hypertension, osteoporosis, and cancer are also on the rise. These diseases are connected

not only to age but also to lifestyle factors such as diet, nutrition, and physical exercise.

The goal of functional foods is to prevent the development of such chronic disease

before treatment requiring drugs becomes necessary.3)

In anticipation of the increase in the senior population in Japan, the Japanese Ministry

of Education initiated research and development projects concerning the functionalities

of food in 1984. The projects employed researchers within the disciplines of nutrition,

pharmacology, psychology, and the medical sciences. These projects defined a

2

functional food for the first time. Foods in general were defined to have 3 functions.

The primary function was identified as nutritional, that is, essential for human survival.

The secondary function was identified as sensory, or sensory satisfaction, such as

‘‘deliciousness’’, flavor, and good texture. The tertiary function was physiological, such

as regulation of biorhythm, the nervous system, the immune system, or body defense. In

the early 1980s the Japanese scientific academy defined a functional food as a food

having a tertiary or physiologically active function. The current Japanese system for

regulation of health foods is called Food with Health Claims (FHC) and is made up of 2

categories:

1) Food with Nutrient Function Claims (FNFC)

2) Food for Specified Health Uses (FOSHU)3)

Belief in the medicinal power of foods is not a recent event but has been a widely

accepted philosophy for generations. Hippocrates, the father of medicine stated almost

2,500 years ago, ‘‘Let food be thy medicine and medicine be thy food’’.4,5)

Today, the belief in the health benefits of selected foods and their components appears

to increase. The consumers are aware of the fact that a healthy diet is important and

necessary for improving human health. An increasing number of scientific studies is

supporting the knowledge that food is an important factor for preventing many of the

chronic disorders and diseases.6)

In 2003, Nohara et al. isolated tomato-saponins, named esculeoside A from

commercial ripe fruits of Japanese Momotaro-tomato and mini-tomato, and esculeoside

B from commercial ripe fruits of Italian San Marzano-Tomato, for the first time, and

3

determined their structures as shown in Fig.1.7,8)

Tomato-Saponins are overwhelming major component in the ripe tomatoes; their

contents are approximately four times that of lycopene. Thus far, the bioactivity of

tomato has been explained only in terms of lycopene. Therefore, in the future,

pharmacological examinations should be carried out with regard to esculeoside A.

O

O

N

3

CH2O

OAc

16

2223

25

18

27

H

H

O

OH

HO

OOH

O

O

OHO

OH

O

OH

HO

HO

OOH

HOHO

HO

OHO

OH

OH

CH2OH

19

5

21

Esculeoside A

O

3

HO

OHHO

OOH

O

O

OHO

OH

O

OH

HOHO

OOH

HOHO

HO

OHO

OHOHCH2OH

5

27

Esculeoside B

O

HN

OH

H

CH2O25

2322

Gal

Glc

Xyl

Glc

Glc

Glc

Gal

Xyl

19

18

Glc

Glc

Fig. 1. Esculeoside A and Esculeoside B

4

Additionally, Fujiwara et al. revealed that esculeogenin A, the sapogenol of esculeoside

A, significantly suppressed the activity of acyl-coenzymeA (CoA): cholesterol

acyl-transferase (ACAT) protein and leads to reduction of atherogenesis.9)

Meanwhile,

through recent studies on the constituents of Solanum plants, Nohara et al. observed the

followings: pregnane glycosides are accompanied by normal spirostanol and furostanol

glycosides;10-18)

esculeogenin A is easily converted into a pregnane derivative by

refluxing with aqueous pyridine (Fig. 2);19)

and a pregnane glycoside was obtained from

the overripe tomato fruit.20)

The above facts strongly suggested that the orally

administered steroidal glycosides could be metabolized into a pregnane derivatives,

which is a type of steroidal hormone. This speculation was actually proved by the

metabolic experiment (Fig. 3).21)

Nohara has proposed a hypothesis that when steroidal glycosides such as spirostane,

spirosolane and furostane glycosides are administered orally, they could be metabolized,

leading to the introduction of a hydroxyl group at C-23, and these intermediates would

then be metabolized into pregnane derivatives. Thus, it is expected that the

tomato-saponins, esculeoside A and esculeoside B would be metabolized into various

steroidal hormones such as pregnane that express various bioactivities such as

anti-osteoporosis, anti-menopausal disorder, and anti-tumor actions in the body.22)

5

HN

HHO

OH

H

O OH OH

HN

OH

H

OH

HO

OH

HHO

O

H+

HN

OH

HHO

OH

pyridine-water

Esculeogenin A

3β,16β-Dihydroxy-5α-pregna-20-one

HOH+

O

H

Fig. 2. Facile Conversion of Esculeogenin A into Pregnane Derivative

6

HGlc A-O

O

O

HN

HHO

CH2OH

OH

O

O

HN

H

O

OHHO

HOH2C

O

OO

HOH2C

O

OH

HO

HOH2C

O

OHHOHO

HO

HO

O

CH2O

OOH

HO

HOH2C

HO

OAc

HGlc A-O

HHO

O

Pregnane

Androstenol 3-O-glc A5α-H: Androsterone 3-O-glc A 5β-H: Etiocholanolone 3-O-glc A

Esculeoside A

Esculeogenin A

Fig. 3. Metabolism of Steroidal Glycoside

7

I have been strongly impressed in the sciences of tomato. Investigation of anti-cancer

activities are now in progress in other Universities, so that I have planned to elucidate

the chemical interrelation between esculeoside A and esculeoside B, which is a rare

naturally occurring substance. Secondly, I have attempted to examine the content

variations of fresh tomato, tomato boiled in water, tomato heated using a microwave

oven or far-infrared light, freeze-dried tomato, and commercially available processed

tomato products contained in plastic bottles and cans in order to develop a health food.

Thirdly, We have planned to develop a tomato-health food with a co-operation of a

company (N.D.R. Co., Ltd.) and we have tried to apply for persons suffering from high

blood pressure, high blood sugar, and atopic dermatitis.

Apart from tomato, I have also interested in onion (Allium cepa L.). It is the most

widely used Allium, and ranks third among produce consumed, after tomatoes and

cabbage.23)

It is a rich source of organosulfur compounds, which are mainly responsible

for many biological activities. I have aimed to isolate and structure elucidate a stable

sulfur compound in order to develop a natural health food.

The scientific research concerning healthful benefits of onion continues to increase.

Promising results have been obtained from epidemiological studies, in vivo research,

and numerous in vitro investigations. In China, onion and garlic tea have long been

recommended for fever, headache, cholera and dysentery. The 1999 World Health

Organization (WHO) monograph states under principle uses for onion supported by

clinical data that onion can be used: “ to prevent age-dependent changes in the blood

vessels, and loss of appetite.” The monograph provides only one study involving rats as

8

documentation. It also notes uses of onion in traditional medicine for “ treatment of

bacterial infections such as dysentery, and as a diuretic… to treat ulcers, wounds, scars,

keloids, and asthma… and adjuvant therapy for diabetes.” Uses described in folk

medicine, not supported by experimental or clinical data, include onion “ as an

antihelminthic, aphrodisiac, carminative, emmenagogue, expectorant and tonic, and for

the treatment of bruises, bronchitis, cholera, colic, earache, fever, high blood pressure,

jaundice, pimples, and sore.”23)

Evidence from several investigations suggests that the

biological activities of onions are mainly due to organosulfur compound. These sulfur

compound and flavonoids possess antioxidant, antidiabetic, anti-inflammatory,

anti-cancer, anti-microbial, antihyperlipidemic, anticholesterolaemic, fibrinolytic,

anthiatherosclerotic, anticataractogenetic, antiplatelet aggregation, immunomodulatory,

neuroprotective in ischemia and reperfusion-induced cerebral injury. Wide spectrum of

biological activities makes onion as potential therapeutic agent.24)

In spite of the numerous studies about the beneficial effects of onions on human health,

certain aspects still need to be investigated. Perhaps, it is necessary to go more in depth

on the structure of other bioactive compounds, which could not yet be identified as well

as new biological properties. More research is still needed to clearly identify in vivo

health benefits of onions.25)

In 1999, Wagner et al. isolated thiosulfinates and α-sulfinyldisulfides from chloroform

extracts of onion.26)

However, these were not genuine constituents and were volatile and

unstable. They also isolated a novel biologically active compound:

2,3-dimethyl-5,6-dithiabicyclo[2.1.1]hexane 5-oxide.27)

9

Me

C

H

C

H

S C

O

S

S

CH2

H2C

Me

CH2

Me

H

Me

C

H

C

H

S C

O

S

CH2

Me

H

S

C C

Me

H H

Me S

O

S C C Me

H

H

Me S

O

S C C Me

HH

S

O

S C C Me

HH

CH2CH2MeS

O

S C C Me

H

CH2CH2Me

H

S+

H3C

H3C

H

H

S

H

HO��

S+

H

H3C

H

CH3

S

H

HO��

trans-Methylsulphinothioic acid-S-1-propenylestercis-Methylsulphinothioic acid-S-1-propenylester

cis-2,3-Dimethyl-5,6-dithiabicyclo [2.1.1] hexane 5-oxidetrans-2,3-Dimethyl-5,6-dithiabicyclo [2.1.1] hexane 5-oxide

cis-Propylsulphinothioic acid-S-1-propenylestertrans-Propylsulphinothioic acid-S-1-propenylester

trans-5-Ethyl-4,6,7-trithia-2-decene 4-S-oxide

Me

C

H

C

H

S C

O

S

CH2

Me

H

S

C C

H

H Me

trans, trans-5-Ethyl-4,6,7-trithia-2,8-decadiene 4-S-oxide

trans, cis-5-Ethyl-4,6,7-trithia-2,8-decadiene 4-S-oxide

Fig. 4. Known Sulfur Compounds Isolated by Wagner et al.

10

I have tried to isolate stable sulfur compounds from acetone extracts of onion, in order

to develop a health food could combat many diseases.

11

Main Issue

I. Tomato-Saponin

1. Chemical Conversion of Esculeoside A into Esculeogenin B

From the ripe fruits of Japanese tomato (pink-type: Momotaro tomato and mini

tomato), the fruits of Solanum lycopersicum, a spirosolane-type glycoside,

esculeoside A (1),7,8)

3-O-β-lycotetraosyl (5α,22S,23S,25S)-23-acetoxy-3β,27-

dihydroxyspirosolane 27-O-β-D-glucopyranoside, was obtained, and its bioactivity

anti-arteriosclerotic has been revealed.9)

On the other hand, from Italian San

Marzano tomato (red-type), a solanocapsine-type glycoside,28,29)

esculeoside B,8)

3-O-β

–lycotetrosyl (5α,22S,23R,25S)-22,26-epimino-16β,23-epoxy-3β ,23,27-trihydroxych-

olestane 27-O-β-D-glucopyranoside, was isolated. Its framework is rare naturally

occurring compound. Tomato-Saponins, esculeoside A and esculeoside B were isomer

to each other, therefore, esculeoside B is also expected to express anti-arteriosclerotic.

