CHAPTER 1

BACKGROUND OF THE STUDY

According to the World Health Organization (2013), cancer is the number one cause of

mortality worldwide. In 2008, it accounted for 7.6 million deaths, and the loss of lives due to

cancer is still expected to increase, reaching up to 13.1 million by 2030. In the Philippines,

cancer ranks third in causing deaths, which implies that for every 1800 Filipinos, 1 is likely to

develop cancer annually (Ngelangel & Wang, 2009).

Today, not a single treatment exists for cancer. However, there are medical processes

available to prevent cancer from metastasizing or spreading any further. These processes may

come in the form of surgery, radiation, chemotherapy, immunotherapy or gene therapy. (World

Health Organization, 2013). But then again, these processes are not cost-effective and may

cause side effects. Such problems have led researchers to find an alternative cure for cancer that

would be operative, safe and cost-effective.

Unfortunately, acquiring cancer cells or even laboratory generated cells (HeLa cells) is

difficult. Keeping these cells alive also needs advanced technology. Because of this, scientists

utilize model organisms as pseudo cancer cells. And on top of the list of reliable model

organisms is Saccharomyces cerevisiae or commonly known as Baker’s yeast. (Mell & Burgess,

2002)

Ashitaba or Tomorrow’s leaf (Angelica keiskei) is a short-lived perennial plant that looks a

lot like celery (Bove, 2013). The plant has its origins in Japan and China, but given the amount

of attention this plant is catching today, agriculturists and local farmers in the Philippines,

especially those in Luzon, Davao and some parts of Visayas, have started propagating the plant

in the country. The Philippines even has products made with Ashitaba in the public market.

1

The plant is known for yielding a yellow sap that contains flavonoids, including a unique

and rare class called chalcones (Hida, 2010) (Akihisa, et al., 2010). According to research,

flavonoids are scientifically proven to be promising anticancer agents (Ren, Qiao, Wang, Zhang,

& Zhu, 2003) (Alcaraz, et al., 2009).

As such, isolated flavonoids from the leaves and stem of Ashitaba may display

cytotoxicity and antiproliferative activity against Saccharomyces cerevisiae which will serve as

pseudo cancer cells.

STATEMENT OF THE PROBLEM

This study aims to answer the question:

Do the flavonoids isolated from the leaves and stem of Ashitaba (Angelica keiskei)

exhibit anti-proliferation and cytotoxic activities on simulated cancer cells (Saccharomyces

cerevisiae)?

Furthermore, it intends to answer the following:

1. Which of the solutions with varying concentration display the greatest effect on

yeast in terms of cytotoxicity and cellular proliferation inhibition?

a. 50%

b. 75%

c. 100%

2

2. Is there a significant difference between the most effective isolated flavonoid

concentration and the negative control, distilled water, in terms of cytotoxicity and

antiproliferative activity?

3. Is there a significant difference between the most effective isolated flavonoid

concentration and the positive control, Hydrogen Peroxide, in terms of cytotoxicity and

antiproliferative activity?

SIGNIFICANCE OF THE STUDY

The results of the proposed study would be of great benefit to the following:

Doctors and Institutes that focus on Giving Aid to Cancer Patients – they would be

provided with a cost-effective, operational and safe way to prevent the cancer from progressing

any further.

Cancer Patients- they would be able to avail a more economical, effective and safe way to

prevent their degenerative illness from progressing any further.

Scientific Community- the study would be able to contribute knowledge to what is

known about alternative ways of controlling the growth of cancer cells

HYPOTHESES

1. Flavonoids isolated from the leaves and stem of Ashitaba (Angelica keiskei) exhibit

anti-proliferation and cytotoxic activities on simulated cancer cells (Saccharomyces

cerevisiae).

2. A solution of 100% isolated flavonoid displays the greatest effect on yeast in terms of

cytotoxicity and cellular proliferation inhibition.

3

3. There a significant difference between the most effective isolated flavonoid

concentration and the negative control, distilled water, in terms of cytotoxicity and

antiproliferative activity.

