Introduction

The Philippines, with its vast wealth of medicinal herbal plants, has surprisingly the

highest breast cancer incidence in Asia. A major concern for society is addressing the prevention

and control of breast, cervical and ovarian cancer. Unfortunately, the cases of real cancer

remissions have been few and far between since most patients are usually diagnosed in the late

stages of the disease. Early, detection of the illness is crucial to the survival of individuals, since

in the early stages the aberrant cells have not yet significantly differentiated and still rely on the

body’s inherent biochemical processes. Sadly, the fact is that most patients die within five years

from their diagnosis. Evidently, there is no panacea and most of medical protocols can only

guarantee the extended survival of conservatively a few months and that is only if the procedure

was successful. Most procedures which include drastic surgery, chemotherapy and radiation may

lead to remission of cancer cells but not without the undeniable risks of future side- effects or

recurrence of the disease.

What is left to most patients is the serendipitous chance that researchers find a

less toxic avenue by which this highly invasive disease can be eradicated. Evidences of the

importance of certain bioactive compounds in produce that could present certain health benefits

have surfaced and these have led to more widespread and extensive scientific investigation. In

fact, the consumption of fresh vegetables and fruits has been clearly advocated by members of

the medical profession which has led to the alteration of the daily nutrition paradigm.

In a number of cases, the presence of glucosinolates in foodstuffs has been the crux of

research. For example glucoraphasatin, a glucosinolate, has been shown to be

present in the tubers of several radish varieties.1 Glucosinolates however are

only useful when hydrolyzed via the enzyme myrosinase which is stored in specialized

vacuoles within the plant structure. Glucosinolates yield a variety of compounds after hydrolysis

depending on factors such as plant species and cultivar, site of hydrolysis, the presence of

cofactors and the environmental conditions.

The presence of peroxidase and isothiocyanates which have been linked to anti-

carcinogenic and tumorogenic activity has been reported in certain radish varieties. The roots

are a rich source of peroxidase, an oxidoreductase, which can scavenge

harmful free radicals. Additionally, 4-(methylthio)-3-butenyl isothiocyanate, a

compound capable of anti-microbial, anti- mutagenic, and anti-carcinogenic

activity has been isolated from radish roots.2 Glucosinolates vary qualitatively and

quantitatively in each radish type and the concentrations will also depend on the extent of

hydrolysis or degradation that has taken place within the plant, and the processing that the

samples have undergone.

1. Statement of the Problem

There has been limited research done on glucosinolates and their corresponding

hydrolysis products in Raphanus sativus. This study focused on the varietal differences in

glucosinolate content, the effect of processing and storage on the major glucosinolates in radish

tubers and the anti-carcinogenic activities of radish extracts against specific cancer cell lines. The

activity of the hydrolytic enzyme, myrosinase and the potential changes in the glucosinolate

hydrolysis products was also investigated on the radish varieties.

2. Significance of the Study

There have been numerous reports suggesting that fruit and vegetable

intake reduces the risk of almost all types of cancer. In many vegetables,

the anticancer properties have been attributed to the hydrolytic products of

glucosinolates. However due to limited investigations, no handling or

processing guidelines or protocols have been established that will ensure

maximum health benefits derived from the consumption of glucosinolate –

rich vegetables. A systematic and careful monitoring of these compounds during

various stages of processing and prolonged storage should be a step in this direction

The proposed research was made on radish tubers, a commonly used local vegetable

known to contain glucosinolates but for which very few in – depth studies have been made. In

addition, few reports have been made on the biological activity of radishes. The observed

changes or variations resulting from varietal differences, processing and storage was used to

suggest appropriate procedures pertaining to the handling and post- harvest treatments that will

provide the maximum health benefits that this specific produce can offer.

3. Objectives

General objective:

This work entailed the investigation of radishes for glucosinolates using HPLC-UV and

LC-MS; myrosinase activity assay via UV-VIS; isothiocyanates utilizing gas chromatography

with either MS or FID detector; and genotoxic activity towards several cell lines employing the

COMET Assay.

Specific objectives:

This study aimed to:

1. determine the concentration of major glucosinolates in locally grown varieties of

radish

2. determine the effects of storage on the

(a) glucosinolate content

(b) myrosinase activity

(c) glucosinolate hydrolysis products

of a selected radish variety

3. determine the

(a) glucosinolate content

(b) glucosinolate hydrolysis products

of a selected radish variety during various phases of processing (boiling,

steaming, pickling)

4. determine the anti – cancer activity of extracts obtained from raw and processed

radish samples.

4. Scope and Limitations

The proposed research envisioned that from the data accumulated the most effective

means of storing and processing be determined so as to minimize loss of glucosinolates and their

hydrolysis products isothiocyanates. The samples was procured immediately before extraction to

ensure reliability of the results. Storage conditions investigated included freezing and ordinary

refrigeration. Culinary processes, meanwhile, covered boiling, steaming and pickling since these

are the common ways by which radishes are prepared in most Filipino homes.

Determination of the farming practices involved in the production of radishes as well as

the amount of fertilizers and the type of soil in which these were grown are beyond the scope of

this research. In spite of this, the samples to be utilized in the experiments was collected from the

same source as soon as possible after harvesting.

Review of Related Literature

Some food constituents may promulgate cancer development, whereas others

have a protective effect. Indoles, phenols, and other phytochemical substances found in

vegetables such as radishes, may reduce the risk for cancer Glucosinolates a type of

phytochemical, which include glucobrassicin, gluconapin, sinigrin, and glucoiberin, are found

abundantly in Cruciferous vegetables of which radishes are a part of. The hydrolysis products

termed isothiocyanates such as ally isothiocyanates, indoles, and sulforaphane, which are derived

from glucosinolates, are present in these foodstuffs.3 On the other hand, studies focusing on these

plants reveal that the compounds of interest may fluctuate according to the combined effect of

several factors. This section presents a review of existing literature about the properties,

occurrence and reactions of glucosinolates as well as the importance of their degradation

products in order to obtain a brief background of what these glucosinolates are and how these

have become one of the subjects of interest in the field of food chemistry.

1. Glucosinolates

1.1 Properties and Occurrence

At the beginning of the 17th century, the unique properties of glucosinolates and

isothiocyanates or mustard oils, as they are commonly known, were investigated to understand

the chemical origin of the sharp taste of mustard seeds. The discovery and early history of

glucosinolates and the participation of the enzyme myrosinase (a thioglucosidase) in their

conversion to isothiocyanates, are the subjects of an interesting and scholarly review by

Challenger (1959).4 The glucosinolates known by the trivial names sinigrin (2-propenyl or allyl

glucosinolate) and sinalbin (4-hydroxybenzyl glucosinolate) were isolated early in the 1830s

from black (Brassica nigra) and white (Sinapis alba) mustard seeds, respectively.4

A striking and characteristic chemical property of cruciferous plants is their high content

of glucosinolates, which often approaches 1% or more of their dry weight and play protective and

evolutionarily important roles in plants. These include allelopathy (suppression of growth of

neighboring plants), specific positive and negative feeding cues for some insects and broad

antibiotic properties including nematocidal, antimicrobial, antifungal, antiprotozoal and

insecticidal activities.

Glucosinolates are -thioglucoside N-hydroxysulfates [also known as (Z)-(or cis)-N-

hydroximinosulfate esters or S-glucopyranosyl thiohydroximates], with a side chain (R) and a

sulfur-linked -D-glucopyranose moiety. The geometrical isomerism at the CN bond was

established to be Z (or cis-) by X-ray crystallographic analysis of sinigrin. Five hundred species

of non-cruciferous dicoty-ledonous angiosperms have been reported to contain one or more of

the over 120 known glucosinolates. Most glucosinolate-containing genera are clustered within

the Brassicaceae, Capparaceae and Caricaceae; of the sixteen families listed in Table 3, these

include the largest number of glucosinolate-containing species.4

The skeleton of glucosinolates consists of a thioglucosidic link to the carbon of a

sulphonated oxime. The R group (side chain) and the sulfate group have anti stereochemical

configuration. The R group is derived from amino acids and is highly variable. It can be aliphatic

such as in the case of alkyl, alkenyl, hydroxyalkenyl, w-methylthioalkyl, aromatic or

heterocyclic. The sulfate group imparts strongly acidic properties and thus the glucosinolates

occur in nature as anions counterbalanced by a cation. The cation is usually potassium, being one

of the most abundant cations in plant tissues. The sulphate group and the thioglucose moiety

impart nonvolatile and hydrophilic properties to all glucosinolates; the R group is variable in

properties from lipophilic to marked hydrophilic. The natural forms of glucosinolates exhibit

levo rotation in solution. Glucosinolates have a large number of homologues and ß-hydroxylated

analogues. As an example -methylthioalkyl side chains range from MeS(CH2)3 to MeS(CH2)8.

The general structure of glucosinolates is shown in Figure 1.

The general structure of glucosinolates

Glucosinolates were originally named according to their plant origin and eventually upon

their side chain structures. Table 1 shows trivial names for some glucosinolates and indicates

their side chain. The semi-systematic name contains the side chain followed by the word

"glucosinolate."

The most extensively studied glucosinolates are the aliphatic, 1-methylthioalkyl, aromatic

and heterocyclic (e.g. indole) glucosinolates, typified by those found in the Brassica vegetables

The most numerous glucosinolates are those containing either straight or branched carbon

chains. Many of these compounds also contain double bonds (olefins), hydroxyl or carbonyl

groups, or sulfur linkages in various oxidation states. The largest single group (one-third of all

glucosinolates) contain a sulfur atom in various states of oxidation (e.g. methyl-thioalkyl-,

methylsulfinylalkyl-, or methylsulfonylalkyl). Another small group of benzyl glucosinolates

have an additional sugar moiety, rhamnose or arabinose, in glycosidic linkage to the aromatic

ring. The presence of these sugars is of unknown significance, although it is intriguing that they

are present in two families of plants (the Moringaceae and Resedaceae) containing certain genera

that are widely exploited for their pharmacological properties. There has been an unconfirmed

report that the 5-carbon sugar, apiose, may be linked to the hydroxybenzyl glucosinolate side

chain in Hesperis matronalis, a member of the Brassicaceae family. Additionally, a number of

sinapoyl and cinnamoyl salts and esters of some of the common glucosinolates are substituted on

the thioglucoside moiety. There have been claims that cinnamoyl derivatives of glucosinolates

predominate in some plants and plant parts, and they present hypothetical structures of p-

coumaroyl, cafeoyl, feruloyl, sinapoyl and isoferuloyl glucosinolates with these phenylpro-penyl

moieties esterified to the S--glucose at positions C-2’ and C-6’.4

Table 1. Chemical structure of the most commonly occurring glucosinolates in cruciferous vegetables

