Anti-Inflammatory Effects of Strawberry

Wine Extracts on LPS-Stimulated RAW

264.7 Macrophage Cells

PRACTICAL TRAINING REPORT

This practical training report is submitted for

the partial requirement for Bachelor Degree

By:

Lukas Terry Boedianto

12.70.0044

DEPARTMENT OF FOOD TECHNOLOGY

FACULTY OF AGRICULTURAL TECHNOLOGY

SEOGIJAPRANATA CATHOLIC UNIVERSITY

SEMARANG

2015

ANTI-INFLAMMATORY EFFECTS OF STRAWBERRY WINE EXTRACTS

ON LPS-STIMULATED RAW 264.7 MACROPHAGE CELLS

Practical Training at Fu Jen Catholic University, Taiwan

By:

LUKAS TERRY BOEDIANTO

Student ID: 12.70.0044

Faculty: Agricultural Technology

This Practical training report has been approved and supported by examiner in

Practical Training Exam on July 13th 2015

Semarang, July 13th 2015

Department of Food Technology

Faculty of Agricultural Technology

Soegijapranata Catholic University

Practical Training Advisor I Practical Training Advisor II

Dr. Tsung-Yu Tsai Dr. Ir. Lindayani, MP.

Dean

i

PREFACE

Praise the Lord because by His grace and blessing, the author would have the opportunity

to undergo the practical training and finish the report. This report is the complete

accountability from the practical training which was done in New Taipei City, Taiwan

that take place from January 12th until March 12th 2015. During the training, the author

did the research with title: Anti-Inflammatory Effects of Strawberry Wine Extracts on

LPS-Stimulated RAW 264.7 Macrophage Cells this report was written as a requirement

to acquire Bachelor Degree of Food Technology. The author would not be able to finish

this task alone and only by support and guidance give by people around the author this

report could be finished. Special thanks for:

1. Jesus Christ that always blessed, saved and guided the author in every step of practical

training in Taiwan.

2. Dr. Victoria Kristina Ananingsih, ST., MSc. for giving me opportunity to join the

internship program.

3. Dr. Tsung-Yu Tsai as my advisor who has advising me and supports me all the time

when I did this research.

4. Dr. Ir. Lindayani, MP. as my advisor for taking care of me during this practical training

in Taiwan.

5. Jenny, Ajheng, Amber, Gina, Tonny, Cindy, Family and Natasha who alywas give

their time to help me to understand the experiment.

6. My family, Mamah, Papah, Raymond, Willy and Alex who always support me in

finance and cheers me every day.

7. Last but not least, I would like to give my gratitude to all my beloved friends in

Taiwan: Andy, Anne Shih, Joyce, Allisa, Cindy Mama, Yvonne, Anne Fang, Wira

Lin, Ian, Garry, Kevin and all other friends that I can not mention one by one that

always support and accompany me when I was in Taiwan.

This report is far from perfect, however the author hope this report can still be an

inspiration and provide useful information for all the reader.

Semarang, July 13th 2015

Author,

Lukas Terry Boedianto

(12.70.0044)

ii

CONTENTS

Aproval Page .............................................................................................................. i

Preface ........................................................................................................................ i

Contents ..................................................................................................................... ii

List of Tables ............................................................................................................ iv

List of Figures ............................................................................................................. v

List of Appendices .................................................................................................... vi

1. INTRODUCTION ................................................................................................ 1

1.1. Background of Practical Training ................................................................... 1

1.2. Purpose of Practical Training ......................................................................... 2

2. INSTITUTE PROFILE ........................................................................................ 3

2.1. Fu Jen Catholic University ............................................................................. 3

2.2. Food Science Department ............................................................................... 3

3. RESEARCH PROJECT ...................................................................................... 4

3.1. Research of Research ...................................................................................... 4

3.2. Literature Review ........................................................................................... 5

3.2.1. Strawberry (Fragaria ananassa) Wine ................................................ 5

3.2.2. Characteristics of Inflammation ........................................................... 6

3.2.3. Raw 264.7 Macrophage Cell ................................................................ 7

3.2.4. Lipopolysaccharide (LPS) .................................................................... 8

3.2.5. Trolox ................................................................................................... 9

3.2.6. MTT Assay ......................................................................................... 10

3.2.7. Nitric Oxide (NO) Production Test .................................................... 10

4. RESEARCH METHODOLOGY ...................................................................... 10

4.1. Time and Place of Practical Training ........................................................... 12

4.2. Strawberry Wine Extraction ......................................................................... 12

a. Materials ................................................................................................... 12