4. There is no significant difference between the most effective isolated flavonoid

concentration and the positive control, Hydrogen Peroxide, in terms of cytotoxicity

and antiproliferative activity.

OBJECTIVES

1. To determine whether or not Flavonoids isolated from the leaves and stem of

Ashitaba (Angelica keiskei) exhibit anti-proliferation and cytotoxic activities on

simulated cancer cells (Saccharomyces cerevisiae).

2. To identify which of the solutions with varying concentration displays the greatest

effect on yeast in terms of cytotoxicity and cellular proliferation inhibition.

a. 50%

b. 75%

c. 100%

3. To determine whether or not there is a significant difference between the most

effective isolated flavonoid concentration and the negative control, distilled water, in

terms of cytotoxicity and antiproliferative activity.

4. To determine whether or not there is a significant difference between the most

effective isolated flavonoid concentration and the positive control, Hydrogen

peroxide, in terms of cytotoxicity and antiproliferative activity.

4

SCOPE AND LIMITATIONS

The proposed study does not cover the antiproliferative and cytotoxic activities of

flavonoids isolated from Ashitaba leaves and stem on live human cancer cells. However, this

limitation will not setback the main purpose of the study since S. cerevisiae can be used as a

pseudo cancer cell. Moreover, the study does not include the difference between how flavonoids

isolated from Ashitaba leaves and stem using Column Chromatography affects normal human

cells and how it affects human cancer cells.

FEASIBILITY

Mature Ashitaba or Tomorrow’s leaf would be bought in Sampaloc, Manila and will be

propagated and harvested in Sta. Margarita, Samar. S. cerevisiae or Baker’s yeast is also very

common since it is used for the production of bread and fermentation of wine and is readily

available in the industrial market or wineries. The time it will take to finish the study is estimated

to be more or less 5-6 months. Isolation of flavonoids from the leaves and stem would make use

of Column Chromotography. Data for cytotoxicity and antiproliferative acivity would be

generated using trypan blue staining and hematocytometer or cell counter. The laboratory

equipment and reagents needed for the determination of the cytotoxicity and antiproliferative

activity of the flavonoid are practically available in DOST 8 laboratory, Visayas State University

and local pharmacies.

5

DEFINITION OF TERMS

Cytotoxicity - the degree to which an agent possesses a specific destructive action on

certain cells or the possession of such action

Antiproliferative – pertaining to a substance used to prevent or retard the spread of cells,

especially malignant cells, into surrounding tissues

Column Chromotography – a method used to purify individual chemical

compounds from mixtures of compounds

Eluent- a fluid used in extracting one material from another

6

CHAPTER II

REVIEW OF RELATED LITERATURE

Cancer

Normal human body cells follow a cell-cycle. This cell-cycle is an organized path of

growth, division and programmed cell death or apoptosis. The human cell is able to effectively

go through this cycle with the help of four different genes. (Costra, 2008)

Cancer begins in genes, bits of biochemical instructions composed of individual segments

of the long, coiled molecule deoxyribonucleic acid (DNA). Genes contain the instructions to

make proteins, molecular laborers that serve as building blocks of cells, control chemical

reactions, or transport materials to and from cells. The proteins produced in a human cell

determine the function of each cell, and ultimately, the function of the entire body. (Peterson,

2008)

In a cancerous cell, permanent gene alterations, or mutations, cause the cell to

malfunction. For a cell to become cancerous, usually three to seven different mutations must

occur in a single cell. These genetic mutations may take many years to accumulate, but the

convergence of mutations enables the cell to become cancerous. (Peterson, 2008)

Simply put, cancer is the general term used for a huge cluster of diseases that can affect

almost any part of the body. It is characterized by the rapid production of irregular cells that do

not die and invade organs close to them. (World Health Organization, 2013)

Today, cancer reaps most humans of their lives worldwide, reaching up to 7.6 million in

2008. What is worse is that this number is still expected to climb up to 13.1 million in the year

7

2030. Also, according to (World Health Organization, 2013), 70% of these mortalities occur in

developing countries. Philippines is one of these so-called developing countries. In an article of

Nelangel and Wang (2009), they stated that cancer is the third cause of mortality in the

Philippines, next to communicable diseases and heart diseases. This suggests that for every

1800 Filipinos, one is likely to acquire cancer every year.

Up to date, there is not a single treatment available for cancer patients. Treatments

usually come in combinations of therapies and palliative care. These therapies include surgery,

chemotherapy, radiotherapy, immunotherapy, hormone therapy and gene therapy (Costra,

2008).