C

S

N

R

-glucose

sulfate

R = Alkyl

C=C-COH-C-

C=C-C-C

C-SO-C-C-C-

C-SO-C-C-C-C-

C-S-C=C-C-C-

C=C-C- Sinigrin

Progoitrin

Gluconapin

Glucoiberin

Glucoraphanin

Glucoalyssin

Glucoraphasatin

C-SO-C-C-C-C-C-

R = Aryl

C-C- Gluconasturitin

R = Indolyl

N

Rn

CH2R4

R4 Rn

H H

HOH

OCH3

OCH3H

H

Glucobrassicin

4-Hydroxy-glucobrassicin

4-Methoxy-glucobrassicin

Neoglucobrassicin

1.2 Biosynthesis

Studies have shown that glucosinolates are derived from amino acids. The biosynthetic

studies have involved feeding experiments with labeled compounds, isolation of intermediates

and isolation of some of the enzymes involved in the pathway. Aliphatic, indole and aromatic

side chains are derived from methionine, tryptophan and phenylalanine respectively, from both

protein and non-protein sources. The initial steps in the formation of most glucosinolates are N-

hydroxylation followed by oxidative decarboxylation to yield an aldoxime. These steps are

common to the biosynthesis of other groups of natural products. The biosynthetic pathways then

diverge at the aldoxime to produce different compounds. The majority of glucosinolates possess

aglycone structures which are not related to protein amino acids. It is generally accepted,

however, that these glucosinolates are also derived from protein amino acids. These protein

amino acids undergo a chain elongation process in which their 2-oxo-acids condense with

acetate. The entire homologous series of glucosinolates with side chains ranging from R=

MeS(CH2)3 to R=MeS(CH2)8 is considered to be derived from repeated chain extensions starting

with methionine. Each time the sequence is traversed a new higher amino acid homologue is

formed. A glyoxylate aminotransferase is believed to be the first enzyme of the chain elongation

process. This enzyme catalyses the formation of the 2-oxo-acid from its corresponding amino

acid.5

    All the intermediates between the amino acid and the glucosinolate are nitrogenous and the

amino acid carbon-nitrogen bond is preserved. The amino acid, whether it has undergone chain

elongation or not, is specifically hydroxylated to the N-hydroxyamino acid in the presence of

oxygen and NADPH. The N-hydroxyamino acid is decarboxylated to give the aldoxime,

followed by a reduction step to a nitro compound which tautomerizes to the acid form. The

thiohydroximate is then formed by introduction of sulfur; feeding experiments have shown that

cysteine is involved as the sulfur donor. The thiohydroximate is transglycosylated to the

desulphoglucosinolate. An enzyme catalysing the transfer of glucose from UDP- glucose to the

thiohydroximate has been isolated. The glucosinolate is obtained by sulphonation and it is known

3`-phosphoadenosine-5`-phosphosulphate (PAPS) is involved as the sulfate donor. A summary

of the biosynthesis of glucosinolates is shown in Scheme 1.

RHC COOH

NH2

Amino Acid

NADPH + O2

H2C C

N

OH

H

Oxime

NADPH + O2

R

H2C C

N+

OH

H

R

-O

Aci-nitro compound

CysH2C C

N

OH

S

R

H2C

HC COOH

NH2

S-alkyl-thiohyroximate

H2C C

N

OH

SH

Thiohydroximate

R UDPG

H2C C

N

OH

S

RGlu

DesulfoglucosinolatePAPS

H2C C

N

O

S

RGlu

SO3-

Glucosinolate

Scheme 1. Proposed pathway for biosynthesis of glucosinolate synthesis5

It has been reported that glucosinolates are evolutionarily related to cyanogenic

glucosides as both groups of natural plant products are derived from amino acids that are

converted into oximes by cytochromes P450 that belong to the CYP79 family. In addition,

CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in

the biosynthetic pathway of glucosinolates by the combined use of a biochemical and a

bioinformatics approach. The data derived from a study a was consistent with the hypothesis that

the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a

'P450mox' that converted the oxime into a toxic compound that the plant detoxified into a

glucosinolate.6

1.3 Hydrolysis

Glucosinolates are invariably accompanied in plant cells by the enzyme myrosinase (a ß-

thioglucosidase), which is normally physically segregated from its glucosinolate substrates but is

released and hydrolyzes glucosinolates to isothiocyanates and other products when plants are

injured by predators or when food is prepared or chewed. This reaction is responsible for the

development of the sharp taste of horseradish, mustard and wasabi. In the absence of myrosinase,

for example, when plants are cooked and myrosinase is heat inactivated, humans can efficiently

convert glucosinolates to isothiocyanates through the action of the microflora of the

gastrointestinal tract. 7

A variety of products which are dependent on pervading conditions are usually formed,

although isothiocyanates are usually obtained. The formation of isothiocyanates is favored at

high pH via a Lossen-type rearrangement reaction where a nitrogen atom migrates followed by a

loss of a sulfate group. At low pH, however, the formation of the nitrile is more favored.

Thiocyanates may also be formed depending on the reaction conditions. The formation of cyano-

epitho alkanes, on the other hand, is catalyzed by a small protein called epithiospecifier protein

(ESP) which co-occurs with myrosinase. ESP interacts with myrosinase to facilitate the transfer

of sulfur from the S-glucose group of a terminally unsaturated glucosinolate to the alkenyl group

eventually forming epithionitriles as shown in Scheme 2.

At pH 6 to 7, the major hydrolysis products are stable ITCs, except for GLSs possessing a

β-hydroxylated side chain or an indole moiety; β-hydroxy-ITCs are unstable and spontaneously

cyclize to oxazolidine-2-thiones (e.g. goitrin) whereas indole ITCs undergo lysis. As a result, the

corresponding alcohol, such as indole-3-carbinol (I3C), is formed, which subsequently

condenses into dimers, trimers or tetramers. In the presence of ascorbic acid, ascorbigen and

thiocyanate are the major products of indole GLSs between pH 4 and 7.15 In some plant

varieties, autolysis of fresh plant material produces nitriles, but the mechanism of this nitrile

formation and the possible role of a ‘nitrile-forming factor’ are still not clear.8

R C

S Glu

NOSO3

Myrosinase

-Thioglucosidase

Glucosinolate

R C

S SH

NOSO3

+ Glucose

Thiohydroximate-O-sulfonate-H2SO4

pH 6-7

N C S

Stable ITC

Unstable ITC

RHC CH2 CN S

H2O

- OH ITC

N

H2C N C S

R2

R1

Indolylmethyl ITC

pH 3-4; pH5-7 tissue dependent; nitrile forming factor? Fe2+

S-

R C N

Indole and Aromatic; indole and OH nitriles

R SH

C NH

Thiocyanates

mechanisim of formation unknown

Epithiospecif ier

n=alkenyl

H2CHC

S

(CH2)n C N

EpithioalkylnitrileDepending on plant tissue& proximal double bonds

SpontaneousCyclization

OS

NH

O

R

5-oxazolidine-2-thioneGoitrin R=CH=CH

+Ascorbic Acid

Ascorbigen

N

H2C OH

R2

R1

Indole-3-carbinol

3,3 Diindolylmethanethen trimers, tetramers,etc.

Fe2+

Scheme 2: Hydrolysis of glucosinolates.8

Multiple forms of myrosinase called myrosinase isoenzymes also exist in different plants

as well as in the different parts of the same plant such as the leaves, stem, roots or seeds. The

species of the plant, developmental stage and age also affect the distinct pattern that the

myrosinase isoenzyme may contain and its activity. Some studies even reported that myrosinase

activity was higher in the outer leaves of the Brussel sprouts than in the inner leaves or stalk.

Researchers have cited that total potential myrosinase activity is high during seedling growth of

plants and that ascorbic acid plays a vital role in the activation of the isoenzymes. Separation of

these isoenzymes had been done using techniques such as analytical gel electrophoresis revealed

that the myrosinase isoenzymes may have different degrees of glycosylation and could degrade

different glucosinolates at different rates.

Since the hydrolysis of glucosinolates only take place after the plant is wounded, an

analysis of the specific location of glucosinolates, myrosinase and ascorbic acid presents three

possible substrate and enzyme locations. The glucosinolates, myrosinase and ascorbic acid may

be located in different cells, in the different compartment of the same cell or inside the same

compartment of the same cell but in an inactive form. Of the three, the possibility that the

glucosinolates, myrosinase and ascorbic acid are located in separate cells or that these are in the

same compartment of the same cell but in an inactive form offer more realistic explanations to

the organization of the myrosinase – glucosinolate system.

As previously mentioned, a number of products can be obtained during the enzymatic

degradation of glucosinolates. Nitriles and epithionitriles are formed by the action of

epithiospecifier protein (ESP) which requires the presence of ferrous ions which is assumed to

facilitate the formation of an intermediate from the thiohydroximate followed by sulfur insertion

producing cyanoepithionitriles and nitriles as seen in Scheme 3. Current studies were even able

to show that overexpressing ESP can possibly divert isothiocyanate formation to nitrile

formation; however, since ESP is even more sensitive to heat than myrosinase, isothiocyanates

may be formed in larger quantities if the plant sample is subjected to short heating.

Scheme 3: Proposed mechanism for cyanoepithioalkanes and nitrile formation.9

Isothiocyanates are inherently considered to form in the presence of myrosinase and a

thiocyanate forming factor only when the glucosinolates can yield stable carbocations after the

enzymes. Examples of stable glucosinolates would include benzyl-, propenyl- and 4-

methylthiobutyl-glucosinolates. Unstable isothiocyanates that undergo solvolysis with water are

considered as the precursors of indolyl glucosinolates. It is important to add that other products

which are very specific of the glucosinolate precursors may also form depending on the reaction

conditions inside the plant cells.8

1.4 Chemical Degradation

Aside from the action of myrosinase, glucosinolates can also undergo chemical and

thermal degradations. Researches have showed the transformation of glucosinolates to a variety

of products without the use of myrosinase such as the chemical degradation of 2-propenyl

glucosinolate to different substances (Scheme 4).10

The parent glucosinolate, 4-(methylsulfinyl) but-3-enylglucosinolate was purified and

converted to the silver derivative and decomposed by sodium thiosulphate, which mainly

resulted in production of the isothiocyanate with some of the corresponding nitrile. Similarly it

was found that the silver derivative of benzylglucosinolate on acid and alkaline hydrolysis gave

benzylcyanide (2-phenylacetonitrile).