However, raw fresh Italian tomato is difficult to acquire. Although Italian canned

tomatoes are imported, it was revealed that esculeoside B was not present in large

amounts probably owing to decomposition during heat treatment of the can procedure.

Therefore I have planned a conversion of the spirosolane derivative esculeoside A (1)

into the solanocapsine derivative esculeogenin B 30)

by acid hydrolysis, because

esculeogenin A,7,8)

the sapogenol of esculeoside A, is an isomer of esculeogenin B.

I have examined various hydrolyses using varieties of acid conditions.

12

(1) Acid Treatment of Esculeoside A (1) with 2 N HCl

First, esculeoside A was treated with 2 N HCl for 1.5 h by refluxing to give compound

2 in a yield of 35% along with compound 3 in a yield of 9% as shown in Chart 1.

Compound 2 has the molecular formula as C33H55NO9 by HR-FAB-MS. Its various

2D-NMR spectra (1H–

1H COSY, HMQC, HMBC) made the following assignments: The

respective signals due to H3-19, H3-18, H3-21, Ha-26, Hb-26, and H-16 appeared at δ

0.74 (3H, s), 1.00 (3H, s), 1.05 (3H, d, J = 7.5 Hz), 2.94 (1H, d, J = 11.6 Hz), 3.23 (1H,

dd, J = 3.2, 11.6 Hz), and 4.44 (1H, m). The signal due to one anomeric proton was also

observed at δ 4.88 (1H, d, J = 7.9 Hz). The 13

C-NMR spectrum showed signals due to

the sapogenol C-1–27, which were almost coincident with those of esculeogenin A

except around C-27, together with the presence of one β-D-glucopyranosyl moiety

C-1–6. The HMBC between the anomeric proton of the glucosyl moiety and the C-27

indicated that the β-D-glucopyranosyl moiety links to the C-27 hydroxyl group.

Therefore the structure of 2 was determined to be esculeogenin A 27-O-β-D-gluco-

pyranoside.

Compound 3, the molecular formula was measured as C35H57NO10 by HR-FAB-MS.

The 1H-NMR signals were assigned as follows: δ 0.56 (1H, m, H-9), 0.76, 0.79 (each s,

H3-18), 0.81 (3H, s, H3-19), 2.14, 2.19 (each s, OAc), 2.96 (d, J = 11.5 Hz, Ha-26), 3.30

(1H, dd, J = 3.5, 11.5 Hz, Hb-26), 3.06 (m, H2-26), 4.77, 4.87 (each d, J = 7.6 Hz, glc

H-1), 5.00 (1H, m, H-16), and 5.18 (1H, dd, J = 3.2, 9.4 Hz, H-23). The 13

C-NMR data

also suggested the existence of the sapogenol C-1−27 together with one β-D-glucoyran-

osyl moiety. Since the above 1H-and 13

C-NMR signals appeared as split pattern at H3-18,

13

H2-26, OAc, and glucosyl H-1, and C-16−C-23, C-25–27, the compound was

conceivably a 1 : 1 mixture of C-22S and C-22R. The HMBC between anomeric proton

and C-27 indicated that the β-D-glucopyranosyl moiety links to the C-27 hydroxyl

group. Coupling constants due to H-23 showed that both acetoxyl groups oriented to

equatorial accompanied by steric inversion. Therefore the structure of 3 was determined

to be a mixture of 23-O-acetyl esculeogenin A 27-O-D-glucopyranoside and 23-O-acetyl

isoesculeogenin A28)

27-O-β-D-glucopyranoside. This acid hydrolysis was regarded as

not completed owing to insolubility of the products without organic solvent; therefore

next, I tried acid hydrolysis by addition of MeOH.

14

O

O

N

GalGlcGlc

Xyl

3

2 43

OGlc

OAc

16

20 2223

2526

27

2 N HCl

H

H

O

HO

N

OGlc

OH

H

H

O

HO

N

OGlc

OAc

H

H

O

HO

N

H

H CH2OGlc

AcO

+

(1)

2

(3)

Chart 1. Acid Treatment of Esculeoside A (1) with 2 N HCl

15

(2) Acid Treatment of Esculeoside A (1) with 2 N HCl-MeOH

Refluxing of esculeoside A (1) with 2 N HCl–MeOH for 1.5 h provided compound 4

and compound 5 in yields 21% and 32%, respectively, as shown in Chart 2.

Compound 4, has the molecular formula C33H55NO9 from positive HR-FAB-MS. In the

1H-NMR spectrum, the signal due to H3-21 appeared at δ 1.51 (3H, d, J = 7.5 Hz) and

the signal due to H-16 at δ 5.30, both of which were lower shifted by +0.46 and +0.86

ppm respectively, by comparing with those of 2. This indicated that the C-23-hydroxyl

group in equatorial configuration approaches to the H3-21 and H-16, causing extreme

lower shifts for H3-21 and H-16.31)

That is, the F-ring was reversed at C-22

configuration. The E-ring once opened to give enamine-imine type intermediates, to

which the 16-OH took place recyclization as shown in Chart 3.

Compound 5, has the molecular formula C33H55NO9 from positive HR-FAB-MS. It

showed a singlet olefinic methyl signal at δ 1.72 in the 1H-NMR spectrum; on the other

hand, the 13

C-NMR spectrum displayed the occurrence of one double bond at δ 95.7 and

165.2, which latter were assigned to C-20 and C-22 by the HMBC. Its chemical

structure was represented as shown in Chart 2. Even the above reaction did not give the

sugar-free compound; thus next, I tried reacting in stronger acid conditions.

16

O

O

N

GalGlcGlc

Xyl

3

2 43

OGlc

OAc

16

20 2223

2526

27

2 N HCl-MeOH

H

H

(1)

O

HO

N

H

HCH2OGlc

HO

(4) (5)

OH

HO

N

H

HCH2OGlc

HO

+

Chart 2. Acid Treatment of Esculeoside A (1) with 2 N HCl-MeOH

17

O

HO

N

OGlc

OH

H

H

(2)

16

20

22 23

2526

27

OH

HO

N

OGlc

OH

H

H+

enamine

imine

OH

HO

N

OGlc

OH

H

H

OH

HO

NH

H

HO

H+

CH2OGlc

O

HO

N

H

H CH2O

HO

(4)

Glc

Chart 3. Recyclization of F-ring

18

(3) Acid Treatment of Esculeoside A (1) with 3 N H2SO4-MeOH

Esculeoside A (1) was hydrolyzed with 3 N H2SO4 in MeOH to provide esculeogenin A

(6) in a yield of 25% and isoesculeogenin A (7)30)

in a yield of 13% along with

compound 2 as shown in Chart 4. Here, we first could obtain free sapogenols; however,

esculeogenin A was not isomerized into esculeogenin B.

O

O

N

GalGlcGlc

Xyl

3

2 43

OGlc

OAc

16

20 2223

2526

27

H

H

(1)

3 N H2SO4-MeOH

O

HO

N

OH

OH

H

H

+

Esculeogenin A (6)

O

HO

N

H

H CH2OH

HO

Isoesculeogenin A (7)

Chart 4. Acid Treatment of Esculeoside A (1) with 3 N H2SO4-MeOH

19

(4) Acid Treatment of Esculeoside A (1) with 2 N HCl in Dioxane and Water (1:1)

Next, to elevate refluxing temperature, we used 2 N HCl in a solution of dioxane and

water (1:1). After refluxing for 1.5 h, the reaction mixture was neutralized and

evaporated under reduced pressure to give a residue, to which water was added and it

was then subjected to polystyrene gel. First it was eluted with water and the products

were recovered with MeOH. Major product was measured with the 1H-NMR spectrum

suggesting it to be a mixture of esculeogenin B analogues. Therefore we separated using

ODS with 65% MeOH to give two kinds of esculeogenin B, named esculeogenin B-1 (9,

16% yield from 1) and esculeogenin B-2 (8, 21% yield from 1) as shown in Chart 5.

Esculeogenin B-2 (8), showed the molecular formula C27H45NO4 by the HR-EI-MS

and [α]D −96.2° (pyridine). In the

1H-NMR spectrum (in pyridine-d5) displayed signals

at δ 0.77 (3H, s, H3-19), 1.01 (3H, s, H3-18), 1.67 (3H, d, J = 6.7 Hz, H3-21), 3.10 (1H,

t-like, J = 10.1 Hz, Ha-26), 3.40 (1H, br d, J = 10.1 Hz, Hb-26), 3.75 (2H, d, J = 10.2

Hz, H2-27), 3.85 (1H, m, H-3), 4.92 (1H, m, H-16). The 13

C-NMR data were assigned

by 1H–

1H COSY, HMQC and HMBC.

Next, for determination of the stereo configuration at C-22, C-23, and C-25:

1- First, as regards the configurations at C-23, remarkable lower shifts were observed at

H-16 by +0.32 ppm and H3-21 by +0.44 ppm by comparing with those of esculeogenin

B-1 (9). This suggested the hydroxyl group at C-23 to be both 1,3-diaxial conformations

against H-16 and H3-21; therefore the configuration of the hydroxyl group at C-23 was

deduced to be a (C-23: R) as shown in Fig. 5.

2- Moreover, NOESY (Fig. 5) between H-16 and H-17 (δ 1.23), and between H-20 (δ

20

2.99) and H-22 (δ 3.57) indicated that the configuration at C-22 was S. Consequently,

esculeogenin B-2 (8) was characterized as (5α,22S,23R,25S)-22,26-epimino-16β,23-

epoxy-3β,23,27-trihydroxycholestane, which was identical to the compound,

esculeogenin B, previously obtained by enzymatic hydrolysis with tomatinase and

β-glucosidase, in turn.32)

Esculeogenin B-1 (9), showed the molecular formula C27H45NO4 by the HR-EI-MS

and [α]D −68.2° (pyridine). In the

1H-NMR spectrum (in pyridine-d5) it displayed signals

at δ 0.77 (3H, s, H3-19), 0.96 (3H, s, H3-18), 1.23 (3H, d, J = 6.7 Hz, H3-21), 3.01 (1H,

d, J =11.1 Hz, Ha-26), 3.30 (1H, d, J = 11.1 Hz, Hb-26), 4.60 (1H, m, H-16). The

13C-NMR data were assigned by the

1H–

1H COSY, HMQC and HMBC. NOESYs (Fig.

5) were observed between H3-18 and H-20, between H-16 and H-17, and between H-17

and H-22, suggesting the configurations at C-22 and C-23 to be R and S, respectively.

The remaining configuration at C-25 was determined to be S by the coupling constants

of H2-26. Therefore the structure of esculeogenin B-1 (9) was characterized as

(5α,22R,23S,25S)-22,26-epimino-16β,23-epoxy-3β,23,27-trihydroxycholestane.

Thus conversion of spirosolane skeleton-type, esculeoside A, into solanocapsine-type

skeleton, esculeogenin B, has successfully been attained. Its mechanism of conversion

is deduced to be as shown in Chart 6.