These therapies, however, are not cost-effective and some therapies even cause side

effects such as nausea, vomiting, hair loss, fatigue, deafness, vulnerability to other diseases,

anemia, blood clotting problems, mucositis and depression (Medical News Today, 2009).

Saccharomyces cerevisiae as Pseudo Cancer Cells

Saccharomyces cerevisiae or Baker’s yeast has been cultivated by the human race since

agriculture flourished to make bread, beer and wine. But Baker’s yeast has not only contributed

much to industry. It also has modern cellular and molecular biology by serving as a model

organism for all eukaryotic organism including human cells (Pray, 2008) (Mell & Burgess,

2002).

Yeast is a simple living being that functions in practically the same way as human cells.

It is an historical subject of fundamental research to understand cellular and genetic phenomena.

It is a representative model of all eukaryotic cells (Lessafre).

8

This member of kingdom fungi has been utilized as a model organism because research

has proven that like human cells, Baker’s yeast is a sexual eukaryote, which means that its

nucleus contains the same chromosomes found in the nucleus of human cells. Also, Baker’s

yeast undergoes a cell-cycle that is very similar to the human cell-cycle. Surprisingly, the genes

controlling these cell-cycles are more or less the same. Furthermore, almost 20% of human gene

diseases have complements in yeast. This means that these diseases result from a disturbance in

basic cell processes like DNA restoration and cell division. These plus the fact that yeast is easily

propagated, unicellular and easily manipulated makes it an ideal pseudo cancer cell (Pray, 2008)

(Mell & Burgess, 2002).

Another important characteristic of yeast essential to their role as “model organisms” is

the fact they are relatively easy to work with. Yeast replicates quickly and are easy to manipulate

genetically. The doubling time for yeast is about 90 minutes, which is about the same as the

doubling time for human cancer cells. There are also well defined genetic methods for yeast that

allow researchers to easily isolate mutants, cross them with other mutants or into other genetic

backgrounds, and map the locations of genes. In fact, genetic maps constructed based on the

genetic distance between genes gave researchers their first view of the genome and its

organization, and were the culmination of genetic studies that date back to the first half of the

twentieth century (SGD Wiki, 2013).

Because studies have proven that yeast can be used to represent higher eukaryotic cells

such as human cells, researchers today are also utilizing Saccharomyces cerevisiae to find new

ways to combat cancer. Ola Czyz, Masters student in biological sciences and assistant professor

in Vanina Zaremberg’s lab, is currently testing anti-tumor lipid drugs against baker’s yeast to

develop a way to kill cancer cells without harming normal healthy human cells. (Allford, 2013)

9

Ashitaba (Angelica keiskei)

According to Bove (2013), Ashitaba or Tomorrow’s leaf is a short-lived perennial plant

that “looks a bit like its cousin, angelica, with ribbed stems and deep green dissected leaves and a

fat, white, tapering root.” Ashitaba got its name for its ability to grow new stems and leaves on a

daily basis. This plant grows well in a tropical country given that it would not be directly

exposed to sunlight and kept moist on healthy soil.

The plant has its origins in Japan and China where it’s leaves are commonly used as an

herbal medicine and a source of food. However, given the amount of attention this plant is

catching today, agriculturists and local farmers in the Philippines, especially those in Luzon,

Davao and some parts of Visayas, have started propagating the plant in the country. The

Philippines even has products made with Ashitaba in the public market just like tea, food,

cosmetics and hygiene care products. Even though this is the case, most locals still do not know

of the plant and so, it is still known by its Japanese name locally.