The reduction of glucosinolates to amines has been demonstrated by using Raney

nickel/water at high temperatures. Here the amino group is at the carbon atom corresponding to

C-0 in glucosinolates, and the products are not the amines proposed to arise from catabolism, in

which the amino group is bonded to C-1 of the glucosinolates. 11

Acid decomposition of glucosinolates leads to the corresponding carboxylic acid together

with hydroxylammonium ion and has been used in the identification of new glucosinolates. Base

hydrolysis of glucosinolates results in the formation of several products. In addition to allyl

cyanide (but-3-enenitrile) and ammonia, thioglucose is obtained from 2-propenylglucosinolate

with aqueous sodium hydroxide.12 Thioglucose has also been reported as a product of the reaction

of 2-propenylglucosinolate with potassium methoxide. While, basic degradation of 4-

hydroxybenzylgluosinolate gives thiocyanate, indol-3-ylmethylglucosinolate gives glucose,

sulphate, H2S, thiocyanate, indol-3-ylacetic acid, indol-3-ylacetamide (2-(1H-indol-3-

yl)acetamide), indol-3-ylmethyl cyanide (2-(1H-indol-3-yl)acetic acid), 3-(hydroxymethyl)indole

(1-H-indol-3-yl)methanol), 3,3′-methylenediindole (di(1H-indol-3-yl)methane), indole-3-

carbaldehyde 2-(1H-indol-3-yl)acetaldehyde, and indole The base hydrolysis of 2-

propenylglucosinolate and benzylglucosinolate with 2 M NaOH has been found to give the

corresponding amino acids.13

It is thought that the amino acids are derived from a Neber rearrangement.

Mechanistically this takes place by the loss of a proton from the carbon atom α to the C N

grouping, with subsequent bond formation between the alpha carbon atom and the nitrogen atom

with the loss of arene sulphonate ion to give an azirine, and addition of an alcohol molecule to

give an alkoxyaziridine.14 Ring opening then takes place by the addition of a second molecule of

alcohol, and hydrolysis gives the α-aminoketone.

Depending on the pH of the solution, metallic salts of aglucones can undergo

decomposition to the cyanide or isothiocyanate. It was found that the silver derivative of the

aglucone of 2-propenylglucosinolate could degrade in the presence of iodide ion to give either

cyanide or isothiocyanate, depending on the pH of the solution. Scheme 4 gives a summary of

some chemical degradation reactions of 2-propenyl glucosinolate.

Scheme 4. Chemical degradation of 2-propenylglucosinolate 15

1.5 Thermal Degradation

Thermal degradation, on the other hand, showed that the degradation of some

glucosinolates is complete at 100°C in as fast as 30 minutes. The disappearance of the

glucosinolates and appearance of the hydrolysis products can be determined by UV analysis and

most of the thermal degradation studies involved the common processes done during cooking

such as microwave treatment and boiling. Some of the analysis on the concentrations of

individual and total glucosinolates showed that about 50% of the total glucosinolates were lost

after cooking. When myrosinase was inactivated before thermal degradation, it was found that

the indolyl glucosinolates were more sensitive to heat compared with the aliphatic

glucosinolates.16

Since the hydrolysis of glucosinolates produces different compounds, some of which

have biological significance, there has been interest in monitoring not only the rate of product

formation but the hydrolysis process as a whole. Comprehensive research that describe in detail

the complete monitoring of the process as well as the products that involve the common methods

of glucosinolate analysis such as gas chromatography (GC) or high performance liquid

chromatography (HPLC) are not yet available.

In a recent study, however, capillary electrophoresis was utilized in the simultaneous

monitoring of enzymatic hydrolysis and its degradation products. Glucosibarin was chosen as the

model for the method which was subsequently validated using other glucosinolates such as

glucobarbarin, progoitrin, glucotropaeolin and gluconasturtiin. The method developed was

shown to be easy, economical and time-bound since it only makes use of small volumes of

enzyme and substrate, can be monitored in a 24 hour period and also covers a considerable pH

range (from 3 to 8).17

1.6 Effects of Processing

Studies focusing on how long the glucosinolates remain intact in the plant and how fast

they are hydrolyzed have also been made using time-trial experiments. Results from one study

revealed that storage of some glucosinolate-containing plants in ambient temperature (12-20ºC)

does not significantly decrease the glucosinolate content; however, storage in a refrigerator with

temperatures from 4-8 ºC decreased the glucosinolate concentration. Losses were attributed to

the destruction of plant cells in the freezing and thawing processes. Significant losses were also

detected after 7 days storage. Other studies focusing on the impact of cold storage on

glucosinolate levels confirmed losses in glucosinolate content after several days of storage,

however, these losses may still depend on the plant samples used as well as the kind of

glucosinolate being monitored.

An investigation conducted to determine the total glucosinolate, vitamin C and

other health-promoting compounds in broccoli as affected by transport and distribution reported

that loss in total glucosinolate occurred even when the samples were refrigerated. Losses after

the whole transport, distribution and retail sale (2-3 days maximum) periods where cold storage

was employed amounted to 71-80% total glucosinolate loss. On the contrary, vitamin C were

only slightly affected.

Most literature report losses in glucosinolate content during cold storage in a

refrigerator with temperature up to 4ºC; other studies involving storage under controlled

atmosphere claimed that total glucosinolate content increased rather than decreased after 7 days

of storage. An investigation of the total and individual glucosinolate content in Marathon

broccoli florets stored for 7 days at 10 ºC under air and other varied conditions such as O.5% O2,

0.5% O2+20% CO2 , 20% CO2, followed by transfer to air for 2 days revealed that the samples

stored under air and 0.5% O2+20% CO2 higher total glucosinolate contents than the freshly

harvested broccoli at the end of the 7 day storage period with a slight decrease at day 9 due to the

deterioration of the sample. However, individual glucosinolate profiles such as glucobrassicin

and 4-methoxyglucobrassicin concentrations gave different results after exposure to the different

controlled atmospheres and subsequent aeration.

Aside from storage, basic culinary processes employed by man before consuming

vegetables that contain glucosinolates also affect the concentrations of these compounds. The

inactivation of myrosinase, loss of enzyme cofactors, leaching of the glucosinolates and thermal

degradation are only some of the factors that needs to be thoroughly investigated. Glucosinolates

appear to be stable with respect to heating, but not myrosinase since common heating

temperatures during cooking inactivates the enzyme. However, loss of glucosinolates, even

without enzymatic hydrolysis, is still evident because the compounds are leached out into the

cooking water after cell lysis. These losses during boiling are affected by the size of cut pieces of

the vegetables, vegetable to water ration and duration as well as extent of heating. Blanching and

microwaving have also resulted in lower total glucosinolate levels with thermal degradation

being speculated to take place during microwaving. On the contrary, thermal degradation studies

utilizing red cabbage instead of broccoli showed that total extractable glucosinolate content even

increased. In this thermal degradation study, myrosinase was inactivated to distinguish

enzymatic from thermal breakdown. Differences in the results may have resulted from the use of

a different vegetable (red cabbgage) other than broccoli or the intensity of the microwaving

conditions. Nevertheless, these studies still provide some insights as to the fate of the

glucosinolates when they are subjected to microwave treatment. A more detailed investigation is

therefore needed to monitor the changes in glucosinolate levels during different intensities and

durations of microwave treatment as applied in different intensities and durations of microwave

treatment as applied in different vegetables that are commonly consumed by man. With this, a

standard set of conditions may possibly be determined for a certain vegetable in order to obtain

the maximum amount of glucosinolates that could be beneficial in the diet.

Individual glucosinolate contents in red cabbage as affected by other thermal

degradation processes such as standardized conditions of blanching, cooking and canning were

also previously investigated. As opposed to experiments conducted by actually blanching,

cooking and canning the red cabbage, which also includes not only thermal breakdown but

leaching as well, this experiment utilized the estimated degradation kinetics obtained for the

individual glucosinolates in red cabbage to perform a processing simulation that would predict

thermal degradation effects alone. Results of the processing simulation indicated that belching

does not significantly affect the glucosinolates while cooking significantly degrades the indole

glucosinolates more as compared with the aliphatic glucosinolates. Canning, on the other hand,

which has a standardized condition of subjecting the sample at 120ºC for 40 minutes, was shown

to significantly affect all the glucosinolates, thus presenting the issue of decreased health benefits

from canned Brassica vegetables. In addition, since the study only utilized estimated degradation

kinetics to account for thermal degradation, losses in the glucosinolate concentrations may even

be higher when these vegetables are actually subjected to the states conditions since leaching

may also take place.

Actual thermal processing studies which utilized not only a single but four

Brassica vegetables in the analysis showed that cooking by steaming, microwaving and stir-

frying methods can preserve glucosinolate contents in brassica vegetables. Cooking methods that

allow the retention of myrosinase activity may also promote more health benefits since this

allows the increased conversion of glucosinolates to isothiocyanates especially during chewing.

2. Description of Raphanus Sativus

Raphanus sativus is a widely cultivated vegetable in the Philippines. It is considered an

anthelmintic, antifungal, antibacterial, antiscorbutic, diuretic, laxative, tonic, carminative,

corrective, stomachic, cholagogue, lithotriptic, and emmenagogue. The juice of the fresh root is

considered powerfully antiscorbutic. In Iranian traditional medicine, seeds are considered

diuretic carminative, antifever, antitussive and gastric tonic. Radishes are rich in ascorbic acid,

folic acid, and potassium. They are a good source of vitamin B6, riboflavin, magnesium, copper,

and calcium. One cup of sliced red radish bulbs provides approximately 20 calories, largely from

carbohydrates. Table 1 lists the botanical taxonomy of Raphanus sativus.18

Table 2. Scientific classification of Raphanus sativus.

Scientific ClassificationKingdom PlantaeUnranked AngiospermsUnranked EudicotsUnranked RosidsOrder BrassicalesFamily BrassicaceaeGenus RaphanusSpecies R.sativus

Binomial NameRaphanus sativus

3. HPLC Glucosinolate Analysis

Cruciferous vegetables and spices such as broccoli, garden cress and black mustard were

analyzed to demonstrate the negative mode methodology for LC–TOF-MS used to confirm the

molecular formula of glucosinolates in these foodstuffs. Figure 2 shows extracted ion

chromatograms of glucosinolates identified in broccoli extracts. Besides minor glucosinolates,

glucoerucin (C12H23NO9S3, calc. 421.0535; found: 421.0535), glucoraphanin (C12H23NO10S3,

calc.: 437.0484; found: 437.0483), glucobrassicin, (C16H20N2O9S2, calc.: 448.0610; found

448.0608), 4-methoxyglucobrassicin (C17H22N2O10S2, calc.: 478.0716 found: 478.0719) and

neoglucobrassicin (C17H22N2O10S2, calc.: 478.0716 found: 478.0715) could be identified.19

Glucotropaeolin (C14H19NO9S2, calc.: 409.0501; found 409.0503) eluted at 16.2 min was

the major glucosinolate in garden cress, followed by 4-methoxyglucobrassicin (C17H22N2O10S2,

calc.: 478.0716; found 478.0715) at 19.9 min as a minor compound. Black mustard seeds

contained sinigrin (C10H17NO9S2, calc.: 359.0345; found 359.0346) as the dominant compound,

and gluconapin (C11H19NO9S2, calc.: 373.0501; found 373.0498), glucoibervirin (C11H21NO9S3,

calc.: 407.0378; found 407.0376), gluconasturtiin (C15H21NO9S2, calc.: 423.0658; found

423.0656) and 4-hydroxyglucobrassicin(C16H20N2O10S2, calc.: 464.0559; found 464.0557) as

minor glucosinolates. The HPLC conditions were as follows 100 × 2.1-mm ODS Symmetry

column with a 10 × 2.1-mm guard column and mobile phase 0.1% TFA in water with a linear

gradient of 0–10% methanol over 0–30 min or (in parentheses) 50 × 2.1-mm graphitic Hypercarb

column and mobile phase 0.1%TFA in water with a linear gradient of 50–100% methanol from 0

to 10 min and isocratic 100% methanol for 20 min. Table 3 gives a summary of the conditions

utilized.