21

O

O

N

GalGlcGlc

Xyl

3

2 43

OGlc

OAc

16

20 2223

2526

27

H

H

(1)


O

HN

O

HN

CH2OH

OH

H

CH2OH

HOHO H H

H

OH

+

Esculeogenin B-2Esculeogenin B-1 (9) (8)

16

20

22

25

26

23

21

3

Chart 5. Acid Treatment of Esculeoside A (1) with 2 N HCl in Dioxane and Water (1:1)

22

O

HHO

H+

N

CH2OH

HHO

OH

N

CH2OH

HHO

OH

HN

HHO

O

HN

OH

OH

H

Esculeogenin A (6)

Esculeogenin B-2 (8)

OH HO

O

HHO

OH

HN

CH2OH

HO

enol

keto

enamine

imine

CH2OH

H

Chart 6. Mechanism of Isomerization

23

O

NH

CH3

H

CH3

OH

HH

H

H

CH2OH

H

H

18

16

21

25

2622

20

23

17

27

O

HN

H

HH

CH3

HH

HO

H

CH2OH

H

CH3

18

14

16

21

27

25

17 23

20

26

22



NOESY

Fig. 5. Key NOESYs of Esculeogenin B-1 (9) and B-2 (8)

24


Processed Tomatoes

Tomato-saponin, esculeoside A, is the main component in the ripe tomato; its content

is approximately four times that of lycopene. Thus far, the bioactivity of tomato has

been explained only in terms of lycopene. Therefore, in the future, pharmacological

examinations should be carried out with regard to esculeoside A.

Recently, Fujiwara et al. revealed the following facts. Esculeogenin A (6), the

sapogenol of esculeoside A (1), significantly inhibited the accumulation of cholesterol

ester (CE) induced by acetylated low-density lipoprotein (acetyl-LDL) in human

monocyte-derived macrophages in a dose-dependent manner without inhibiting

triglyceride accumulation; however, it did not inhibit the association of acetyl-LDL to

the cells. Esculeogenin A also inhibited CE formation in Chinese hamster ovary cells

overexpressing acyl-coenzyme A (CoA): cholesterol acyl-transferase ACAT-1 or

ACAT-2, suggesting that esculeogenin A suppressed the activity of both ACAT-1 and

ACAT-2. Furthermore, esculeogenin A (6) prevented the expression of ACAT-1 protein,

but it did not suppress the expression of scavenger receptor A and SR-BI. Oral

administration of esculeogenin A (6) to apoE-deficient mice significantly reduced the

levels of serum cholesterol, triglycerides, LDL-cholesterol, and the areas of

atherosclerotic lesions without any detectable side effects.9)

Through recent studies on the constituents of Solanum plants, Nohara et al. have

observed the following: pregnane glycosides are accompanied by normal spirostanol

and furostanol glycosides;10-18)

esculeogenin A is easily converted into a pregnane

25

derivative by refluxing with aqueous pyridine (Fig. 2),19)

and a pregnane glycoside was

obtained from the overripe tomato fruit.33)

The above facts strongly suggest that the

orally administered steroidal glycosides could be metabolized into a pregnane

derivatives, which is a type of steroidal hormone. This speculation was actually proved

by the metabolic experiment (Fig. 3).21)

That is, the A tomato-saponin, esculeoside A (1),

might be metabolized into various steroidal hormones such as pregnane derivatives that

are expected to express various bioactivities such as anti-osteoporosis, anti-menopausal

disorder, and anti-tumor actions in the body.

Therefore, I have attempted to examine the content of tomato-saponin, esculeoside A

in the fresh tomato, tomato boiled in water, tomato heated using a microwave oven,

freeze-dried tomato, and commercially available processed tomato products contained

in plastic bottles and cans in order to develop a health food (Fig. 6).

Commercial Momotaro, mini, and middy tomatoes [All species are classified as

Solanum lycopersicum L., and they were purchased at Kumamoto city (cultivated at

Kumamoto Prefecture) during June and August] were used.

As listed in Table (1a–1d), all test specimens were classified into four groups:

(A) water-blended [except for (4)], (B) freeze-dried, (C) heated, and (D) processed

groups.

In the case of (A), specimens (1)–(3) were individually homogenized with water

(approximately 5 times amount of tomato weight) using a mixer for a short time (10–20

s) and filtered using filter paper to obtain a filtrate. The filtrate was then passed through

a highly porous polystyrene gel (Diaion HP-20; bore 45 mm × length 350 mm or 30 mm

26

× 330 mm) and first eluted with water. The water eluate was discarded, and elution was

then carried out using MeOH to obtain an eluate. This eluate was evaporated to obtain a

residue. The residue was subjected to was subjected to reversed-phase silica gel column

chromatography (ODS column; bore 35 mm × length 330 mm or 20 × 227 mm) eluting

with 60% MeOH, the eluate of which was evaporated to obtain the tomato-saponin,

esculeoside A (Fig. 7). In (4), tomatoes were homogenized with MeOH (ca. 4 times

amount of tomato weight) and refluxed with MeOH for 2.5 h. Then, the mixture was

filtered using filter paper to obtain the filtrate, which was evaporated to dryness. The

residue was subjected to Diaion HP-20 column chromatography with excess water. The

column was then eluted with MeOH and the eluate was evaporated to obtain a residue.

This residue was then subjected to ODS with 60% MeOH to afford esculeoside A.

Specimens (5) and (6) were incubated at 38℃ for ca. 33 days or left to stand at rt for 10

days, and the action of the included tomato enzyme in these specimens was checked.

The after-treatment of these specimens was carried out in a manner analogous to that for

(1)–(4) (Table 1a).

In the case of (B), tomato bodies were crushed and then freeze-dried, in order to

develop a health food without change of ingredients, to give a powder in ca. 5% yield

amount weight of the original tomato weight. The subsequent treatment was carried out

in the same manner as described above (water was added 5 times amount of the original

tomato weight) (Table 1b).

In the case of (C), Momotaro (9) and mini tomatoes (11) were boiled in water for

approximately 20 min. Momotaro tomato (10) was heated under far-infrared light for 1

27

day, and mini tomato (12), was heated using a microwave oven at 500 W for 15 min.

The after-treatment of these specimens was carried out in the same manner as that

described above (Table 1c).

In the case of (D), we analyzed commercially available processed tomato specimens

(13)–(19) that were contained in PET bottles, juice, jars, and cans. The contents of

esculeoside A were measured in a manner analogous to that employed for fresh

tomatoes (Table 1d).

Lycopene was isolated by the following method. Mini tomato (719 g) was blended

with water in a mixer, and the mixture was filtered using filter paper to obtain the

residue; The residue was then dried to obtain the final residue (19 g). This residue was

subsequently refluxed with CHCl3 for 130 min. The CHCl3-soluble portion was

subjected to silica gel chromatography with n-hexane-CHCl3 (6 : 1) to afford lycopene

(132.4 mg).34)

28

Fig.6. Tomato Products Used in Our Study

29

Fig.7. Extraction and Isolation Method of Esculeoside A (1)

30

Table 1a. Various Preparation Methods and Yields of Tomato-Saponin (Esculeoside A)

The yields of mini and middy tomatoes were thus three times that of Momotaro

tomatoes. The blend exhibited no change after incubation at 38℃ for 787 h or left to

stand at rt for 247 h.

Procedure Crude Material

Weight

(g)

Diaion HP-20

MeOH Eluate

Yield

(mg) (w/w%)

Esculeoside A

Yield

(mg) (w/w%)

(A)

Water-

blended

(1) Mini 719 1300.0 0.181 311.9 0.043

(2) Middy 2361 4479.9 0.190 1094.2 0.046

(3) Momotaro 472 379.2 0.080 69.2 0.015

(4) Momotaro (Extracted with

MeOH) 475 420.3 0.089 80.4 0.017

(5) Momotaro (Incubated for

787 h) 130 119.0 0.092 14.1 0.011

(6) Momotaro (Left stand for

243 h at rt) 342 327.4 0.096 51.5 0.015

31

Table 1b. Various Preparation Methods and Yields of Tomato-Saponin (Esculeoside A)

for Freeze-dried Tomato

The freeze-dried tomato give a powder in ca. 5% yield amount weight of the original

tomato weight.

Procedure Crude Material Weight (g)

Diaion HP-20

MeOH Eluate

Yield

(mg) (w/w%)

Esculeoside A

Yield

(mg) (w/w%)

(B)

Freeze-dried

(7) Momotaro 257 229.8 0.089 29.7 0.012

(8) Mini 2504 3115.9 0.124 815.6 0.033

32

Table 1c. Various Preparation Methods and Yields of Tomato-Saponin (Esculeoside A)

for Heated Tomato

Upon heating by boiling in water for approximately 20 min, the yields of esculeoside

A in blended Momotaro and mini tomatoes did not change. This fact indicated that the

tomato-saponin does not decompose or change upon heating.

No change occurred even upon heating under far-infrared light or using a microwave.


Weight

(g)

Diaion HP-20

MeOH Eluate

Yield

(mg) (w/w%)

Esculeoside A

Yield

(mg) (w/w%)

(C)

Heated

(9) Momotaro (Boiled for 20 min) 1809 1451.9 0.080 285.0 0.016

(10) Momotaro (Heated under

far-infrared for 1 day) 1137 900.0 0.079 127.7 0.011

(11) Mini (boiled for 23 min) 299 739.4 0.248 113.3 0.038

(12)

Mini (Heated under microwave

oven at 500 W for 15 min)

292 523.0 0.179 154.5 0.053

33

Table 1d. Various Preparation Methods and Yields of Tomato-Saponin (Esculeoside A)

for Processed Tomato

In commercial juices and cans, tomato-saponin could not be found.

Procedure

Crude Material

Weight

(g)

Diaion HP-20

MeOH Eluate

Yield

(mg) (w/w%)

Esculeoside A

Yield

(mg) (w/w%)

(D)

Processed

(13)

Juice

Straight, PET

bottle 1800 1617.1 0.090 0.0 0.000

(14) Concd. Red.,

PET bottle 600 582.5 0.097 0.0 0.000

(15) PET bottle 920 657.0 0.071 0.0 0.000

(16) Can, Hokkaido 570 942.7 0.165 0.0 0.000

(17) Jar, Hokkaido 1000 655.1 0.066 0.0 0.000

(18) Jar, Kumamoto 500 600.0 0.120 43.0 0.009

(19) Can Italian Tomato 800 894.1 0.112 0.0 0.000

34

By analyzing specimens (1)–(12), belonging to the four groups mentioned above, I

have observed the followings.

1) When tomatoes were homogenized in water, the yields of the tomato-saponin,

esculeoside A, in the mini and middy tomatoes were found to be 0.043% and 0.046,

respectively, as listed in Table 1a. A thin-layer chromatogram (solvent:

CHCl3:MeOH:water = 7:3:0.5) of the MeOH eluate from the Diaion HP-20 column

showed almost one spot of esculeoside A (1). The yield of esculeoside A is

approximately four times that of lycopene in mini tomatoes.