Nutritional analysis shows that Ashitaba contains eleven different types of vitamins and

thirteen minerals. One of these vitamins is Vitamin B12, a vitamin that is usually produced in

animals and marine plants, not in terrestrial plants. It is this feature that places Ashitaba in the

same class as marine products (Hida, 2010). Vitamin B12 has been known to promote the

production of red blood cells, increase production of growth hormone, increase attention span

and concentration and boost the immune system.

A certain mineral found in Ashitaba also generates the production of interferon since it is

normally produced in the body in small amounts. Interferon aids in protecting the body from

certain types of cancer and malignant tumors.

10

The plant is also known for having plant fibers, proteins, germanium, coumarins,

sapponins, strong antioxidants and flavonoids. These active ingredients give rise to the plants

ability to purify blood, improve skin and hair quality, cleanse the colon, relieve muscle, joint and

nerve aches, enhance respiratory functions, cure ulcers, inhibit neurological damage, lower blood

sugar, inhibit HIV and lessen effects of degenerative diseases such as cancer (Natural Health

Organic Farm, 2013).

The flavonoids in Ashitaba are responsible for the yellow color of the sap. Just like most

plants, Ashitaba contains more than just one type of flavonoid. One of the these types is a rare

and unique class called chalcones (Gitoglu & Acikara) (Hida, 2010).

Chalcones are a class of flavonoids rarely found in plants, especially to the strain

angelica. Aside from being insecticidal, anti-inflammatory, insulin-like, antimalarial,

bactericidal, fungicidal, antiviral, antifilarial, antimicrobial, anticonvulsant, antitumor and

antiulcer, chalcones are also able to stimulate the production of Nerve Growtth Factor which in

turn prevents neurological diseases such as Alzheimer’s Disease (Natural Health Organic Farm,

2013). Furthermore, certain types of chalcones such as Xanthoangelol found in Ashitaba are

proven to be able to kill cancer cells without harming normal human cells (Syam, Abdelwahab,

Al-Mamary, & Mohan, 2012) (Akihisa, et al., 2010). Most of the chalcones in Ashitaba is found

in the sap. (Bove, 2013)

Also, a recent study shows that two types of chalcones found in ashitaba induce apoptosis

on stomach cancer cells (Takaoka, Hibasami, Ogasawara, & Imai, 2008).

11

Flavonoids

Flavonoids consist one the largest groups of secondary metabolites found in barks,

leaves, seeds and barks of plant. It has 4000 different structures which are subdivided in terms of

chemical structures, namely; flavones, flavonols, flavonones, dihydroflavonols, isoflavones,

anthocyanins, catechins and chalcones. Human diet actually consists of flavonoids. (Alcaraz, et

al., 2009) (Ren, Qiao, Wang, Zhang, & Zhu, 2003) (Gitoglu & Acikara) (Akihisa, et al., 2010) .

Flavonoids are one of the most important groups of bioactive compounds in plants, which

exist in the free aglycones and the glycoside forms showing a diverse structure and a broad range

of biological activities. Flavonoids include several classes of compounds with similar structure

having a C6-C3-C6flavone skeleton. They are differentiated on the degree of unsaturation and

oxidation of the three carbon segment. Within different subclasses further differentiation is based

on the number and nature of substituent groups attached on the rings. Mostly they occur in O-

glycosidic forms with a number of sugars such as glucose, galactose, rhamnose, arabinose,

xylose and rutinose but they are also present as C-glycosides. Flavonoid glycosides have many

isomers with the same molecular weight but different aglycone and sugar component at different

positions attaching on the aglycone ring (Ana Plazonić , Franz Bucar , et. al., 2009).

Flavonoids have protective role in carcinogenesis, inflammation,

atherosclerosis, thrombosis and have high antioxidant capacity.

Furthermore, flavonoids have been reported as aldose reductase inhibitors

blocking the sorbitol pathway that is linked to many problems associated

with diabetes. Flavonoids interact with various enzymatic systems. Their

inhibition of the enzymes cyclooxygenase and lipooxygenase results in a

decrease of platelet activation and aggregation, protection against

12

cardiovascular diseases, cancer chemoprevention and their anti-

inflammatory activity. Many other biological activities are attributed to

flavonoids: antiviral, antimicrobial, antihepatotoxic, antiosteoporotic,

antiulcer, immunomodulatory, antiproliferative and apoptotic activity (Ana

Plazonić , Franz Bucar , et. al., 2009).