The mass spectrometer was tuned by direct infusion of standard sinigrin producing

maximum abundant precursor ion m/z 358 ([M−H]−) and fragment ion m/z 97 ([SO3H]−) signals

(approximately in equivalent abundance) during MS/MS. The mass spectrometric conditions

were as follows: capillary, 2.35 kV; cone voltage, −35 V (RF-1, 50 V); desolvation gas

temperature, 450 °C at a flow of 16.5 L/min; source temperature, 120 °C; collision energy,

18 eV. The following transitions were used to assay 10 individual glucosinolates: glucoiberin

(422 > 97), sinigrin (358 > 97), progoitrin (388 > 97), glucoerucin (420 > 97), glucoraphanin

(436 > 97), gluconapin (372 > 97), glucoalysin (450 > 97), glucobrassicin (447 > 97),

neoglucobrassicin (477 > 97), and 4-methoxy glucobrassicin (477 > 97).20

Quantitative determination of glucosinolates in cruciferous vegetables can be achieved by

a combination of adequate component separation and appropriate detection selectivity. The

discriminatory character of LC-MS/MS using SRM detection is its superior sensitivity and

selectivity. Previous studies using negative ion tandem mass spectrometry showed that MS/MS

of the deprotonated molecule ([M−H]−) of intact glucosinolates produced a characteristic

fragment of m/z 97 ([SO3H]−), which facilitates conducting MS/MS SRM experiments. Figure 3

shows representative SRM mass chromatograms from analysis of glucosinolates in broccoli

sprouts using LC-ESI/MS/MS with SRM detection. Ten glucosinolates and the internal standard

were determined simultaneously during the MS/MS analysis using SRM detection, providing

improved sensitivity and selectivity in comparison to the commonly used full scan and selected

ion monitoring techniques. Because neoglucobrassicin and 4-methoxyglucobrassicin exhibit

identical molecular masses and fragment ion (m/z 97), they were differentiated by comparison

with reported elution sequence during reversed-phase HPLC. Co-elution of glucoiberin,

progoitrin, and sinigrin, as previously reported as observed during LC-MS/MS analysis which

may affect the quantification of these compounds by HPLC.

ESI ion-trap mass spectrometry has proved to be an important technique for the

classification and quantification of glucosinolates. Several studies have utilised the MS

fragmentation behavior of glucosinolates for targeted analysis. Glucosinolates undergo consistent

neutral losses during MS fragmentation. Some works have utilized the loss of the glucose moiety

(−162) in a LC-ESI-tandem mass spectrometry study on various plants of the family Brassicacea.

Other authors have used the m/z 96 or 97 ion ([SO3H]−) as being indicative for glucosinolates 21

The fragmentation of a wide range of aliphatic, alkenyl and indole glucosinolates has

demonstrated the utility of a sulfated glucose moiety fragment (m/z 259) for both targeted and

untargeted analysis of glucosinolates using ESI-ion trap mass spectrometry. Also described, is a

novel parent ion mapping approach that allows rapid assessment of glucosinolate content. This

method takes advantage of the fragment (m/z 259) which is consistently produced by the

disassociation of glucosinolates in the ion trap mass spectrometer. This fragmentation can also be

employed for quantification via LC-ion trap-MS.21

3.1 Mechanism of Gas Phase Fragmentation

It has been identified that the MS/MS fragmentation of glucosinolates gives a number of

generic ions. The most discussed is the HSO4 - ion at m/z 97. However, the more diagnostic ion is

the m/z 259 ion, since the loss of sulfate may occur in other sulfated metabolites, such as sulfated

sterols. The larger m/z 259 ion is due to a sulfated glucose moiety and has been observed by

several authors.1 Although the structure has been described as a sulfated glucose, there has been

no attempt to describe the gas phase reaction leading to this ion. Figure 4 offers a possible

explanation for the formation of m/z 259. Further fragmentation of the m/z 259 ion resulted in the

spectrum shown. These fragmentations support the ring-opened structure proposed. Accurate

mass measurements found in Table 4 support the fragmentations depicted in Figures 4. The m/z

259 ion was consistently produced from glucosinolates, though the relative abundance of this ion

was not uniform. For example, although the MS2 spectrum of sinigrin was dominated by the m/z

259 ion, glucoraphanin preferentially fragmented to m/z 372 (the loss of the methyl sulfoxide

moiety). While the m/z 259 was present in the MS2 spectrum of glucoraphanin, it was more

abundant in the MS3 spectrum. 22

3.2 Ion mapping experiments

LC-MS techniques have been employed to investigate the glucosinolates content of many

plant extracts. Ion mapping offers a sensitive, qualitative technique to rapidly and simultaneously

assess a wide range of glucosinolate contents. The aqueous plant/seed extract can be infused

directly into the mass spectrometer. Analysis using this technique required less than three

minutes for a mass range of 300–900 and less than 2 min for a mass range of 300–600. The ion

mapping experiments identified any parent ion that gives rise to the daughter ion at m/z 259, an

ion consistently generated in the ion trap fragmentation of glucosinolates. Infusion of the

broccoli seed extract generated an intensity map of the parent ions and a spectrum view of the

parent ions detected as seen in Figure 1. The glucosinolates were identified from the [M–H]− ion

as confirmed by LCMSn analysis. Studies have shown the following results: m/z 358 sinigrin, m/z

372 gluconapin, m/z 388 progoitrin, m/z 406 glucoiberverin, m/z 420 glucoerucin, m/z 422

glucoiberin, m/z 436 glucoraphanin, m/z 463 4-hydroxyglucobrassicin, m/z 477

neoglucobrassicin and/or 4-methoxyglucobrassicin.22

3.3 LC/MSn analysis

The apparently ubiquitous nature of the 259 fragmentation ion allows the rapid

identification of glucosinolates in a complex mixture by LCMSn analysis. Research done on a

mixture of glucosinolates containing glucoiberin (m/z 422), glucoraphanin (m/z 436),

glucosinalbin (m/z 424), glucotropaeolin (m/z 408), glucoerucin (m/z 420) and neoglucobrassicin

(m/z 477) was analyzed by LC-ESI-ion trap mass spectrometry. The LC-ion trap ms analysis

allowed identification of all these compounds. An extraction of the dependant scans at m/z 259

correlated to each metabolite in the standard mix. Figure 5 shows the targeted analysis of

brassica sprouts which depicts the parent ion and MSn identification of glucosinolates via

extraction of the m/z 259 ion.

To confirm that this ion was due to the sulphated sugar moiety, the mixed standards were

subjected to enzymatic desulfation. This desulphated mixture was analysed by both the unbiased

and targeted methods, with no detection of intact glucosinolates or the ion at m/z 259. The

method was also applied to a mustard seed extract, broccoli seed extract and an extract of

commercially available mixed brassica sprouts. In each case the m/z 259 ion was observed. The

results are summarised in Table 5. 22

4. Gas Chromatographic Analysis of Isothiocyanates

Derivatization of isothiocyanates with ammonia to the corresponding thiourea with

positive ion electrospray ionization LC–MS/MS gave detection with an approximately >1000-

fold increase in sensitivity. The detection involved neutral fragment loss of ammonia. Although

this is a common fragment in biological analytes, specific parent mass detection for the various

isothiocyanates eliminated other interferences in the samples. When progoitrin was hydrolyzed

to the corresponding isothiocyanate, 2-hydroxybut-3-enyl isothiocyanate, the isothiocyanate

initially formed undergoes an intramolecular and reversible cyclization to form the oxazolidine,

5-vinyl-oxazolidine-2-thione. This still gave the expected MRM transitions for the

isothiocyanate in LC–MS/MS analysis when analyzed with and without derivatization.

Analytical recoveries were 57–84%, and interbatch coefficients of variation were 9%. a HPLC

conditions: 100 × 2.1-mm ODS Symmetry column with a 10 × 2.1-mm guard column and mobile

phase 0.1% TFA in water with a linear gradient of 0–95% methanol over 0–30 min. Table 6 lists

the isothiocyanates quantitation of isothiocyanates by negative ion MRM.23

The amine degradation products of isothiocyanates were detected by acetylation to N-

acetamides and positive ionization electrospray LC–MS/MS. The N-acetamide derivatives had

retention times of 4.5–26.3 min on the ODS column and LODs of 1–2 pmol as seen in Table 1

and Figure 1 The optimized MRM transitions for methylsulfinylalkyl N-acetamides derived from

glucoiberin, glucoraphanin, and glucoalyssin gave fragment ions formed by loss of

methylsulfonic acid CH3-S( O)H. 5-Vinyl-oxazolidine-2-thione was stable to hydrolysis;

hence, the detection of a related amine derivative was not investigated. For allylamine and 3-

butenylamine derivatives, the optimized MRM transition gave fragment ions with loss of the

acetyl-derived fragment CH2 C O. In the MRM transition of phenethyl N-acetamide, the

optimized MRM transition gave a fragment ion with loss of N-methylacetamide. Analytical

recoveries were 64–72%, and interbatch coefficients of variation were <9%. 23

4.1 Gas Chromatographic Analysis of Isothiocyanates with and without Exogeneous

Myrosinase

Analyses were performed on a Hewlett–Packard GC–MS system (GC model 5890 with a

mass selective detector model 5971A) using two columns with different polarities of stationary

phases: HP-20M (polyethylene glycol; 50 m × 0.2 mm i.d., film thickness 0.2 μm) or HP-FFAP

(polyethylene glycol-TPA modified; 50 m × 0.32 mm i.d., film thickness 0.52 μm) and HP-101

column (dimethylpolysiloxane, 25 m × 0.2 mm i.d., film thickness 0.2 μm). GC operating

conditions for polar columns (HP-20M or HP-FFAP) were: oven temperature was kept at 70 °C

for 4 min and programmed to 180 °C at a rate of 4 °C/min. For the HP-101 column, oven

temperature was programmed from 70 °C isothermal for 2 min, then to 200 °C at a rate of

3 °C/min. Carrier gas was helium with flow rate 1 ml/min, injector temperature 250 °C, volume

injected 1 μl, split ratio 1:50. MS conditions: ionisation voltage 70 eV, ion source temperature

280 °C, mass range 35–350 mass units.24

Mass spectra were obtained using a Quattro Ultima triple quadrupole ion-tunnel mass

spectrometer (Micromass, Manchester, UK) coupled to a Waters 2696 HPLC system equipped

with a 996-photodiode array detector (Waters Associates). HPLC separation was performed on a

250 × 4.6-mm (5-μm) Luna C18 (2) reversed-phase column (Phenomenex, Torrance, CA) with a

Phenomenex security guard column. A linear-gradient mobile phase from 100% A (water

containing 0.5% trifluoroacetic acid) to 15% B (acetonitrile) in 10 min, to 40% B in 5 min, to

50% B in 5 min, and returned to 100% A in 5 min was used to elute the analytes at a flow rate of

1 mL/min. Approximately 100 μL/min of the HPLC eluant separated by a microsplitter was

delivered to the Z-spray ESI source. Negative ion tandem mass spectrometry (MS/MS) was

conducted to detect glucosinolates with selected reaction monitoring (SRM).25

A. sinuata volatiles were isolated by different methods: hydrodistillation (without and

upon autolysis) and dichloromethane extraction upon exogenous myrosinase hydrolysis. The

major components in all samples, obtained either by hydrodistillation or extraction, were

sulphur- and/or nitrogen-containing compounds derived from glucosinolate degradation (Table

1). Structure and mass spectral data of the major isothiocyanates and nitriles formed by

glucosinolate degradation are given in Table 2. Also, to identify volatile O-aglycones, O-

glycosidically bound volatiles were water-extracted and hydrolysed by β-glucosidase (Table 9).