2) On the other hand, the yield of Momotaro tomatoes was 0.015%; the yields of mini

and middy tomatoes were thus three times that of Momotaro tomatoes.

3) As being apprehensive of enzymatic reaction, after homogenizing tomatoes with

water using a mixer, the mixture was incubated at 38℃ for 787 h or left to stand at

rt for 247 h, however, the amount of esculeoside A exhibited no change. In this case,

no particular measure was taken to sterilize the mixture, fermentation did not occur.

4) Upon heating by boiling in water for approximately 20 min, the yields of

esculeoside A of blended Momotaro and mini tomatoes did not change. This fact

indicated that the tomato-saponin does not decompose or change upon heating.

5) No change occurred even upon heating under far-infrared light or using a

microwave oven.

6) In commercial tomato juices and canned tomatoes, tomato-saponin could not be

found. Because we did not analyze all commercial products, it was not possible to

conclude whether or not they include no tomato-saponin; however, in our study, we

35

could not detect tomato-saponins, sapogenol and prosapogenins of esculeoside A.

This issue is currently under investigation.

7) By using the freeze-dried tomato powder, we are treating persons suffering from

high blood pressure, high blood glucose level, and atopic dermatitis. We are

accumulating the data for future use.

36

3. Development of Tomato Health Food

To develop a health food from tomato fruits, in a large scale, we tried the production in

N.D.R. Co. Ltd. The tomato fruits (ca. 200 kg) taken off from stem were instantly

disinfected on hot bath (ca. 80℃), crushed mechanically, and freeze-dried at −20℃ for

30 hr to give freeze-dried powder. Then, by using mixer without additive, we obtained

(ca. 10 kg) powder tomato foods. The powder had a flavor so rich and sweet in taste.

The trademark of this tomato product is shown in Fig. 8.

Fig. 8. A Freeze-dried Tomato Product

37

SBP DBP

4.Treatment Trials of Tomato Product for Some Volunteers

Next, by using this product (the powder includes esculeoside A ( 0.124%), Table 1b),

the powder was administered by some volunteers who suffering from hypertension,

hyperglycemia, and atopic dermatitis. The trials are still in progress. Just now, we are

collecting data from many cases. I introduced only one example for each.

(1) For High Blood Pressure

62 Years old female suffering from hypertension (before systolic blood pressure

(SBP): 158 mm Hg, diastolic blood pressure (DBP): 112. By regular administration of 3

g/day of tomato food with no medication, her blood pressure gradually keeps at normal

range as shown in Fig. 9 and Table 2.

Fig. 9. Effect of Administration of Tomato Powder on Blood Pressure Values

７７７７/14 begining

38

Table 2. Transition of Blood Pressure after Administration of Tomato Product

Duration Systolic Blood Pressure

mmHg

Diastolic Blood Pressure

mmHg

1 month before~beginning

(09/6/14~09/7/14)

158 112

After 1 month

(09/7/14~09/8/14)

149 105

After 2 month

(09/8/14~09/9/14)

122 78

After 3 month

(09/9/14~0/10/14)

119 78

39

(2) For High Blood Glucose

70 Years old male suffering from high blood glucose level. By regular administration

of (3g/day) of tomato food beside his prescribed medication, his blood glucose level

keeps at normal range as shown in Fig. 10.

Fig. 10. Effect of Administration of Tomato Powder on Blood Glucose Level

151151151151169169169169

154154154154

8080808070707070

90909090

30

80

130

180

mg/dlBlood Glucose Level

1/16 begining

40

Fig. 11. Effect of Administration of Tomato Powder on Haemoglobin A1C

6.2

5.3

5.8

5.24.9

4.7

3

4

5

6

7

％Haemoglobin A1C

1/16 begining

41

(3) For Atopic Dermatitis

24 Years old male has been suffering from atopic dermatitis from his birth. He began

administration of (3–5 g/day) tomato powder concomitant with his prescribed

medication:

1- mequitazine tablet: 1.5 mg/dose, 2 times/day.

2- oxatomide tablet: 15 mg/dose, 2 times /day.

3- difluprednate ointment 0.05% is mixed with crotamiton ointment 10%: 2~3 times/day,

for body.

4- hydrocortisone butyrate ointment 0.1% is mixed with heparinoid from animal organs

ointment: 2 times/day, for face.

5- betamethasone 17-valerate, gentamycin sulfate ointment and heparinoid from animal

organs ointment: 2~3 times/day, for ear.

6- betamethasone 17-valerate, gentamycin sulfate ointment: once/day, for back.

After 1 month, itching stopped and gloss appearance of the skin. After 2 months,

reddish eruption almost disappeared and the symptoms of atopic-dermatitis improved

as shown in Fig.12.

42

Fig.12. Recovery from Atopic Dermatitis After Administration of Tomato Powder

beforebeforebeforebefore

after 8 days

after 22 days

after 53 days

after 68 days

43

5. Consideration of the Efficacy of Steroidal Glycosides

Here, I would like to introduce the actual benefits of health food and cosmetics

containing steroidal glycosides. Firstly, in the USA, there is a health food named

Wild Mexican Yam, whose description explains that it is beneficial to women. Yam

contains diosgenin, which is changed into progesterone by the internal metabolism

and transports Ca+2

into the cell. Osteoporosis and menstruation syndromes are not

alleviated by the daily intake of the diosgenin in yams. Wild yam has been used as a

medicinal treatment for several centuries (Fig. 13).

The root of Trillium erectum, Beth root, has been used by women to ease childbirth as

a root preparation named Rydea-Pincas that assists in the process of childbirth

preparation and to treat irregular menstruation, uterus hemorrhage, and various female

diseases. This plant also contains a large amount of diosgenin. Diosgenin could be

metabolized into compounds similar to female hormones. Beth root is a synonym for

birth root (Fig. 13).

In Thailand and India, Solanum fruit is sometimes used as a vegetable in soups. The

daily consumption of Solanum fruit, from which we isolated new 22-β-O-spirostanol

glycosides, in daily life is also considered useful for the prevention of cancer (Fig. 13).

In China, a popular medicine named Yunnan Baiyao is used to improve blood

circulation, to dissipate stagnation, to reduce swelling and to relieve pain. This medicine

is composed of two crude drugs. This plant also contains a large amount of diosgenin

(Fig. 13).

44

Trillium erectum

Recently, a mix of ecdysterone has been used for a French cosmetic for skin beauty

care. Ecdysterone may also be metabolized into a type of pregnane, thus operating as a

female hormone.22)

Fig. 13. Examples of Health Foods Containing Steroidal Glycosides

Solanum torvum Fruits

Wild Mexican Yam

45

Here, I would like to summarize the efficacy of steroidal glycosides. The use of

steroidal glycosides is classified into internal and external uses. Regarding the former,

there are two cases: one is action on the surface of the digestive tract, and the other is

action after assimilation and metabolism. Unassimilated steroidal glycoside acts on the

nervous system or its mediator or receptors to suppress the rise in blood sugar levels.

On the other hand, the assimilated glycoside is first metabolized into C-23-hydroxylated

spirostane or furostane, and further into pregnane derivatives, which demonstrate

various bio-activities. In external use, steroidal glycoside is absorbed via the skin and

demonstrates anti-herpes and anti-skin-cancer activities as mentioned before (Fig. 14).

Steroidal glycosides are regarded as natural pro-drugs of steroidal hormone.22)

O

O

O

Sugar

Internal Use

Surface ondigestive organ

Assimilated, Metabolized

O

HO

O

23-Hydroxylation

HO

O

Steroidal Glycosides may be Pro-drug for Steroid Hormone

External Use

Steroidal Glycosides

Skinabsorption Anti-herpes, Anti-skin cancer

reacts to the nervous system orits mediator, or receptorsuppression of rise of bloodsugar value

Pregnane derivativesanti-osteoporosis, anti-menopausal disorder

Saponin Sugar part

Sapogenol Pregnane

OH

Saponin Sugar part

23

Fig. 14. Effectiveness of Steroidal Glycosides

46

II. Onion Sulfide

Fig. 15. Allium cepa bulb

A blend of onion (Allium cepa L.; Liliaceae) mixed with honey and vinegar, is

sometimes used as an antidiabetic agent and to control blood pressure. Moreover, A.

cepa is known to exhibit anticarcinogenic activities via enzymatic inhibition, enzymatic

induction, and apoptosis. In addition, it possesses anti-inflammatory, antioxidant,

antimicrobial, antifungal, antiparasitic, and antispasmodic properties. Further, ingestion

of onions may prevent certain cardiovascular diseases.23–25,35–37)

In order to develop natural, healthy foods that can prevent and combat disease, I have

tried to isolate a stable sulfur-containing substance from an acetone-extract of onion.

47

1. Extraction and Isolation

Onions were roughly chopped and blended in a mixer along with acetone;

subsequently, the mixture was soaked in acetone for three days at room temperature.

The filtrate was evaporated at 40 ℃ in vacuo to obtain a residue, which was subjected

to polystyrene gel (Diaion HP-20) column chromatography and then repeatedly

chromatographed on silica gel to yield a new compound named onionin A (1) as shown

in Chart 7. The results of a qualitative analysis using the sodium nitroprusside test

confirmed the presence of sulfur in this compound (Fig. 16).

Fig. 16. Positive Color for Sodium Nitroprusside Test

48

Peeled Onion BulbA. cepa (yellow variety)

(10.74 kg)

1. chopped and blended in a mixer along with acetone2. soaked in acetone at room temperture for 3 days3. evaporated under 40 °C

syrup (959.9 g)

100%MeOH eluate(12.4 g)

Diaion HP-20

H2O eluate

silica gel column

CHCl3:MeOH = 100:1 → 50:1

CHCl3:MeOH:H2O = 7:3:0.5→ 6:4:1

Fr-177.0 mg

Fr-270.0 mg

Fr-3100.3 mg

Fr-41370.0 mg

Fr-5502.1 mg

Fr-4-468.84 mg

Fr-4-2527.8 mg

Fr-4-364.0 mg

Fr-4-574.3mg

Fr-4-1265.7 mg

Onionin A

silica gel column

CHCl3:MeOH = 50:1

silica gel columnn-Hexane :Acetone = 4:1

Fr-6410.7 mg

Fr-7664.9mg

Fr-92393.0 mg

Fr-85373.1 mg

Fr-4-2-121.3 mg

Fr-4-2-29.6 mg

Fr-4-2-37.8 mg

Fr-4-2-411.0 mg

Fr-4-2-55.4 mg

Fr-4-2-65.0 mg

Fr-4-2-712.3 mg

Fr-4-2-88.4 mg

Fr-4-2-96.6 mg

Fr-4-2-103.2 mg

Fr-4-2-1175.5 mg

Fr-4-2-1232.5 mg

Fr-4-2-1320.7 mg

Fr-4-2-11-C2.3 mg

Fr-4-2-11-B42.2 mg

Fr-4-2-11-D9.9 mg

Fr-4-2-11-A2.8 mg

silica gel columnn-Hexane :Acetone = 4:1

1

Chart 7. Extraction and Isolation of A. cepa

49

2. Chemical Structure

Onionin A (1), The positive HR-FAB-MS of 1 showed a peak corresponding to

[M + Na]+

at m/z 243.0489 (calcd for C9H16O2S2Na, 243.0489), and a base peak

corresponding to [C6H11OS]+ at m/z 131.0525 (calcd for C6H11OS, 131.0531) (Fig. 25).