13

CHAPTER III

METHODOLOGY

Research Design

The research was intended to test the cytotoxicity and antiproliferative activity of the

methanolic extract of the leaves and stem of Angelica keiskei against Saccharomyces cerevisiae

that served as simulated cancer cells.

Methods

Collection and Preparation of the Plant Sample

The leaves and stem of Angelica keiskei were used as a source of flavonoid for the study.

The needed plant materials were collected at Sta. Margarita, Samar at day break. The stem was

cut 10 centimeters from the bottom and the leaves were clipped from the stem. The collected

stem and leaves were air-dried for two weeks for easy powdering. The plant materials were

ground into fine powder using mortar and pestle.

Preparation of the Ashitaba Crude Extract

Solvent-solid extraction was carried out on 256.0 grams of air-dried and pulverized

leaves and stem of Angelica keiskei. The weighed sample was soaked with 900 mL of 80%

methanol for 48 hours in an Erlenmeyer flask that was covered. The residue was separated from

the filtrate through the use of filter paper in a funnel. This was followed by the concentration of

14

the filtrate using a rotary evaporator where the solution was permitted to evaporate at 60°C,

which is the boiling point of 80% methanol.

Preparation of the Culture Medium

Sabouraud Dextrose Broth was utilized as a culture media for this study. 2 grams of SDB

powder was added to 50 mL of distilled water in an Erlenmeyer flask. The mixture was then

mixed thoroughly. To completely dissolve the SDB powder, the mixture was heated with

frequent stirring and was allowed to boil for 1 minute. The mixture was then covered with

aluminum foil and was autoclaved at 121 degrees Celsius for 15 minutes. The mixture was

allowed to cool and placed inside the refrigerator until use.

Preparation of the Different Treatments

The different treatments that were used in the study were be prepared as follows:

a. 100 % concentration – 20.0 µL of Ashitaba leaf and stem extract

b. 75% concentration – 15.0 µL of Ashitaba leaf and stem extract and 5.0 µL of distilled

water

c. 50% concentration – 10.0 µL of Ashitaba leaf and stem extract and 10.0 µL of water

d. Negative control – 20.0 µl of distilled of distilled water

e. Positive control – 20.0 µl of Canesten

Adjusting the Turbidity of the Inoculum

Twelve mL of SDB agar was transferred to a sterile culture tube using a

mechanical pipette and sterile pipette tips. Then using an inoculating loop that was sterilized by

heating, a pure colony of Saccharomyces cerevisiae was picked out from a slant that was

15

acquired from the Department of Science and Technology Regional Office VIII Microbiology

Laboratory. This was then transferred into the aforementioned culture tube that was immediately

agitated afterwards. The inoculum was then immediately compared to the 0.5 MacFarland

Standard. If the inoculum is more turbid than the 0.5 MacFarland Standard, then sterile saline

solution was added to the inoculum. If it the inoculum becomes less turbid than the 0.5

MacFarland Standard, then more Saccharomyces cerevisiae cells were added to the inoculum.

Preparation of the Assay Tubes

Using a mechanical pipette and sterile pipette tips, 2 mL of the inoculum adjusted to the

0.5 MacFarland Standard was transferred to a sterile test tube. Four other test tubes were also

filled with 2 mL of the inoculum through the same process.

Testing of Cytotoxicity and Antiproliferative Activity

The 5 assay tubes that each contains two mL of the inoculum were then labeled as

follows:

Test tube 1: (+) for the positive control Canesten

Test tube 2: (-) for the negative control distilled water

Test tube 3: 100 for the 100% Ashitaba leaf and stem extract

Test tube 4: 75 for the 75% Ashitaba leaf and stem extract

Test tube 5: 50 for the 50% Ashitaba leaf and stem extract

16

The assay tube labeled (+) was treated with 10 µL of Canesten and this set up served as

the positive control. The assay tube (-) was treated with 10 µL of distilled water and it served as

the negative control. The assay tubes labeled 100, 75 and 50 were treated with 10 µL of 100%

Ashitaba leaf and stem extract, 10 µL of 75% Ashitaba leaf and stem extract and 10 µL of

Ashitaba leaf and stem extract respectively. The assay tubes were then placed inside an

improvised shaking incubator that was made by placing a shaker inside a Styrofoam. The shaker

was set to 300 rotations per minute and the temperature was kept at 30⁰C with constant

monitoring with the use of an infrared surface thermometer.