The compounds in Table 1 and Table 3 are listed in order of their elution on polar column (FFAP

and HP-20M, respectively).25

Hydrolysis of the fresh plant material by exogenous myrosinase liberated compounds was

then extracted with dichloromethane. The most abundant compounds in all hydrodistillates

originated from degradation of two glucosinolates, glucoberteroin and glucobrassicanapin.

Glucoberteroin degradation products were 6-(methylthio)hexanenitrile (36.6–51.5%) and 5-

(methylthio)pentyl isothiocyanate (0.4–9.5%), while 5-hexenenitrile (2.6–14.6%) and 4-pentenyl

isothiocyanate (0.7–8.1%) originated from glucobrassicanapin degradation. 5,6-

Epithiohexanenitrile, that can be formed from glucobrassicanapin, was detected only among

volatiles of fresh plant material obtained by hydrodistillation without autolysis but in a small

percentage (0.6%). In general, all hydrodistillates were nitrile-type. The contents of nitriles in

these samples were 49.5% and 67.6%, depending on whether fresh (without or upon autolysis) or

dried plant material was used. However, the volatiles isolated from fresh plant material also

contained significant percentages of isothiocyanates, 18.3% and 8.7%, respectively, against 1.2%

in hydrodistillate isolated from dried plant material. These results are in accordance with our

previous findings; i.e. drying of plant material favored nitrile formation.25

Two main glucosinolate degradation products identified among volatiles isolated by

extraction upon exogenous myrosinase hydrolysis were 6-(methylsulfinyl) hexanenitrile (11.5%)

and 5-(methylsulfinyl) pentyl isothiocyanate (trivial name alyssin; 10.2%). These compounds

originated from glucoalyssin. Other, quantitatively important volatiles, 4-pentenyl isothiocyanate

(8.9%), 5-hexenenitrile (4.8%) and 5,6-epithiohexanenitrile (3.4%), are degradation products of

glucobrassicanapin. It is interesting that 6-(methylsulfinyl) hexanenitrile was identified in

hydrodistillate of fresh plant material without autolysis (5.2%), while 5-(methylsulfinyl) pentyl

isothiocyanate was absent.

It was also reported that sulforaphane, identified from broccoli, degraded in an aqueous

solution at 50 and 100 °C into dimethyl disulphide, S-methyl methylthiosulfinate, S-methyl

methylthiosulfonate, methyl (methylthio)methyl disulphide, 1,2,4-trithiolane, 4-isothiocyanato-1-

(methylthio)-1-butene, and 3-butenyl isothiocyanate. Alyssin is an analogue of sulforaphane and

is probably thermolabile, so it may degrade to 4-pentenyl isothiocyanate. Furthermore, alyssin

may subsequently degrade to other volatiles, such as dimethyl disulphide and S-

methylmethyltiosulfonate. However, these compounds, as well as other high volatile compounds,

will distill the most rapidly and many of them can be lost in the atmosphere during

hydrodistillation.

In contrast to hydrodistillates, this volatile sample contained epithionitriles in higher

percentage (6.0%). In general, formation of nitriles, and especially epitionitriles, is influenced by

epithiospecifier protein (ESP) present in plant material. ESP is relatively heat-sensitive in

relation to myrosinase, and even brief boiling should inactivate it, but leave the myrosinase

active. Other volatiles, that probably originate from glucosinolate degradation, such as

benzeneacetonitrile, 7-(methylthio) heptanenitrile, sec-butyl isothiocyanate, 3-butenyl

isothiocyanate, octanenitrile and 5-(methylthio) pentanenitrile, were present in smaller

percentages.25

All volatile samples, except the above-mentioned compounds, contained compounds

without nitrogen and sulphur, mostly fatty acids and esters (0.3–19.2%), phenols, phenylpropane

derivatives and related compounds (0.4–15.8%), aliphatic alcohols and carbonyl compounds

(4.2–11.6%). It is interesting to note the presence of five aliphatic alcohols and aldehydes, such

as (E)-2-hexenal, (E)-2-hexenol, (E)- and (Z)-3-hexenol and 1-hexanol, known as “green notes”.

These compounds are considered to be connected with the lipoxygenase biosynthetic pathway

(Margetts, 2005). Another interesting observation was the high content of fatty acids and esters,

especially among volatiles hydrodistillated from fresh plant material subjected to autolysis

(17.1%) and among volatiles extracted with dichloromethane (19.2%). The dominating

compound belonging to this class of organic compounds was, in both samples, hexadecanoic

acid (8.2% and 11.2%, respectively).25

Volatiles identified after isolation and enzymatic treatment of O-glycosides are given in

Table 9. Compounds with sulphur and/or nitrogen, which are characteristic for glucosinolate

were not identified between these compounds. The main aglycone was eugenol (73.0%),

followed by benzyl alcohol (3.7%), 2-phenylethyl alcohol (3.6%), (Z)-3-hexenol (3.1%) and 4-

vinyl-2-methoxyphenol (2.0%). Comparing the chemical composition of O-aglycones with the

composition of volatiles shown in Table 1, nine compounds were found to be identical: eugenol,

benzyl alcohol, 2-phenylethyl alcohol, (Z)-3-hexenol, 4-vinyl-2-methoxyphenol, 2-

phenylacetaldehyde, phenol, vanilline and benzaldehyde. Most aglycones are phenols and

phenylpropane derivatives, 14 compounds representing 85.3% of total aglycones. Almost the

same aglycones were recently reported as the major ones in other Brassicaceae plants. These

aglycones are ubiquitous in aglycone fractions of many plant families. Eugenol and other p-

hydroxyphenylpropanes which were identified in many plants as main aglycones, can be

connected with lignin biosynthesis via the peroxidase-hydrogen peroxide system. The aliphatic

volatiles (alcohols and carbonyls) probably originate from fatty acid catabolism, and aromatic

volatiles (alcohols and carbonyls) from cinnamic acid catabolism.25

5. Analysis of Myrosinase Activity

A myrosinase-producing fungus, Aspergillus sp. NR-4201, was newly isolated from

decayed mustard seed meal samples obtained in Lamphun, Thailand. When preincubated in a

medium containing sinigrin, myrosinase was expressed intracellularly whereas none was

detected in sinigrin-free medium. Sinigrin degradation was closely related to the presence of

myrosinase. Induced mycelium consumed both glucose and sinigrin competitively, while non-

induced myceliun exhausted glucose first and then sinigrin, with no myrosinase being produced

during the glucose consumption period. The product allylcyanide was detected in incubation

mixtures but its accumulation was delayed. Cell-free extracts incubated with sinigrin produced

allyl isothiocyanate at pH 5.6 and 7.2 but not at pH 4.0.26

Extracellular myrosinase activity in cell-free supernatants collected during incubation

experiments was measured by the method described by Palmieri et al .17 One ml of potassium

phosphate buffer, pH 5.6 containing 0.1 M sinigrin and 100 µl of sample were mixed gently and

measured at 227.5 nm using a double beam spectrophotometer (UV/VIS Hitachi U 2000).

Enzyme activity was calculated from the decrease in absorbance with time.

Myrosinase activity in partially purified extracts of 12 cruciferous vegetables and an

acetone powder preparation of Sinapis alba L. (white mustard) was determined by the initial rate

of glucose formation from glucosinolate hydrolysis using a coupled assay. Of the species studied

Raphanus sativus L. (radish, 12.8±0.7 μmol min−1g−1 powdered tissue) had the greatest

myrosinase activity, and Brassica campestris L. ssp. rapifera (turnip) and Nasturtium officinalis

R.Br. (watercress) (0.6±0.1 and 0.8±0.03 μmol min−1g−1 powdered tissue respectively) the least.

The sub-species of Brassica oleracea studied all had similar myrosinase activity (ca

2.5±0.2μmolmin−1g−1 powdered tissue) except B. oleracea L. var. gemmifera D.C. (Brussels

sprouts) and B. oleracea L. var. capitata L. (white cabbage) which had higher activities (7.6±0.1

and 5.2±0.2μmol min−1g−1 powdered tissue respectively). The effect of ascorbate concentration

upon the myrosinase activity of six of the crucifers studied and the white mustard preparation

revealed that the ascorbate concentration necessary to promote maximal activity varied with

species. A concentration of 0.9mM ascorbate maximally activated radish and turnip myrosinase,

while red cabbage, watercress, white mustard and Brussels sprouts were maximally activated at

2.0, 3.0, 5.0 and 0.7–1.0mM ascorbate respectively. Two peaks of maximal myrosinase activity,

occurring between 0.9 and 1.0mM and at 3.0mM ascorbate, were found for B. oleracea L. var.

botrytis L. subvar. cauliflora D.C. (cauliflower).26

6. Glucosinolate and Isothiocyanate analysis in Raphanus Sativus

Volatile compounds, isolated from the leaves and roots of Raphanus sativus var. black,

white and red, as well as O-aglycones obtained by hydrolysis with β-glucosidase from O-

glycosides, were subjected to a detailed GC and GC–MS analyses. Structure and mass spectral

data of isothiocyanates and nitriles formed by glucosinolate degradation identified in the leaves

and/or roots of different varieties of Raphanus sativus L. are given in Table 2. Roots were found

not to contain volatiles bound as O-glycosides. The yields of obtained volatiles were from fresh

leaves 510–1880 mg/kg with identification from 91.7% to 97.8%; from root 359–2440 mg/kg

with identification from 89.9% to 96.8%; from leaves as O-glycosidically bound volatiles 69–

112 mg/kg with identification from 80.5% to 84.5%.27

6.1 Leaves

Corresponding aliphatic or aromatic volatiles formed by known degradation of

glucosinolates found in leaves constituted only 0.3–5.7% of the isolated volatiles. They were 4-

methylpentyl isothiocyanate, benzyl isothiocyanate, 4-(methylthio)-3-butenyl isothiocyanate, 4-

(methylthio)butyl isothiocyanate, 5-(methylthio)-4-pentenenitrile, 2-phenylethyl isothiocyanate

and benzenepropanenitrile. Obtained mass spectral data also notes the presence of 4-

(Methylthio)-3-butenyl isothiocyanate.27

It was reported, previously, that radish leaves contain glucobrassicin (3-indolylmethyl

GLS).It was shown that indole glucosinolates showed higher degradation rates than aliphatic

glucosinolates. However, volatile indole degradation products of corresponding glucosinolate

after 3 hours of hydrodistillation were not detected among volatiles isolated from the leaves.