The IR spectrum of 1 showed absorption bands at 1027 and 2366 cm-1

, which

corresponded to sulfoxide and SH groups, respectively. In the 1H-NMR spectrum of 1

(Fig. 19), three secondary methyl groups appeared at δ 1.05 (3H, d, J = 6.3 Hz), 1.28

(3H, d, J = 6.9 Hz), and 1.90 (3H, dd, J = 1.7, 6.9 Hz), along with signals from two

olefinic protons at δ 6.03 (1H, dd, J = 1.7, 13.8 Hz), and 6.47 (1H, dq, J = 6.9, 13.8 Hz),

and four methine protons at δ 1.97 (1H, m), 2.16 (1H, m), 4.01 (1H, d, J = 5.8 Hz), and

4.99 (1H, dd, J = 3.4, 10.9 Hz). The 13

C-NMR spectrum (Fig. 20) exhibited three

methyl signals at δ 13.9, 18.1, and 18.3, four methine carbons signals at δ 42.9, 55.0,

79.2, and 83.5, and two olefinic carbon signals at δ 131.7 and 139.6 (Fig. 17). The

1H-

1H COSY spectrum (Fig. 21) showed the presence of a sequential correlation from

the S–H at δ 4.31 to the methine proton at δ 4.99, to the methine proton at δ 1.97, to the

methine proton at δ 2.16, to the methine proton at δ 4.01, to the olefinic proton at δ 6.03,

to the olefinic proton at δ 6.47, and to the methyl protons at δ 1.90. Also observed here

vicinal correlations between the methine proton at δ 1.97 and the methyl protons at δ

1.05, and between the methine proton at δ 2.16 and the methyl protons at δ 1.28 (Fig.

17). The HMBC spectrum (Fig. 23) also exhibited correlations from the methine proton

at δ 4.99 to the carbon at δ 79.2, from the methyl protons at δ 1.05 to the three methine

carbons at δ 42.9, 55.0, and 83.5, from the methyl protons at δ 1.28 to the three methine

50

carbons at δ 42.9, 55.0, and 79.2, from the methine proton at δ 4.01 to the methine

carbon at 83.5, from the olefinic proton at δ 6.03 to the olefinic carbon at δ 139.6, from

the olefinic proton at δ 6.47 to the olefinic carbon at δ 131.7, and from the methyl

protons at δ 1.90 to two olefinic carbons at δ 131.7 and 139.6 (Fig. 17).

The configuration at C-1' was determined to be Z from the 1H-NMR signal of H-1' at δ

6.03 (1H, dd, J = 1.7, 13.8 Hz) and from the NOESY correlation between H-1' and H-2'.

The 1H-

1H COSY and HMBC spectra revealed the planar structure of 1 to be

3,4-dimethyl-5-(1Z-propenyl) tetrahydrothiophen-2-sulfoxide-S-oxide, as shown in Fig.

17. Furthermore, the NOESY spectrum (Fig. 24) showed the following 1H-

1H

correlations: SH and H-2; CH3-3 and H-2; CH3-3 and H-3; CH3-4 and H-4; CH3-4 and

H-3; CH3-4 and H-5; H-5 and SH; H-5 and H-1′; and H-1′ and H-2′ (Fig. 18).

51

S+

C

H

S+

H

H

H

H3C

H3C

C CH3

H

O-1

23

45

HMBC

131.7139.6

18.31.90, dd, J = 1.7, 6.9

6.03, dd, J = 1.7, 13.8

1.28, d, J = 6.9

1.05, d, J = 6.3

4.99, dd, J = 3.4, 10.9

18.1

13.9

42.9

55.0

79.2

83.51.97, m

2.16, m

4.01, d, J = 5.8

6.47, dq, J = 6.9, 13.8

4.31, d, J = 10.9

O-

H

H

1H-1H COSY

Fig. 17. Structure of Onionin A (1)

52

S+

H

H3C

H

H

S+

H

O-

H3C

C C CH3

HH

O-

NOESY

1b

H

S+

H3C

H

H3C

H

S+

H

O-

H

1

2

3

4

5

C C CH3

HH

O-

1a

1' 2' 3'

H

Fig. 18. Predicted structures of Onionin A (1) with NOESY

桧日

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Fig.19.'H-NMRSpectrumofOnioninA(1)

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Fig.21.1H-1HCOSYSpectrumofOnioninA(1)

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EI-MSSpectrum

PositiveHRFAB-MSSpectrum

Fig25､MSSpectraofOnioninA(1)

■ヅ面

5９

60

3. Configuration of Sulfoxide

On the basis of the above-mentioned NOESY results, two relative structures of 1 are

possible (1a and 1b) (Fig. 18). Moreover, to determine the configuration of the S+–O

- in

1, first, an aromatic solvent-induced shift reagent (C6D6) was used. Then, Eu(fod)3 shift

reagent was used.

(1) Aromatic Solvent-Induced Shift

Comparison of the 1H-NMR spectrum of 1 in CDCl3 with that in C6D6 (Table 3)

showed that most of the signals in the latter case were shifted upfield by approximately

0.45 ppm. The only signals that remained relatively unchanged were those of CH3-3,

H-2, and H-4. These induced shifts indicate the formation of a collision complex

between the aromatic solvent and the sulfoxide (1a), which has an S+–O

- axial

configuration.38,39)

S+

H3C

H

H3C

H

S+

H O-

H

C C CH3

HH

O-H

Fig. 26. Formation of a Collision Complex Between Aromatic Solvent

and Axial Conformer of (1a)

61

Table 3. 1H-NMR Chemical shifts (δ) for 1 in CDCl3 and C6D6

CDCl3 C6D6 ∆ δ

CH3-3 1.05 0.84 0.21

CH3-4 1.28 0.93 0.35

H3-3′ 1.90 1.30 0.60

H-3 1.97 1.63 0.34

H-4 2.16 2.12 0.04

H-5 4.01 3.52 0.49

SH 4.31 5.01 0.71

H-2 4.99 4.91 0.08

H-1′ 6.03 5.45 0.58

H-2′ 6.47 6.18 0.29

62

(2) Eu(fod)3 Shift Reagent

Second, greater changes were observed in the 1H-NMR chemical signals by ∆ δ 0.17,

0.51, and 0.35 owing to CH3-3, H-4 and H-2, respectively, after sequential addition of

the Eu(fod)3 shift reagent in CDCl3 than in the signals of CH3-4, H-3 , and H-5 by ∆ δ

0.06, 0.18, and 0.16, respectively (Table. 4). Thus, it was considered that the 1-oxide

group on the tetrahydrothiophene skeleton is in an axial arrangement,38)

and therefore, 1

may be proposed as shown in Fig. 27.

Table 4. Lanthanide-Induced Shifts (LIS) (δ) on 1H-NMR of 1 after Addition

of Eu(fod)3 in CDCl3

Eu(fod)3, equiv. H-2 H-3 CH3-3 H-4 CH3-4 H-5 H-1′ H-2′ H3-3′

0 4.99 1.97 1.05 2.16 1.28 4.01 6.03 6.47 1.90

0.01 5.09 2.06 1.07 2.52 1.27 4.04 6.05 6.52 1.86

0.02 5.20 2.09 1.12 2.59 1.30 4.09 6.10 6.58 1.86

0.03 5.34 2.15 1.22 2.66 1.34 4.17 6.18 6.60 1.87

∆ δ 0.35 0.18 0.17 0.51 0.06 0.16 0.15 0.13 0.03

63

S+

H3C

H

H3C

H

S+

H O-

H

1

2

3

4

5

C C CH3

HH

O-

1' 2' 3'

H

Fig. 27. Structure of Onionin A (1)

4. Plausible Biosynthetic Pathway for Production of Onionin A

The formation of onionin A (1) could be estimated as shown in Chart 8:

1-propenesulfenic acid (ii) derived by analogy from (+)-S-propenyl-L-cysteine-S-oxide

(i) present in garlic would yield 1-propenyl-1-propenethiosulfinate (iii), which would

then get converted to 2,3-dimethyl-butanedithial 1-oxide (iv) by [3,3]-sigmatropic

rearrangement.23,27)

Next, protontion to sulfoxide in compound (iv) leads to hydroxyl

group formation (v). Then propanethial S-oxide approaches to the double bond in (v) to

make cycloaddition (vi) followed by realease of sulfur and formation of onionin A (1).

64

Chart 8. Plausible Biosynthetic Pathway for Production of Onionin A (1)

S+

NH2

COOH

O- H O

S

S

S+

O-

- H2O

S

S+

O-

Allinase

(i) (ii) (iii) (iv)

H+

S+

(v)

S OHS+

(v)

S OH

O-

S+

S+ S OH

O

S

H

H3C H

-S

S+ S OH

O-

S+ S+ O-

O- H

H O

S:

Onionin A(vii)

(vi)

65

5. Effect of Onionin A on Macrophage Activation

Macrophages that infiltrate cancer tissues are referred to as tumor-associated

macrophages (TAMs) and are closely involved in the development of tumor

microenvironment.40-42)

Because TAMs have anti-inflammatory functions, they are

considered to be a type of alternatively activated macrophages (M2).43,44)

In the case of

certain types of tumors, the presence of TAMs is associated with poor prognosis in

patients.45-47)

Therefore, inhibition of M2-macrophage polarization is known to

suppress tumor-cell proliferation.

GM-CSF

LPS, IFNγ

Corticosteroids

Bacterial products

M-CSF

IL-4, IL-13, IL-10

Corticosteroids

PGE, VitD3

Tumor Suppression

Atherogenesis

Tumor Promotion

Anti-atherogenesis

Onionin A?

?Monocyte

M1Mφ

M2Mφ

Fig. 28. M1 and M2 Macrophages

66

Incubation of human monocyte-derived macrophages with interleukin (IL)-10 for two

days increased CD163 expression. Under the same conditions, the effect of 1 on

IL-10-induced CD163 expression were measured as shown in Chart 10. It was found

that 1 significantly inhibited CD163 expression; this finding suggests that onionin A

suppresses polarization of M2 macrophages (Fig. 29).

Method

2% PFA fixation

5 min

Blocking (Block ace)

20 min

1-st antibody (anti-CD163 antibody : AM-3K 2µg/ml)

4˚C, Overnight

2-nd antibody (anti-mouse IgG antibody )

1 h

Development

IL-10 (final : 30 ng/ml)

Human macrophage

24 h Onionin A (1)

Isolation of human monocyte

4 days

M2 Macrophage

IL-10

Onionin A

We examined the inhibitory effect of

Onionin A (1) on CD163 expression,

by a cell-ELISA

CD163↑M2 M ф marker

4 days

24 h

Monocyte

48 h

48 h

Chart 9. Examination of Inhibitory Effect of Onionin A (1) on

CD163 Expression by Cell-ELISA

67


Fig. 29. Effect of Onionin A on CD163 Expression

Human monocyte-derived macrophages (5 × 104 cells per well of a 96-well plate) were

incubated with IL-10 (20 nM) in the presence of indicated concentration of onionin A

for two days, followed by determination of CD163 expression by cell-ELISA.