After 18 hours, serial dilution was then carried out on the different samples. One mL of a

sample was diluted in 9 mL of sterile saline solution. One mL of the diluted sample was also

then transferred into another culture tube containing 9 mL of sterile saline solution for further a

second round dilution. Using a mechanical pipette and a sterile pipette, 20 µL of the diluted

sample was mixed with 20 µL of filtered 0.4% Trypan Blue solution by pipetting the solution up

and down slowly to avoid bubbles. The solution was then left to stand at room temperature for 5

minutes. After 5 minutes, the each chamber of the cell counter was then filled carefully and

continuously with 10 µL of the solution. The same procedure was followed in preparing all the

other four samples for cell counting.

The cells were then counted under the microscope in four 1 by 1 mm squares of one

chamber. Non-viable cells stain blue. Viable cells and non-viable cells were counted separately.

Cells on top and left touching the middle line of the perimeter of each square were counted while

cells touching the middle line at bottom and right sides will not be counted. This procedure was

repeated for the second chamber.

The following equation will be used to determine the number of cells per mL:

17

Cells per mL = the average count of viable cells per square x dilution factor x 104

Decontamination

A pressure cooker filled with water will be used for decontaminating the assay tubes and

other glassware used. They will be placed inside the pressure cooker with boiling water for 30

minutes to subject the microorganisms to heat.

Replication

Three replicates will be made for the trial, with each replica following the exact same

steps used to perform the first trial.

Analysis and Interpretation of Data

All results and observations will be tabulated and evaluated. One-way Analysis of

Variance will be used with a 0.05 level of significance.

Appendix

A. Certifications

Below is the certification from Mr. Rod Angler, Horticulturist, the website of the supplier

of the trypan blue stain and the website of the supplier of TLC plates, complete with office

18

address and contact number. Also, below is the reply of DOST-VIII to our request regarding the

use of their laboratory, reagents and yeast culture.

19

20

21

22

B. Budget

Name of Product Price per Unit No. of units to be Bought Total Cost of Product

Trypan Blue P405.00 1 P405.00

Baker’s Yeast P50.00 1 P50.00

Ashitaba P500 2 P1000

Silica gel, mesh 100-230

P300 1 P300

SDA P2000 1 P2000

TLC Plates P2000 1 P5000

Miscellany P1000 P1000

Total Cost: P6255.00

C. Gantt Chart

Nature of Work W1 W2 W3 W4 W5 W6 W7 W8 W9

Preparation of the plant extract

Flavonoid Extraction

Analyzing the fractions obtained from Column ChromatographyPreparation of the Yeast Culture

Testing the Antiproliferative Activity

of the isolatedFlavonoids

Testing the Cytotoxicity of the

isolated Flavonoids

Data Processing and Interpretation

D. Primary Sources

23

PRIMARY SOURCESChalcones, coumarins, and flavanones from the exudate ofAngelica keiskei and their chemopreventive effectsAbstract From an ethyl acetate-soluble fraction of the exudate obtained from the stems of Angelica keiskei (Umbelliferae), 17 compounds, viz. five chalcones (1–5), seven coumarins (6–12), three flavanones (13–15), one diacetylene (16), and one 5-alkylresorcinol (17), were isolated. These compounds were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, which is known to be a primary screening test for antitumor-promoters. With the exception of three compounds (10, 16, and 17), all other compounds tested showed potent inhibitory effects on EBV-EA induction (92–100% inhibition at 1×103 mol ratio/TPA). In addition, upon evaluation of these compounds for the inhibitory effects against activation of (±)-(E)-methyl-2-[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for antitumor-initiators, two chalcones (2 and 3) and six coumarins (6-11) exhibited potent inhibitory effects.Anti-tumorigenic chalconesAbstractOn the basis of our recent findings that licochalcone A isolated from Xin-jiang licorice showed anti-