Also, thiocyanates, epithionitriles and oxazolidine-2-thiones, which can originate from

glucosinolate degradation, were not identified in these oils.

6.2 Roots

4-(Methylthio)-3-trans-butenyl isothiocyanate, a breakdown product of

glucodehydroerucin, was reported to be the main volatile constituent of radish root responsible

for its pungency. However, it was reported previously that radish roots can contain several

glucosinolates such as 4-methylpentyl GLS, hexyl GLS, 5-hexenyl GLS, 4-(methylthio) butyl

GLS (glucoerucin), 5-(methylthio) pentyl GLS (glucoberteroin) and 4-(methylthio)-3-butenyl

GLS (glucodehydroerucin).27

It was also found that after volatile isolation in a Clevenger-type apparatus, that major

components in the oil of all roots, were sulphur and/or nitrogen containing compounds, such as

4-(methylthio)butyl isothiocyanate (17.9–25.7%), 5-(methylthio)pentyl isothiocyanate (1.5–

2.0%), dimethyl trisulfide (0.8–3.8%), 4-(methylthio)-(E, Z)-3-butenyl isothiocyanate (0.2–

1.8%), 2-phenylethyl isothiocyanate (0.1–1.5%), 5-(methylthio)-(E, Z)-4-pentenenitrile (0–

6.9%). Other sulphur and/or nitrogen containing compounds detected were 4-methylpentyl

isothiocyanate, dimethyl disulfide, dimethyl tetrasulfide, 2-acetylthiazole and 1-(methylthio)-3-

pentanone.27

In contrast, it was also found 4-(methylthio) butyl isothiocyanate (erucin) to be the most

abundant compound isolated from roots by hydrodistillation in the Clevenger apparatus. It is

formed by Loosen rearrangement of an unstable thiohydroximate intermediate that originates

from glucoerucin degradation, a glucosinolate previously reported in radish root Among isolated

volatiles from ground roots after hydrodistillation without prior autolysis, 4-(methylthio)-3-

butenyl isothiocyanate (E, Z)-isomers (0.2–1.8%) were also detected. Previously, 4-(methylthio)-

3-(E)-butenyl isothiocyanate, produced by hydrolysis of 4-(methylthio)-3-(E)-butenyl

glucosinolate by the enzyme myrosinase, was reported as the compound primarily responsible

for the characteristic sulfurous, pungent flavour and aroma of radish root.27

The (E)-isomer of 4-(methylthio)-3-butenyl isothiocyanate in particular has been reported

to be labile enough to form a variety of 3-substituted 2-thioxopyrrolidines and dithiocarbamates. 1

It was also reported the presence of the formation of enolated 2-thioxo-3-

pyrrolidinecarbaldehyde, a degradation product of radish 4-(methylthio)-3-butenyl

isothiocyanate in aqueous media. They considered that this compound can be formed in crushed

or salted radish root by enzymatic hydrolysis of its corresponding glucosinolate and can be

stabilised by internal molecular hydrogen bonds between the enolic hydroxyl group and sulphur

atoms.

It has also been suggested that some 4-(methylthio)-3-butenyl isothiocyanate, released

from 4-(methylthio)-3-butenyl glucosinolate via myrosinase (thioglucosidase) spontaneously

converts to raphanusanins. Examined formation of raphanusanins using different extraction

procedures and solvents and demonstrated that raphantin or raphanusanin is an artifact formed

during extraction of radish with methanol. 4-(methylthio)-3-butenyl glucosinolate has been

found to decompose to its corresponding isothiocyanate and nitrile (E, Z)-isomers, 4-

(methylthio)butyl isothiocyanate (erucin) after hydrodistillation. 5-(Methylthio)pentyl

isothiocyanate (berteroin) was isolated as a hydrolysis product of glucoberteroin, an analogue of

glucoerucin. Thiocyanates, epithionitriles and oxazolidine-2-thiones were not identified in these

oils.27

7. Overview of Cancer

A once unknown disease, cancer, has currently escalated in occurrence to near pandemic

levels. In fact, it has caused severe anguish to many families due to the utter helplessness that is

felt when the diagnosis is revealed. Although many researches are now being done for the

realization of a cure; only physiological drugs and diagnostic protocols such as computed

tomography and such positron emission tomography (PET) or the technetium-based stress tests

are presently available. CT and nuclear medicine tests do have a downside, however: they

deliver doses of ionizing radiation from 50 to over 500 times that of a standard x-ray, such as a

chest x-ray or mammogram. Scientists have raised concerns that such large doses of radiation

plus the widespread and increasing use these diagnostic procedures may, in a small but

significant way, pose a cancer risk in the general population.1 In addition, the many toxic effects

of current drugs, mostly synthetic have paved the way for natural product scientists to investigate

the wisdom behind what was deemed as folklore and quackery.

7.1 Definition of Cancer

Cancer or its medical term, malignant neoplasm, is a class of genetic diseases that is

caused by gene mutations. These mutations or abnormalities in genes result in uncontrolled cell

growth or division beyond the normal limits, invasion or intrusion on and destruction of adjacent

tissues, and sometimes metastasis which is the spreading to other locations in the body via lymph

or blood.2

7.2 Statistical Information on Cancer

Cancer is the second most common cause of mortality worldwide. Annual estimated

deaths dues to cancer is 6.2 million and this figure outnumbers death due to AIDS, Tuberculosis

and Malaria combined.3

In 2006, the 10 most commonly diagnosed cancers among men in the United States 2006

are prostate, lung, colon and rectum, and bladder; melanomas of the skin; non-Hodgkin

lymphoma; kidney cancer, mouth and throat cancer, leukemias, and pancreatic cancer. Overall,

708,769 men were told they had cancer and 290,064 men died from cancer.4

Based on incidence, the following accounts for leading causes of cancer-related deaths in

the Philippines: lung cancer, breast cancer, cancer of the cervix, liver cancer, colon and rectum

cancer, prostate cancer, stomach cancer, 'cancer of the oral cavity, cancer of the ovaries, and

leukemia. In the most recent 2009 Philippine data, six out of ten deaths were cancer related.

According to DOH, based on a five year study done in 100,000 people, malignant neoplasm is

the number 3 killer. In fact from 2000-2004, 38,578 people died at a rate of 48.14% per year

(per 100,000 population); whereas on 2005 alone 41,697 suffered mortality at a rate of 48.9% per

year (per 100,000 population).5 Moreover, the Department of Health says that breast cancer is

now the most common cancer in the Philippines, accounting for 16 percent of the 50,000 cases of

the dreaded disease in the country.6

7.3 General Types Of Cancer

There are three general types of cancer: sporadic, familial, and hereditary. Cancers that

are likely due to nonhereditary causes are termed sporadic. Mutations are all acquired and occur

in affected sites/organs so this is not passed on to children. Sporadic cancer usually has late onset

as compared with the inherited ones. Familial cancers occur within a family more than

statistically expected, but no specific pattern of inheritance is manifested. Also, the age of onset

of the cancer is usually variable which may have been influenced by common genetic

background, similar environment or possible lifestyle factors. Even though there is clustering of

cases within families, these do not exhibit classical features of hereditary cancer syndromes. On

the other hand, inherited type of cancer is traced to gene mutations passed on from parents to

offspring. Mutations occur in all cells of the body including the cells of the reproductive organs

(ovaries and testis), so the defective gene is transmitted from parents to children. If a child

inherits the mutation, he or she will more likely develop the disease more than somebody who

does not carry it. Many inherited cancers are of autosomal dominant inheritance, meaning that

only one copy of the defective gene is needed for the disease/trait to be manifested. First degree

relatives of mutation carriers are at 50% risk to have the same mutation. Earlier age of onset of

cancers than is typical may occur with multiple primary cancers in an individual.7 Included under

this inherited type are colorectal cancer, cancer of the breast and ovary.

7.4 Characteristics of Normal and Cancer Cells

Normal cells are in communication, which allows for the smooth repair and replacement

of tissues and other aspects of cell behavior. Communication takes place either indirectly, via

exchange of messenger compounds such as hormones and growth factors, or directly, via cell-to-

cell contact. Contact allows cells to respond to the “feel” of neighboring cells, via cell adhesion

molecules, and to exchange messenger molecules through cell-to-cell portals called gap

junctions. With the help of proper communication, appropriate cells proliferate when new cells

are needed, and when enough new cells have been produced, cell division stops.8

All of these properties have been perturbed by cancer cells. The standard characteristics

of cancer cells are loss of regulation of mitotic rate, loss of specialization and differentiation of

the cell and the ability to move from the original site and establish new malignant growth at

other tissue sites (metastasis), and capacity to invade and destroy normal tissue. These three

malignant properties of cancers differentiate them from benign tumors, which are self-limited,

and do not invade or metastasize. Most cancers form a tumor but some, like leukemia, do not.

The branch of medicine concerned with the study, diagnosis, treatment, and prevention of cancer

is oncology.9

Cancers are named by the type of cell that resembles the tumor and, therefore, the tissue

presumed to be the origin of the tumor. Carcinomas are malignant tumors derived from epithelial

cells. This group represents the most common cancers, including the common forms of breast,

prostate, lung and colon cancer. Sarcomas are the malignant tumors derived from connective

tissue, or mesenchymal cells. Lymphoma and leukemia are malignancies derived from

hematopoietic or blood-forming cells. Germ cell tumors are derived from totipotent cells. Blastic

tumor or blastoma is usually malignant and resembles an immature or embryonic tissue.10

7.5 Genes and Cancer

A gene can be defined as a region of DNA that controls a hereditary characteristic. It

usually corresponds to a sequence used in the production of a specific protein or RNA. A gene

carries biological information in a form that must be copied and transmitted from each cell to all

its progeny. This includes the entire functional unit: coding DNA sequences, non-coding

regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as

several hundred thousand base pairs. It can even be carried by more than one chromosome.