* *

0

0.5

1

1.5

2

2.5


Onionin A

1 µM

Onionin A

3 µM

Onionin A

10 µM

Onionin A

30 µM

Onionin A

Abso

rbance a

t 45

0 n

m

68

Conclusion

There is an ancient saying that food and medicine are from the same source (synonym),

which is also the foundation of functional foods today.

With the rapid increase in the senior population in Japan and the development of a

senior society, chronic diseases of aging such as diabetes, cardiovascular diseases,

hypertension, osteoporosis, and cancer are also on the rise. These diseases are connected

not only to age but also to lifestyle factors such as diet, nutrition, and physical exercise.

The goal of functional foods is to prevent the development of such chronic disease

before treatment requiring drugs becomes necessary.

Under this circumstance, I have been strongly interested the science of tomato and

onion. Regarding to tomato, I have clarified a chemical correlation between esculeoside

A and esculeogenin B. That is, a chemical conversion of spirosolane skeleton-type,

esculeoside A, into solanocapsine-type skeleton, esculeogenin B, has successfully been

attained by acid hydrolysis using 2 N HCl in a solution of dioxane and water (1:1)

yielded two kind of esculeogenin B, named esculeogenin B-1:

(5α,22R,23S,25S)-22,26-epimino-16β,23-epoxy-3β,23,27-trihydroxycholestane, and

esculeogenin B-2: (5α,22S,23R,25S)-22,26-epimino-16β,23-epoxy-3β,23,27-trihydr-

oxycholestane. Its mechanism of conversion has been deduced. Now it is possible to

prepare esculeogenin B for animal experiments.

69

O

O

N

GalGlcGlc

Xyl

3

2 4

OGlc

OAc

H

H

Esculeoside A


O

HN

O

HN

CH2OH

OH

H

CH2OH

HOHO H H

H

OH

+


Chart 5. Acid Treatment of Esculeoside A with 2 N HCl in Dioxane and Water (1:1)

Next, I have determined the content variation of tomato-saponin, esculeoside A in the

fresh tomato, tomato boiled in water, tomato heated using a microwave oven, freeze-

dried tomato, and commercially available processed tomato products contained in

plastic bottles and cans in order to develop a health food. The yields of the tomato

saponin, esculeoside A, in the mini and middy tomatoes were approximately four times

that of lycopene. The yields of mini and middy tomatoes were thus three times that of

Momotaro tomatoes. The tomato-saponin does not decompose or change upon heating

or upon heating under far-infrared light or using a microwave oven. In commercial

tomato juices and cans, tomato-saponin could not be found.

70

Table 5. Various Preparation Methods and Yields of Tomato-Saponin (Esculeoside A)


Weight

(g)

Diaion HP-20

MeOH Eluate

Yield

Esculeoside A

Yield

(mg) (w/w%) (mg) (w/w%)

(A)

Blended with

water

1 Mini 719 1300.0 0.181% 311.9 0.043%

2 Middy 2361 4479.9 0.190% 1094.2 0.046 %

3 Momotaro 472 379.2 0.080% 69.2 0.015%

4 Momotaro (Extracted with MeOH) 475 420.3 0.089% 80.4 0.017 %

5 Momotaro (Incubated for 787 h) 130 119.0 0.092% 14.1 0.011%

6 Momotaro (Left stand for 243 h at rt) 342 327.4 0.096% 51.5 0.015%

(B)

Freeze-dried

7 Momotaro 257 229.8 0.089% 29.7 0.012%

8 Mini 2504 3115.9 0.124% 815.6 0.033%

(C)

Heated

9 Momotaro (Boiled for 20 min) 1809 1451.9 0.080% 285.0 0.016%

10

11

Momotaro (Heated under far-infrared

for 1 day)

1137 900.0 0.079% 127.7 0.011%

Momotaro (Boiled for 20 min) 299 739.4 0.248% 113.3 0.038%

12

Momotaro (Heated under far-infrared

for 1 day)

292 523.0 0.179% 154.5 0.053%

(D)

Processed

13

Juice

Straight, PET bottle 1800 1617.1 0.090% 0.0 0.000%

14 Concd. Red., PET bottle 600 582.5 0.097% 0.0 0.000%

15 PET bottle 920 657.0 0.071% 0.0 0.000%

16 Can, Hokkaido 570 942.7 0.165% 0.0 0.000%

17 Jar, Hokkaido 1000 655.1 0.066% 0.0 0.000%

18 Jar, Kumamoto 500 600.0 0.120% 43.0 0.009%

19 Can Italian Tomato 800 894.1 0.112% 0.0 0.000%

71

Next, I have tried to isolate a stable sulfur compound from onion. Onionin A, a novel,

stable, sulfur compound has been isolated from an acetone-extract of bulbs of onion

(Allium cepa), and its structure has been characterized as:

3,4-dimethyl-5-(1Z-propenyl)-tetrahydrothiophen-2-sulfoxide-S-oxide.

S+

H3C

H

H3C

H

S+

H O-

H

1

2

3

4

5

C C CH3

HH

O-

1' 2' 3'

H

Onionin A

Fig.27. Structure of Onionin A (1)

The biosynthetic pathway for production of onionin A could be estimated.

Chart 8. Plausible Biosynthetic Pathway for Production of Onionin A (1)

S+

NH2

COOH

O- H O

S

S

S+

O-

- H2O

S

S+

O-

Allinase

(i) (ii) (iii) (iv)

H+

S+

(v)

S OHS+

(v)

S OH

O-

S+

S+ S OH

O

S

H

H3C H

-S

S+ S OH

O-

S+ S+ O-

O- H

H O

S:

Onionin A(vii)

(vi)

72

Then, we have examined the inhibitory effect of onionin A on CD163 expression by

cell- ELISA.

Method

2% PFA fixation

5 min

Blocking (Block ace)

20 min

1-st antibody (anti-CD163 antibody : AM-3K 2µg/ml)

4˚C, Overnight

2-nd antibody (anti-mouse IgG antibody )

1 h

Development

IL-10 (final : 30 ng/ml)

Human macrophage

24 h Onionin A

Isolation of human monocyte

4 days

M2 Macrophage

IL-10

Onionin A

We examined the inhibitory effect of

Onionin Aon CD163 expression,

by a Cell-ELISA

CD163↑M2 Mф marker

4 days

24 h

Monocyte

48 h

48 h

Chart 9. Examination of Inhibitory Effect of Onionin A (1) on

CD163 Expression by Cell-ELISA

We found that onionin A significantly inhibited CD163 expression; this finding

suggests that onionin A has a potential to suppress tumor cell proliferation by inhibition

of M2 macrophage polarization.


Fig.29. Effect of Onionin A (1) on CD163 Expression

* *

0

0.5

1

1.5

2

2.5


Onionin A

1 µM

Onionin A

3 µM

Onionin A

10 µM

Onionin A

30 µM

Onionin A

Ab

sorb

ance

at

45

0 n

m

73

In Fresh tomato, tomato-saponin is mainly concentrated in the gel like substance

surround the seeds. Therefore, ingestion of fresh tomatoes could not liberate enough

tomato-saponin to exert its desired health activity. We can overcome this problem by

using fresh tomato juice or freeze-dried powder to get enough concentration of

esculeoside A. On the other hand, onion should be eaten raw (uncooked) since onion

contains beneficial sulfur compounds which are destroyed by cooking.

74

Experimental

I. Tomato-Saponin

1. Chemical Conversion of Esculeoside A into Esculeogenin B

General Procedure

The optical rotations were measured with a JASCO DIP-1000 (l = 0.5) automatic

digital polarimeter. The 1H- and

13C-NMR spectra were measured with JEOL-α-500

NMR spectrometers, and the chemical shifts are given on a δ (ppm) scale with

tetramethylsilane as the internal standard. The HR-FAB-MS spectra were measured

with a JEOL JMS-DX303HF spectrometer and taken in a glycerol matrix containing

NaI. HPLC was carried out using the Mightysil RP-18 (10.0 mm i.d. × 250 mm,

Kanto Chemical Co., Ltd., Tokyo, Japan); column with a Tosoh CCPM pump, Tosoh

RI-8010 detector, and JASCO OR-2090 detector. TLC was performed on silica gel

plates (Kieselgel 60 F254, Merck) and RP C18 silica gel plates (Merck).The spots on TLC

were visualized by UV light (254/366 nm) and sprayed with 10% H2SO4, followed by

heating. Column chromatography was carried out on a Diaion HP-20 (Mitsubishi

Chemical Ind.), ODS (Wako Pure Chemical Industries, Ltd., Fuji Silysia Chemical, Ltd.,

Japan), and silica gel 60 (spherical, 40–100 mm, and 230–400 mesh ASTM, Kanto

Chemical Co., Inc.).

(1) Acid Treatment of Esculeoside A (1) with 2 N HCl

A solution of esculeoside A (1, 920 mg) in 2 N HCl (25 ml) was refluxed for 1.5 h.

After neutralization with 3% KOH, the mixture was then concentrated, water (100 ml)

75

was added, and passed through Diaion HP-20 eluted first with water, then MeOH. The

MeOH eluate was evaporated to give a residue, which was chromatographed on silica

gel column with CHCl3-MeOH-water = 9:2:0.1 to 8:2:0.2 to give compound 2 (154 mg,

35%) as a major component and compound 3 (42 mg, 9%).

Compound 2: An amorphous powder, HR-FAB-MS (m/z): 632.3793 (Calcd for

[C33H55NO9 + Na]+ 632.3774).

1H-NMR (in pyridine-d5) δ: 0.74 (3H, s, H3-19), 1.00 (3H, s, H3-18), 1.05 (3H, d, J =

7.5 Hz, H3-21), 1.81 (1H, d-like, J = 8.2 Hz, H-17), 2.94 (1H, d, J = 11.6 Hz, Ha-26),

2.99 (1H, t-like, J = 7.3 Hz, H-20), 3.23 (1H, dd, J = 3.2, 11.6 Hz, Hb-26), 4.36 (1H, dd,

J = 4.9, 11.6 Hz, glc Ha-6), 4.44 (1H, m, H-16), 4.88 (1H, d, J = 7.9 Hz, glc H-1).