inflammatory and anti-tumorigenic activities, we synthesized more than 40 chalcone derivatives to

examine their anti-tumorigenic activities. In vitro inhibitory activity against phosphorylation of

phospholipids promoted by tetradecanoylphorbol-13-acetate (TPA) in HeLa cells was adopted as a

screening test for anti-tumor-promoting effect. In vivo experimental mouse skin tumors initiated by

dimethylbenz[a]-anthracene (DMBA) and promoted by TPA were used to test the anti-tumor-promoting

effect of chalcones. In the results, 3′- and 4′-methyl-3-hydroxychalcone showed the highest potency in

inhibiting tumorigenesis. They also showed a remarkable inhibitory effect on the proliferation of HGC-27

cells derived from human gastric cancer. We discuss the structure-activity relationship, including stereo-

chemical phototransformation, of some chalcone derivatives with reference to their ultraviolet (UV) and

nuclear magnetic resonance (NMR) spectroscopic data.

Cytotoxicity and Antiproliferative Activities of Several Phenolic Compounds Against Three Melanocytes Cell Lines: Relationship Between Structure and Activity

AbstractAbstract: Polyphenolic compounds are widely distributed in the vegetable kingdom and are therefore consumed regularly in the human diet. Epidemiological studies suggest that foods rich in polyphenolic compounds contribute to reducing the risk of cancer. The purpose of our work is to: 1) study the possible cytotoxicity and antiproliferative effects of 13 polyphenolic compounds on 3 cell lines of melanocytes, 2 of melanoma (B16F10 and SK-MEL-1), and 1 of nontransformed melanocytes (Melan-a); and 2) identify the possible relationship between the chemical structure of the tested compounds and their effect on cellular viability. The said polyphenolic compounds corresponded to 8 flavonoids with varying hydroxyl and methoxyl substituents, related structurally through the oxidation state of their flavonoid skeleton, a catechin polymer and 4 phenolic acids. The cytotoxic activity of all the studied compounds was modest or not apparent. The flavonoids luteolin, tangeretin, baicalein, quercetin, and myricetin, and gallic acid showed antiproliferative effects on the tested lines. Our results suggest that a correlation exists between

24

the structural oxidation state and the position, number, and nature of substituents of the polyphenolic compounds studied and their antiproliferative effects.

Chalcones from Angelica keiskei Induce Apoptosis in Stomach Cancer Cells

ABSTRACTApoptosis was observed in human stomach cancer KATO III cells exposed to two chalcones isolated from the stems of ashitaba (Umbelliferae, Angelica keiskei). Exposure of the KATO III cells to the chalcones, identified by mass spectrometry (MS) and 1H-NMR to be xanthoangelol and 4-hydroxyderricin, produced oligonucreosomal-sized fragments, a characteristic of apoptosis. The DNA fragmentation of the KATO III cells could be observed at concentration of 10 μg L-1 at 2 days after the addition of the chalcones to a culture of KATO III cells, fragmented DNA of human stomach cancer KATO III cells. These findings suggest that growth inhibition by xanthoangelol and 4-hydroxyderricin results from the induction of apoptosis by these chalcones.

Flavonoids: Promising anticancer agents

AbstractFlavonoids are polyphenolic compounds that are ubiquitously in plants. They have been shown to possess

a variety of biological activities at nontoxic concentrations in organisms. The role of dietary flavonoids in

cancer prevention is widely discussed. Compelling data from laboratory studies, epidemiological

investigations, and human clinical trials indicate that flavonoids have important effects on cancer

chemoprevention and chemotherapy. Many mechanisms of action have been identified, including

carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis and differentiation,

inhibition of angiogenesis, antioxidation and reversal of multidrug resistance or a combination of these

mechanisms. Based on these results, flavonoids may be promising anticancer agents. © 2003 Wiley

Periodicals, Inc. Med Res Rev, 23, No. 4, 519–534, 2003

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