Humans are thought to have between 30,000 and 40,000 genes.11

7.5.1 Oncogenes

Genes are important as they produce proteins with specific function(s). There are 3

classes of genes often mutated in cancer: oncogenes, tumor suppressor genes and mutator genes.

An oncogene is a gene that causes the transformation of normal cells into cancerous tumor cells,

especially a viral gene that transforms a host cell into a tumor cell. Whereas, tumor suppressor

genes are a class of genes which, when mutated, predispose an individual to cancer by causing

the loss of function of the particular tumor suppressor protein encoded by the gene. Mutator

genes increases the rate of mutation of one or more other genes which is also called mutator.12

Proteins produced by genes are very important in the survival of organism. Functions of

these proteins, especially those involved in human cancer are best understood within the context

of their role in signal transduction.

Cells are continuously exposed to various environmental factors/agents. Cells respond

differently to various stimuli and signal transduction is the mechanisms by which a cell converts

a mechanical/chemical stimulus to a specific cellular response.13 Each protein/molecule involved

in these cascading events in signal transduction is synthesized by a gene. Mutation in this gene

may alter the structure of the protein such that it loses its function or the altered protein may have

an altered function. Sometimes the mutation may also cause the over-expression of that protein.

Signal transduction starts with a signal, recognition of the cell of this signal, processing of this

signal and ends with a cellular response.12 The movement of signals can be simple, like that

associated with receptor molecules of the acetylcholine class: receptors that constitute channels

which, upon ligand interaction, allow signals to be passed in the form of small ion movement,

either into or out of the cell. These ion movements result in changes in the electrical potential of

the cells that, in turn, propagates the signal along the cell. More complex signal transduction

involves the coupling of ligand-receptor interactions to many intracellular events. These events

include phosphorylations by tyrosine kinases and/or serine/threonine kinases. Protein

phosphorylations change enzyme activities and protein conformations. The eventual outcome is

an alteration in cellular activity and changes in the program of genes expressed within the

responding cells. Moreover, within multicellular organisms, numerous small molecules and

polypeptides serve to coordinate a cell's individual biological activity within the context of the

organism as a whole. 12

The change of an oncogene from normal to cancerous function can be caused by a point

mutation in the sequence of a gene. For example, a change in the ras oncogene, located on

human chromosome 11, from guanine to cytosine is frequently associated with bladder cancer.

This simple change results in glycine at amino acid #12 being substituted with a valine. This

dramatically changes the function of the G-protein encoded by the ras gene. Normally, the

protein cycles from an inactive to active state by change the bound GDP to GTP. The mutation

does not allow the release of GTP, and the protein is continuously active. Because the signal

delivered by the ras oncoprotein is continuously delivered, the cell continues to grow and divide.

This unabated growth leads to the bladder cancer.13

Deletions of the ligand binding domain of the EGFR oncogene, located on human

chromosome 7, results in continuous signal transduction by the epidermal growth factor receptor

it encodes. The deletion protein can form a dimer even in the absence of the epidermal growth

factor. Dimerization leads to continuous tyrosine kinase activity and uncontrolled activation of

the signal transduction pathway associated with this gene.14

7.5.2 Tumor Suppressor Genes

Tumor suppressor genes generally follow the 'two-hit hypothesis', which implies that

both alleles that code for a particular gene must be affected before an effect is manifested.

Tumor-suppressor genes, or more precisely, the proteins for which they code, either have a

dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and

sometimes do both. For example, the product of the tumor suppressor gene p53 is a protein of 53

kilodaltons which prevents a cell from completing the cell cycle if DNA is damaged or if the

cell has suffered other types of damage. When the damage is minor, p53 halts the cell cycle or

cell division until the damage is repaired. If the damage is major and cannot be repaired, p53

triggers the cell to commit suicide by apoptosis.15

Loss of heterozygosity (LOH) on chromosome 18 is frequently observed in colorectal

carcinomas (73%) and in advanced adenomas (47%), but only occasionally in earlier-stage

adenomas (11 to 13%). The area of chromosome 18 which is observed to be lost resides between

18q21.3 and the telomere. A 370 kbp stretch of DNA from the region of 18q suspected to contain

the tumor suppressor gene was cloned. Expressed exons were used as probes for screening

cDNA libraries to obtain clones that encoded a gene which was given the name DCC (deleted in

colorectal carcinomas). DCC has been shown to induce apoptosis in the absence of ligand

binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase

substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic

effect of DCC completely.16

7.5.3 Mismatch Repair Genes

Mismatch repair (MMR) genes are involved in numerous cellular functions including: (1)

repairing DNA synthesis errors; (2) repairing double-strand DNA breaks; (3) apoptosis; (4) anti-

recombination; and (5) destabilization of DNA. These responsibilities make MMR proteins

extremely important in the basic maintenance of the genetic material, the regulation of the

cellular cycle, and in the last instance, development of an effective immune system. When MMR

is lost or defective there is a decrease in apoptosis, an increase in cell survival, and a potential

increase in damage-induced mutagenesis. This can provide a selective growth advantage to the

cell, thus causing an increased susceptibility to tissue-specific cancers.16

Mismatch repair (MMR) proteins is a group of nuclear enzymes, which in all

proliferating cells participate in repair of base-base mismatch, that occur during DNA

replication. The proteins form complexes (heterodimers) that bind to areas of abnormal DNA and

initiates its removal. Loss of MMR proteins leads to an accumulation of DNA replication errors

in the proliferating cells, particularly in areas of the genome with short repetitive nucleotide

sequences, a phenomenon known as microsatellite instability (MSI). Hence, MMR protein

deficiency in cells is closely related to a high degree of MSI (MSI-H), in contrast to cells with a

low degree of MSI (MSI-L) and cells that are MSI stable (MSS).17

The most common inherited form of colorectal cancer is Lynch syndrome. Individuals

carrying mutation in their mismatch repair gene are predisposed to colorectal cancer, particularly

HNPCC C or Hereditary nonpolyposis colorectal cancer. Among the 7 MMR genes already

identified, mutation in MSH2 and MLH1 accounts for 70-80% of HNPCC cases. HNPCC or

Lynch syndrome is an autosomal dominant cancer susceptibility syndrome characterized by

predominantly right-sided proximal colon cancer. In summary, if these genes are mutated they

cannot do their function, thus the errors accumulate and which many believed is the starting

point of cancer or the accumulation of mutations.18

7.6 Other Plants that Effect Signal Transduction

Cancer is often described as diseases that arise from the mis-regulation of signal

transduction. Extensive research on the effect of some plant secondary metabolites has been

honed towards the signal transduction mechanisms involved in cancer therapy. In fact, a class of

promising metabolites are classified as phytoanticipins. When plant tissue in which they are

present is disrupted, the phytoanticipins are bio-activated by the action of β-glucosidases such as

found in the hydrolysis of glucosinolates by myrosinase.18

Various phytochemicals can inhibit pathways of signal transduction and gene expression

that play critical roles in carcinogenesis and tumor growth. Epidemiological studies have

described the beneficial effects of dietary polyphenols (flavonoids) on the reduction of the risk of

chronic diseases, including cancer. Moreover, it has been shown that flavonoids, such as

quercetin in apples, epigallocatechin-3-gallate in green tea and genistein in soya, induce

apoptosis. Potential cancer preventive and therapeutic effects of green tea and its polyphenolic

mixture termed GTP. It has become clear that much of these effects of GTP are mediated by its

most abundant catechin, epigallocatechin gallate (EGCG).18

Phytochemicals have been found to have inhibitory effects on the erbB family of RTKs

and their downstream signaling pathways, and the inhibitory effects of these chemicals on the

transcription factors AP-1 and NF-κB. The phytochemicals EGCG, resveratrol, genistein,

curcumin, and capsaicin caused inhibition of the AP-1 transcription factor, which normally

stimulates cell proliferation, as well as inhibition of the NF-κB transcription factor, which

normally enhances cell survival. These combined effects appear to play an important role in the

antitumor effects of these compounds, although other cellular effects of these compounds

probably also play a role. Proposed pathways have stated that EGCG, genistein, and curcumin

can inhibit activation of the EGFR and/or the HER2 receptor. 18

7.7 Apoptosis

Apoptosis, or programmed cell death, is a normal component of the development and

health of multicellular organisms. Cells die in response to a variety of stimuli and during

apoptosis they do so in a controlled, regulated fashion. This makes apoptosis distinct from

another form of cell death called necrosis in which uncontrolled cell death leads to lysis of

cells, inflammatory responses and, potentially, to serious health problems. Apoptosis, by

contrast, is a process in which cells play an active role in their own death, which is why

apoptosis is often referred to as cell suicide. 19

Upon receiving specific signals instructing the cells to undergo apoptosis a number of

distinctive changes occur in the cell. A family of proteins known as caspases are typically

activated in the early stages of apoptosis. These proteins breakdown or cleave key cellular

components that are required for normal cellular function including structural proteins in the

cytoskeleton and nuclear proteins such as DNA repair enzymes. The caspases can also activate

other degradative enzymes such as DNases, which begin to cleave the DNA in the nucleus.

Apoptotic cells display distinctive morphology during the apoptotic process. This can be seen

in the image below which shows a trophoblast cell undergoing apoptosis.19

7.8 Selection of Cell Lines

Four cell lines were chosen to evaluate the anti-carcinogenic properties of radish extracts.

PAE or Human Pulmonary Artery Endothelial Cells served as the control to observe the

cytotoxicity of selected radish extracts on normal cells. In the Philippines, breast

adenocarcinoma is the most prevelant cancer for women. The increasing occurrence of chronic

mylegeneous leukemia and colorectal adenocarcinoma are common to both genders. Due to

persistent pharmacological and medical research, so-called targeted chemotherapeutic drugs are

now available in the Philippines to help patients with these degenerative genetic diseases.

7.8.1 Human Pulmonary Artery Endothelial Cells: HPAEC

Figure 1. Human pulmonary artery endothelial cells: HPAEC

Human Pulmonary Artery Endothelial Cells (HPAEC) are isolated from normal human

pulmonary arteries. They are cryopreserved at second passage and can be cultured and

propagated at least 15 population doublings. HPAEC possess an array of enzymatic activities.

They respond to a wide range of vasoactive substances, commensurate with their control of

blood pressure and blood pH in vivo. HPAEC have been used for the study of vascular

permeability4 and inflammatory responses. HPAEC in co-culture with HPASMC have been used

as a model for pulmonary angiopathy.20

7.8.2 Breast Adenocarcinoma : MCF-7

Figure 2. Michigan cancer foundation – 7: MCF720

MCF-7 is a breast cancer cell line isolated in 1970 from a 69-year-old Caucasian woman.