13C-NMR (in pyridine-d5): sapogenol C-1–27 at δ 37.5 (C-1), 32.3 (C-2), 70.6 (C-3),

39.3 (C-4), 45.2 (C-5), 29.1 (C-6), 32.6 (C-7), 35.2 (C-8) , 54.6 (C-9), 35.9 (C-10), 21.4

(C-11), 40.8 (C-12), 41.5 (C-13), 56.5 (C-14), 32.4 (C-15), 79.3 (C-16), 63.0 (C-17),

17.3 (C-18), 12.5 (C-19), 34.7 (C-20), 15.1 (C-21), 101.6 (C-22), 65.7 (C-23), 33.2

(C-24), 35.9 (C-25), 40.3 (C-26), 71.3 (C-27); (glc C-1–6) δ: 105.0, 75.3, 78.4, 71.8,

78.4, 62.9.


[C35H57NO10 + Na]+ 674.3880).

1H-NMR (in pyridine-d5) δ: 0.56 (1H, m, H-9), 0.76, 0.79 (each s, H3-18), 0.81 (3H, s,

H3-19), 2.14, 2.19 (each s, OAc), 2.96 (1H, d, J = 11.5 Hz, Ha-26), 3.06 (m, H2-26),

3.30 (1H, dd, J = 3.5, 11.5 Hz, Hb-26), 4.77, 4.87 (each d, J = 7.6 Hz, glc H-1), 5.00

(1H, m, H-16), and 5.18 (1H, dd, J = 3.2, 9.4 Hz, H-23).

76


39.2 (C-4), 45.3 (C-5), 29.1 (C-6), 32.4 (C-7), 35.2 (C-8), 54.5 (C-9), 35.9 (C-10), 21.4

(C-11), 40.3 (C-12), 41.2 (C-13), 56.6 (C-14), 32.4 (C-15), 79.8, 82.4 (C-16), 62.8, 62.9

(C-17), 16.9, 17.2 (C-18), 12.4, 12.5 (C-19), 35.2, 45.3 (C-20), 15.9, 16.6 (C-21), 97.6,

100.8 (C-22), 73.1, 75.0 (C-23), 36.5 (C-24), 35.3, 37.5 (C-25), 41.1, 45.2 (C-26), 63.5,

65.1 (C-27), 21.2 (CH3CO), 170.7 (CH3CO); glc C-1–6 at δ 105.0, 75.3, 78.4, 71.8,

78.4, 62.9.

(2) Acid Treatment of Esculeoside A (1) with 2 N HCl-MeOH

A solution of esculeoside A (1, 542 mg) in 2 N HCl-MeOH (22 ml) was refluxed for

1.5 h. After neutralization with 3% KOH, the mixture was then concentrated, water (100

ml) was added, and passed through Diaion HP-20 eluted first with water, then MeOH.

The MeOH eluate was evaporated to give a residue, which was chromatographed on

silica gel column with CHCl3-MeOH-water = 9:2:0.1 to 8:2:0.2 to give compound 4

(54.6 mg, 21 %) and compound 5 (83.2 mg, 32%).

Compound 4: an amorphous powder, HR-FAB-MS (m/z): 632.3781 (Calcd for

[C33H55NO9 + Na] + 632.3774).

1H-NMR (in pyridine-d5) δ: 0.76 (3H, s, H3-19), 0.90 (3H, s, H3-18), 1.51 (3H, d, J =

7.5 Hz, H3-21), 3.10 (1H, t-like, J = 11.5 Hz, Ha-26), 3.16 (1H, dd, J = 3.1, 11.5 Hz,

Hb-26), 4.78 (1H, d, J = 7.6 Hz, glc H-1), 5.30 (1H, m, H-16).


39.1 (C-4), 45.5 (C-5), 28.9 (C-6), 32.4 (C-7), 35.1 (C-8), 54.5 (C-9), 35.7 (C-10), 21.4

(C-11), 40.8 (C-12), 40.5 (C-13), 55.7 (C-14), 34.1 (C-15), 82.6 (C-16), 63.5 (C-17),

77

16.6 (C-18), 12.4 (C-19), 45.0 (C-20), 16.3 (C-21), 102.2 (C-22), 72.7 (C-23), 27.7

(C-24), 43.8 (C-25), 41.0 (C-26), 71.5 (C-27); (glucosyl C-1–6) δ: 104.5, 74.9, 78.4,

71.7, 78.4, 62.6.


[C33H55NO9 + Na]+ 632.3774).

1H-NMR (in pyridine-d5) δ: 0.79 (3H, s, H3-19), 0.81 (3H, s, H3-18), 1.72 (3H, s, H3-21),

4.88 (1H, d, J = 7.9 Hz, glc H-1).


39.1 (C-4), 42.0 (C-5), 28.9 (C-6), 33.3 (C-7), 34.9 (C-8), 54.8 (C-9), 35.6 (C-10), 20.8

(C-11), 39.8 (C-12), 39.8 (C-13), 54.5 (C-14), 31.7 (C-15), 73.2 (C-16), 53.9 (C-17),

12.3 (C-18), 13.1 (C-19), 95.7 (C-20), 19.4 (C-21), 165.2 (C-22), 66.5 (C-23), 24.9

(C-24), 45.1 (C-25), 47.1 (C-26), 70.2 (C-27); (glc C-1–6) δ: 104.9, 75.0, 78.5, 71.8,

78.5, 62.7.

(3) Acid treatment of Esculeoside A (1) with 3 N H2SO4-MeOH

A solution of esculeoside A (1, 850 mg) in 3 N H2SO4 (45 ml) was refluxed for 1.5 h.

After neutralization with 3% KOH, the mixture was then concentrated, water (140 ml)

was added, and passed through Diaion HP-20 eluted first with water, then MeOH. The

MeOH eluate was evaporated to give a residue, which was chromatographed on silica

gel column with CHCl3-MeOH-water = 9:2:0.1 to 8:2:0.2 to give compound 6 (75 mg,

25%) and compound 7 (53 mg, 13%), which were identified as esculeogenin A and

isoesculeogenin A, respectively, as a major component and compound 2 (45 mg, 11%).

78

(4) Acid Treatment of Esculeoside A (1) with 2 N HCl in Dioxane and Water (1:1)

Esculeoside A (1, 1200 mg) in 2 N HCl (55 ml) in a solution of dioxane and water (1:1)

was refluxed for 1.5 h. After neutralization with 3% KOH, the mixture was then

concentrated, water (160 ml) was added, and passed through Diaion HP-20 eluted first

with water, then MeOH. The MeOH eluate was evaporated to give a residue, which was

chromatographed on silica gel column with CHCl3-MeOH-water = 9:2:0.1 to 8:2: 0.2 to

give a mixture of compound 8 and compound 9 (220 mg, 52%).

Next, separation by using ODS with 65% MeOH led to the isolation of two kind of

Esculeogenin B, named esculeogenin B-1 (9, 67 mg, 16% yield from 1) and

esculeogenin B-2 (8, 89 mg, 21% yield from 1).

Esculeogenin B-1(9): an amorphous powder, HR-EI-MS (m/z): 447.3256 (Calcd for

C27H45NO4 : 447.3349) and [α]D –68.2 ° (c = 0.1, pyridine).

1H-NMR spectrum (in pyridine-d5) δ: 0.77 (3H, s, H3-19), 0.96 (3H, s, H3-18), 1.23

(3H, d, J = 6.7 Hz, H3-21), 2.99 (1H, m, H-20), 3.01 (1H, d, J = 11.1 Hz, Ha-26), 3.30

(1H, d, J = 11.1 Hz, Hb-26), 4.60 (1H, m, H-16).

13C-NMR (in pyridine-d5) δ: 38.5 (C-1), 32.5 (C-2), 70.6 (C-3), 37.6 (C-4), 45.4 (C-5),

29.2 (C-6), 32.5 (C-7), 35.4 (C-8), 55.3 (C-9), 36.0 (C-10), 21.0 (C-11), 37.5 (C-12),

43.0 (C-13), 53.6 (C-14), 33.9 (C-15), 69.1 (C-16), 58.3 (C-17), 15.0 (C-18), 12.5

(C-19), 27.3 (C-20), 18.0 (C-21), 62.2 (C-22), 94.1 (C-23), 39.3 (C-24), 42.0 (C-25),

48.8 (C-26), 65.0 (C-27).

Esculeogenin B-2 (8), an amorphous powder, HR-EI-MS (m/z): 447.3298 (Calcd for

C27H45NO4 : 447.3349) and [α]D –96.2° (c = 0.1, pyridine).

79

1H-NMR spectrum (in pyridine-d5) δ: 0.77 (3H, s, H3-19), 1.01 (3H, s, H3-18), 1.67 (3H,

d, J = 6.7 Hz, H3-21), 3.10 (1H, t-like, J = 10.1 Hz, Ha-26), 3.40 (1H, br d, J = 10.1 Hz,

Hb-26), 3.75 (2H, d, J = 10.2 Hz, H2-27), 3.85 (1H, m, H-3), 4.92 (1H, m, H-16).

13C-NMR data were assigned as follows: δ 37.5 (C-1), 32.5 (C-2), 70.6 (C-3), 37.6

(C-4), 45.4 (C-5), 29.2 (C-6), 32.5 (C-7), 35.4 (C-8), 55.3 (C-9), 36.0 (C-10), 21.0

(C-11), 37.5 (C-12), 43.0 (C-13), 55.6 (C-14), 33.7 (C-15), 69.1 (C-16), 58.3 (C-17),

15.2 (C-18), 12.5 (C-19), 27.3 (C-20), 19.8 (C-21), 60.0 (C-22), 96.5 (C-23), 39.3

(C-24), 41.1 (C-25), 48.8 (C-26), 64.2 (C-27).


Processed Tomatoes

Extraction and Isolation of Esculeoside A

Commercial Momotaro, mini, and Middy tomatoes [All species are classified as

Solanum lycopersicum L., and they were purchased at Kumamoto city (cultivated at

Kumamoto Prefecture ) during June and August] were used.

As listed in Table (1a–1d), all test specimens were classified into four groups:

(A) water-blended [except for (4)]

(B) freeze-dried,

(C) heated

(D) processed groups.

In the case of (A), specimens (1)–(3) were individually homogenized with water

(approximately 5 times amount of tomato weight) using a mixer for a short time (10–20

s) and filtered using filter paper to obtain a filtrate. The filtrate was then directly passed

80

through a highly porous polystyrene gel column (Diaion HP-20 column; bore 45 mm ×

length 350 mm or 30 mm × 330 mm) and first eluted with water. The water eluate was

discarded, and elution was then carried out using MeOH to obtain an eluate. This eluate

was evaporated to obtain a residue.

Furthermore, the residue was subjected to reversed-phase silica gel column

chromatography (ODS column; bore 35 mm × length 330 mm or 20 × 227 mm),

eluting with 60% MeOH, the eluate of which was evaporated to obtain the

tomato-saponin, esculeoside A. In (4), tomatoes were homogenized with MeOH (ca. 4

times amount of tomato weight) and refluxed with MeOH for 2.5 h. Then, the mixture

was filtered using filter paper to obtain the filtrate, and this filtrate was evaporated to

dryness. The residue was subjected to Diaion HP-20 column chromatography with

excess water. The column was then eluted with MeOH and the eluate evaporated to

obtain a residue. This residue was then subjected to ODS with 60% MeOH to afford

esculeoside A. Specimens (5) and (6) were incubated at 38℃ for ca. 33 days or left to

stand at rt for 10 days, and the action of the included tomato enzyme in these specimens

was checked. The after-treatment of these specimens was carried out in a manner

analogous to that for (1)–(4). In the case of (B), tomato bodies were crushed and then

freeze-dried, in order to develop a health food without change of ingredients to give a

powder in ca. 5% yield amount weight of the original tomato weight. The subsequent

treatment was carried out in the same manner as described above (water was added 5

time amount of the original tomato weight). In the case of (C), Momotaro (9) and mini

tomatoes (11) were boiled in water for approximately 20 min. Momotaro tomato (10)

81

was heated under far-infrared light for 1 day, and mini tomato (12) was heated using a

microwave oven at 500 W for 15 min. The after-treatment of these specimens was

carried out in the same manner as that described above. In the case of (D), we analyzed

commercially available processed tomato products specimens (13)–(19) that were

contained in PET bottles, juice, jars, and cans. The contents were measured in a manner

analogous to the previous manner.

Lycopene was isolated by the following method. Mini tomato (719 g) was blended

with water in a mixer, and the mixture was filtered using filter paper to obtain the

residue; The residue was then dried to obtain the final residue (19 g). This residue was

subsequently refluxed with CHCl3 for 130 min. The CHCl3-soluble portion was

subjected to silica gel chromatography with n-hexane-CHCl3 (6 : 1) to afford lycopene

(132.4 mg).

82

Fig. 6. Tomato Products Used in Our Study

83

II. Onion Sulfide

General Procedure

The optical rotation was measured with a JASCO P-1020 (l = 0.5) automatic digital

polarimeter. The IR spectrum was measured with Fourier Transform FT/IR-4200

spectrometer (JASCO). The 1H and

13C- NMR spectra were measured in CDCl3 and

C6D6 with JEOL alpha 500 spectrometer at 500 and 125 MHz, respectively, and

chemical shifts are on the δ (ppm) scale. The HR-FAB-MS were measured with a JEOL

JMS-DX303HF mass spectrometer and taken in a glycerol matrix containing NaI.

Column chromatography was carried out on Diaion HP-20 (Mitsubishi Chemical

Industries), and silica gel 60 (230–400 mesh, Merck). TLC was performed on silica gel

plates (Kieselgel 60 F254, Merck). TLC spots were visualized by UV light (254/366

nm) and sprayed with 10% H2SO4 and anisaldehyde sprays followed by heating.

Plant Material

Onion bulbs (Allium cepa L. family Liliaceae) (yellow variety) cultivated in

Kumamoto prefecture, Japan, were purchased in October 2009 from the market at

Kumamoto city.

1. Extraction and Isolation

The fresh peeled bulbs (10.74 kg) of onions were roughly chopped and blended in a

mixer along with acetone. Subsequently, the mixture was soaked in acetone for three

days at room temperature. The filtrate was concentrated at 40℃ in vacuo to obtain a

syrup residue (959.9 g), which was then subjected to passage over a polystyrene gel

(Diaion HP-20), eluting with H2O and MeOH. The eluted fraction with MeOH (12.4 g)

84

was subjected to silica gel column chromatography (CHCl3:MeOH = 100:1→50:1→

CHCl3:MeOH:H2O = 7:3:0.5 →6:4:1) to provide nine fractions (fr.1–9). Fr. 4 [1.37 g,

CHCl3:MeOH = 100:1 eluate] was subjected to silica gel column chromatography

(CHCl3;MeOH = 50:1) to afford five subfractions (fr.4-1 to fr.4-5). Fr. 4-2 (527.8 mg)

was then repeatedly chromatographed on silica gel (n-hexane:acetone = 4:1) to afford a

new compound named onionin A 1 (42.2 mg).

2. Chemical Structure

Onionin A (1)

Pale yellow amorphous powder; [α]24

D +16.7°(c = 0.2, CHCl3). Positive HR-FAB-

MS m/z: 243.0489 [M + Na]+

(calcd for C9H16O2S2Na, 243.0489) (70%); and base peak

at m/z 131.0525 [C6H11OS]+ (calcd for C6H11OS, 131.0531). IR νmax (KBr) 1027 and

2366 cm-1

.

1H-NMR (CDCl3) δ: 1.05 (3H, d, J = 6.3 Hz, CH3-3), 1.28 (3H, d, J = 6.9 Hz, CH3-4),

1.90 (3H, dd, J = 1.7, 6.9 Hz, H3-3′), 1.97 (1H, m, H-3), 2.16 (1H, m, H-4), 4.01 (1H, d,

J = 5.8 Hz, H-5), 4.31 (1H, d, J = 10.9 Hz, SH), 4.99 (1H, dd, J = 3.4, 10.9 Hz, H-2),

6.03 (1H, dd, J = 1.7, 13.8 Hz, H-1′), 6.47 (1H, dq, J = 6.9, 13.8 Hz, H-2′).

1H-NMR (C6D6) δ: 0.84 (3H, d, J = 6.9 Hz, CH3-3), 0.93 (3H, d, J = 6.9 Hz, CH3-4),

1.30 (3H, dd, J = 1.7, 6.9 Hz, H3-3′), 1.63 (1H, m, H-3), 2.12 (1H, m, H-4), 3.52 (1H, d,

J = 5.8 Hz, H-5), 4.91 (1H, dd, J = 3.2, 10.7 Hz, H-2), 5.01 (1H, d, J = 10.9 Hz, SH),

5.45 (1H, dd, J = 1.5, 15.1 Hz, H-1′), 6.18 (1H, dq, J = 6.9, 13.8 Hz, H-2′).

1H-NMR (CDCl3 and 0.01 equiv. Eu(fod3)) δ: 1.07 (3H, d, J = 6.9 Hz, CH3-3), 1.27 (3H,

d, J = 6.9 Hz, CH3-4), 1.86 (3H, d, 6.9 Hz, H3-3′), 2.06 (1H, m, H-3), 2.52 (1H, m, H-4),

85

4.04 (1H, d, J = 5.8 Hz, H-5), 5.09 (1H, brs, H-2), 6.05 (1H, d, J = 14.8 Hz, H-1′), 6.52

(1H, m, H-2′); 1

H-NMR (CDCl3 and 0.02 equiv. Eu(fod3)) δ: 1.12 (3H, d, J = 6.9 Hz,

CH3-3), 1.30 (3H, d, J = 6.9 Hz, CH3-4), 1.86 (3H, dd, J = 1.4, 6.6 Hz, H3-3′), 2.09 (1H,

m, H-3), 2.59 (1H, m, H-4), 4.09 (1H, d, J = 5.7 Hz, H-5), 5.20 (1H, brs, H-2), 6.10 (1H,

dd, J = 14.8 Hz, H-1′), 6.58 (1H, m, H-2′); 1

H-NMR (CDCl3 and 0.03 equiv. Eu(fod3))

δ: 1.22 (3H, d, J = 6.3 Hz, CH3-3), 1.34 (3H, d, J = 6.9 Hz, CH3-4), 1.87 (3H, d, J = 6.9

Hz, H3-3′), 2.15 (1H, m, H-3), 2.66 (1H, m, H-4), 4.17 (1H, d, J = 5.8 Hz, H-5), 5.34

(1H, brs, H-2), 6.18 (1H, d, J = 14.9 Hz, H-1′), 6.60 (1H, brs, H-2′).

13C-NMR (CDCl3) δ: 13.9 (CH3-3), 18.1 (CH3-4), 18.3 (C-3′), 42.9 (C-4), 55.0

(C-3),79.2 (C-5), 83.5 (C-2),131.7 (C-1′) and 139.6 (C-2′).

3. Effect of Onionin A on Machrophage Activation

Determination of the Inhibitory Effect of Compound 1 on CD163 Expression

Human monocyte-derived macrophages (5 × 104 cells per well of a 96-well plate) were

incubated with onionin A (1, 30 µM) for 24 h after treatment with IL-10 (20 nM) for

two days, followed by the determination of CD163 expression by cell-ELISA.

Cell Enzyme-linked Immunosorbent Assay (Cell-ELISA)

Expression of CD163 on human monocyte-derived macrophages was evaluated using

a cell-ELISA procedure, as described previously.48)

Briefly, each well of a 96-well plate

was blocked with Block Ace, and washed three times with PBS containing 0.05%

Tween 20 (washing buffer). The wells were incubated with anti-CD163 antibody,

AM3K (2 µg/mL) and dissolved in washing buffer for 1 h. The wells were then washed

with washing buffer three times and reacted with HRP-conjugated anti-mouse IgG

86

antibody, followed by reaction with Ultrasensitive TMB (Moss, Inc. Pasadena, MD).

The reaction was terminated by the addition of 1 M sulfuric acid, and the absorbance at

450 nm was read on a micro-ELISA plate reader.

Statistics

All data are representative two or three independent experiments. Data are expressed

as means ± SD. Mann-Whitney’s U-test was used for two-group comparison. A value of

p < 0.05 was considered statistically significant.

87

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Acknowledgements

To ALLAH, everything in life is resumed. In this work, he has helped me a lot. He

offered me what I did not know and which I have to know. Hence, if only one to be

thanked, God is the first the last. Then, those offered by God to advise and guide have to

be thanked.

My sincere thanks and gratefulness are expressed to the Egyptian Government

(Ministry of Higher Education and State for Scientific Research) for my scholarship and

financial support.

I would like to express my deepest gratitude and appreciation to Prof. Toshihiro

Nohara for his encouragement, valuable supervision, scientific guidance and continuous

support during the process of this work. It was great honor and pleasure to work under

his kind supervision.

Additionally, I would like to special thanks to Prof. Akira Yagi who introduced me to

Prof. Toshihiro Nohara and I got this opportunity to study in Kumamoto University.

It is my great pleasure to express my deep everlasting gratitude and most sincere

thanks to the associate Prof. Dr. Tsuyoshi Ikeda for her kind assistance and unlimited

help during this research.

I wish to express my hearty appreciation and sincere thanks the associate Prof. Dr.

Hitoshi Yoshimitsu (Sojo University), Dr. Hiroyuki Miyashita (Sojo University) for

their helpful assistants and advice.

92

My sincere thanks and gratefulness are expressed to Dr. Yukio Fujiwara for helping me

in biological activity measurement.

I am grateful to Prof. Junei Kinjo (Fukuoka University) for the measurements of

HR-FAB-MS.

I would like to thank N.D.R. Co., Ltd. for their co-operation in the developing of

freeze-dried tomato product.

I would like to express my sincere appreciation to Associate Prof. Dr. Mikako Fujita

and Associate Prof. Dr. Masaharu Sugiura, for inspection of my doctoral thesis.

I would like to thank all members of natural medicine laboratory in Kumamoto

University and Sojo University, for their warms help and encouragements.

Finally, I would like to express my great appreciation and most sincere thanks to all

members of my family for their valuable help and continuous encouragement.

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