MCF-7 is the acronym of Michigan Cancer Foundation - 7, referring to the institute in Detroit

where the cell line was established in 1973 by Herbert Soule and co-workers. The Michigan

Cancer Foundation is now known as the Barbara Ann Karmanos Cancer Institute.

Prior to MCF-7, it was not possible for cancer researchers to obtain a mammary cell line that was

capable of living longer than a few months. 21

The patient, whose name is unknown to the vast majority of cancer researchers, died in

1970. Her cells were the source of much of current knowledge about breast cancer. Her name

was Frances Mallon and, at the time of sampling, she was a nun in the convent of the Immaculate

Heart of Mary (Monroe, Michigan) under the name of Sister Catherine Frances.21

Table 3. Characteristics of MCF-7

Primary tumor: invasive breast ductal carcinoma

Origin of cells : pleural effusionPresence of estrogen receptors : yesProliferative response to estrogens : yes yesPresence of progesterone receptors : yes yesERBB2 gene amplification (with Her2/neu protein overexpression) :

no

Tumorigenicity in mice : yes, but only with estrogen supplementation

Phenotype : luminal epithelial

This cell line retained several characteristics of differentiated mammary epithelium

including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of

forming domes.Tumor necrosis factor alpha (TNF alpha) inhibits the growth of MCF-7 breast

cancer cells. Treatment with anti-estrogens can modulate the secretion of insulin-like growth

factor binding proteins.21

7.8.3 Human Immortalised Myelogenous Leukaemia Line :K562

Figure 3. Human immortalised myelogenous leukaemia line :K56222

K562 cells were the first human immortalised myelogenous leukaemia line to be

established. K562 cells are of the erythroleukamia type, and the line is derived from a 53 year

old female CML patient in blast crisis. The cells are non-adherent and rounded, are positive for

the bcr:abl fusion gene, and bear some proteomic resemblance to both undifferentiated

granulocytes and erythrocytes.22

In culture, they exhibit much less clumping than many other suspension lines,

presumably due to the downregulation of surface adhesion molecules by bcr:abl. K562s can

spontaneously develop characteristics similar to early-stage erythrocytes, granulocytes and

monocytes102 and are easily killed by natural killer cells as they lack the MHC complex required

to inhibit NK activity. They also lack any trace of Epstein-Barr virus and other herpesviruses. In

addition to the Philadelphia chromosome they also exhibit a second reciprocal translocation

between the long arm of chromosome 15 with chromosome 17.22

7.8.4 Colorectal Adenocarcinoma HT-29.

Figure 4. Colorectal adenocarcinoma HT-29

Human HT-29 cells are adherent and the morphology is that of epithelial origins. These

cells were originally derived from a colon affected by a malignant disease termed colorectal

adenocarcinoma. Although deposited with the ATCC as a colon adenocarcinoma line established

from a 78 year old female, DNA fingerprinting has shown this line to be a derivative of HT-29

(ATCCHTB-38). The cells are negative for Colon Antigen 3 expression but are positive for

keratin by immunoperoxidase staining. The cells expressed p53 antigen (the p53 produced has a

G > A mutation resulting in Arg > His at position 273). Growth of WiDr cells is inhibited by

tumor necrosis factor alpha (TNF alpha). Inhibitors of dihydrofolate reductase are highly

cytotoxic to WiDr cells.23

7.9 COMET Assay

To determine the bioactivity of the radish samples, the comet assay was utilized because

it is a highly standardized microscopic technique that can easily detect the degree of DNA

damage. In fact, the COMET Assay also known as the Single Cell Gel Electrophoresis assay is a

sensitive method for the detection of DNA damage at the level of the individual eukaryotic cell.

It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells

in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells.

The comet assay or single cell gel electrophoresis assay is based on the alkaline lysis of

labile DNA at sites of damage. The unwound, relaxed DNA is able to migrate out of the cell

during electrophoresis and can be visualized using SYBR Green I nucleic acid gel stain. Cells

that have accumulated DNA damage appear as fluorescent comets with tails of DNA

fragmentation or unwinding, whereas, normal undamaged DNA does not migrate far from the

origin. Each slide provides two sample surfaces specially treated to promote agarose adherence,

and a hydrophobic barrier to allow treatment with one of Trevigen’s DNA repair enzymes. Cells

were then added to the low melting point Comet LMAgarose, and pipet onto the slide. This is

followed by visual analysis with staining of DNA and calculating fluorescence to determine the

extent of DNA damage. This can be performed by manual scoring or automatically by imaging.24

Three parameters in the measurement of DNA damage can be derived through

epifluorescence microscopic evaluation of the slides with the appropriate free software

downloaded. The following are the specific values that can deduce using the freeware

“COMETSCORE”:

a) Tail length is the distance of the DNA migration from the nuclear core and it is used to

evaluate the extent of DNA damage. Measured by subtracting the head diameter from

comet length.

Tail Length = Comet Length – Head Diameter

b) Percent DNA in Tail is the amount of DNA found on the cells tail. It is measured by

dividing the Total Tail Intensity by the Total Comet Intensity multiplied by a hundred.

Tail DNA intensity and Comet DNA intensity are measured through a scoring program

which reads the intensity of the stain.

% DNA in Tail = 100 X Tail DNA intensity Total Comet Assay

c) Total Moment incorporates a measure of both the smallest detectable size of both the of

migrating DNA, reflected in the comet tail length, and the number of relaxed or broken

pieces as represented by the intensity of DNA in the tail. This is measured by multiplying

Tail length and the percent DNA in tail.

Tail Moment = Tail Length X % DNA in Tail

The application of the single-cell gel electrophoresis or comet assay has revolutionized the

field of genetic ecotoxicology or eco-genotoxicology. It is a rapid, sensitive and relatively

inexpensive method providing the opportunity to study DNA damage (including oxidative

damage), repair and cell death (apoptosis) in different cell types without prior knowledge of

karyotype and cell turnover rate. Moreover, cell death could lead to degradation of DNA, hence

all tests that evaluate primary DNA damage, including comet assay, have the potential to detect

agents that are cytotoxic rather than genotoxic. However, the assay has been criticized for its

lack of ecotoxicological relevance. In addition, in contrast to genetic toxicology where rapid

technical progress has been made to improve cell- and tissue-specific adoption of the assay, only

limited advancement has been made to transfer the methodologies to ecotoxicological studies.25

The cell lines that were used in this research include normal endothelial cells from the

pulmonary artery , PAE; human breast adenocarcinoma cells. MCF7; chronic myelogenous

leukemia cells, K562 and colorectal cancer cells, HT-29. The aforementioned cells will be

incubated with several radish extracts for twenty-four hours to determine the bioactivity of the

isothiocyanates found in these extracts.

8.0 Biomonitoring of Cell Lines Incubated with Radish Extracts Using the COMET Assay

Isothiocyanates have been found to protect normal cells from DNA damage and to induce

malignant cancer cells to undergo apoptosis. Cell biologists exposed human liver cancer cells to

rocket juice extracts and later to a specific rocket isothiocyanate and found that a large

proportion of cancer cells switched on the cellular suicide programme. Isothiocyanates from

rocket and other cruciferous plants induce apoptosis in cancer cells. Investigations revealed that

genes were switched on in the cancer cells upon exposure to isothiocyanates,

Lamy et al stated that the tumour suppressor gene p53, which was discovered as far back

as 80 years ago halted cell division cycle when cell damage occured in order to initiate repair

mechanisms; in the case of irreparable cell damage, p53 induces apoptosis. Genes that prevented

apoptosis were suppressed in the cells exposed to isothiocyanate. The Freiburg researchers thus

found that the plant substances also released the breaks that protected the cancer cells from the

onset of apoptosis. The COMET Assay used were stained DNA with fluorescent ethidium

bromide . As a result, DNA, which was damaged due to the cells’ exposure to carcinogenic

substances(not specified), exhibited significant migration of DNA due to fragmentation.

They also found that isothiocyanates also limited the concentration of the telomerase

enzyme, which catalyses a reaction in which specific DNA repeats are added to the ends of

eukaryotic chromosomes. This prevents the cells from ageing and dying. Telomerase activity is

suppressed in the majority of human cells; however, 85 to 90 per cent of all tumour cells express

telomerase and are able to divide continuously.26

The comet assay was adapted for quantifying the degree of photo-induced DNA damage

by using radish sprouts exposed to varied light conditions. An index, IND, was defined to express

the DNA intactness, based on image-analyzing of nuclei in protoplasts prepared from the plant

leaves. The IND value gradually decreased with increasing light intensity (22–430 W m−2) and

exposure time (0–6 h), and ultimately fell to 21% at 6 h under a light intensity of 430 W m−2, as

compared to a reference level in the plants virgin of the exposure. Furthermore, the DNA

damage was found to be restored to an appreciable extent when the plants were fed with

antioxidants such as ascorbic acid and green tea extract, suggesting that DNA damage from

strong light can be caused by photo-oxidative stress generated by the excess energy over a

scavenging capacity of antioxidative defense mechanisms in the plant cells.27

A hexane extract of root and methanolic extract of stem and leaf exhibited negligible

cytoxicity and genotoxicity to normal lymphocytes and displayed significant protective effect

against cell death and oxidative DNA damage induced by H2O2 in lymphocytes under ex vivo

conditions. Cytoprotective and genoprotective effect could be related to the presence of

isothiocyanates and polyphenolics in Raphanus sativus which may act in a synergistic and/or in

an additive manner and exert their protective effect by virtue of their of their ability to remove

reactive oxidants by enhancing the antioxidant enzyme system.28

Oxidative damage to lymphocytes was found at concentrations from 100 – 500 mM with

the majority of the cells showing a tailed DNA. Considerable cytoprotective properties, after

lymphocytes were pre-incubated with radish tuber extracts, were shown at a concentration of 25

g/mL. In this study, a significant decrease in % DNA in tail was noticed from 32.71% in H2O2

treated cells to 7.18% in R. Sativus treated cells. Also, the overall tail moment (OTM) was

reduced from 9.36 to 2.94 in radish treated cells. It was also found that hexane extracts of roots

showed the most potent genoprotection as compared to methanolic extracts of stems and leaves.28

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9http://staryweb.fmed.uniba.sk/patfyz/ANGL/cancer3.pdf

10http://www.answers.com/topic/oncogene

11http://www.answers.com/tumor%20suppressor%20genes

12http://ghr.nlm.nih.gov/glossary=mutatorgene

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14http://p53.free.fr/Database/p53_cancer/p53_Colon.html

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16http://en.wikipedia.org/wiki/Exonuclease_1

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20 http://www.cellapplications.com/product_desc.php?id=57

21http://mcf7.com/

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26http://www.biopro.de/standort/5_bioregionen/bioregio_freiburg/index.html?lang=en&art

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27 http://linkinghub.elsevier.com/retrieve/pii/S1369703X0900134X

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