b. Methods .................................................................................................... 12

4.3. Raw 264.7 Macrophage Cell Preparation ..................................................... 13

a. Materials ................................................................................................... 13

b. Methods .................................................................................................... 13

4.4. Raw 264.7 Macrophage Cell Counting and Seeding .................................... 13

a. Materials ................................................................................................... 13

b. Methods .................................................................................................... 14

4.5. Sample Preparation and Injection ................................................................. 15

a. Materials ................................................................................................... 15

b. Methods .................................................................................................... 15

b.1. Control, LPS and T100 Sample Preparation ....................................... 15

b.2. Strawberry Wine Extract Sample Preparation .................................. 15

b.3. Sample Injection ............................................................................... 15

4.6. MTT Assay ................................................................................................... 16

a. Materials ................................................................................................... 16

b. Methods .................................................................................................... 16

iii

b.1. MTT Reagent Preparation ................................................................. 16

b.2. MTT Test .......................................................................................... 16

4.7. Nitric Oxide Production Test ........................................................................ 17

a. Materials ................................................................................................... 17

b. Methods .................................................................................................... 17

b.1. Griess Reagent Preparation ............................................................... 17

b.2. NO Standard Curve Preparation ....................................................... 17

b.3. NO Production Test .......................................................................... 17

4.8. Statistical Analysis ........................................................................................ 18

5. RESULT AND DISCUSSION ........................................................................... 19

5.1. Cell Viability Test ......................................................................................... 19

5.2. Cell Morphology ........................................................................................... 20

5.3. NO Production Test ...................................................................................... 21

6. CONCLUSION AND SUGGESTION .............................................................. 24

6.1. Conclusion .................................................................................................... 24

6.2. Suggestion .................................................................................................... 24

8. REFERENCES ................................................................................................... 25

9. APPENDICES ..................................................................................................... 29

iv

LIST OF TABLES

Table 1. Composition of Strawberry in 100 gram ...................................................... 5

Table 2. Characteristics of Acute and Chronic Inflammation .................................... 7

v

LIST OF FIGURES

Figure 1. Effect of strawberry wine water extract on the cell viability in LPS-

stimulated RAW 264.7 cells. Normal cells as control (C). LPS at 100

ng/mL (LPS). Trolox at 100 μg/mL (T100). Strawberry wine extract at 25-

250 μg/mL (25, 50, 75, 100, 250)..................................................................19

Figure 2. Raw 264.7 Macrophage Cell Morphology. Normal cells as control (C).

LPS at 100 ng/mL (LPS). Trolox at 100 μg/mL (T100). Strawberry wine

extract at 25-250 μg/mL (25, 50, 75, 100, 250).............................................21

Figure 3. Effect of strawberry wine water extract on the NO production in LPS-

stimulated RAW 264.7 cells. Normal cells as control (C). LPS at 100

ng/mL (LPS). Trolox at 100 μg/mL (T100). Strawberry wine extract at 25-

250 μg/mL (25, 50, 75, 100, 250).................................................................22

vi

LIST OF APPENDICES

Appendices 1. Research Schedule.................................................................................29

1

1. INTRODUCTION

1.1. Background of Practical Training

The most important things in sustainability of human life is food. Nowadays, people not

only expect to have delicious food, but they also expect to have a nutritious food to

maintain their health. Many food industries with their great technology concern about

food quality and safety to search for the best to satisfy consumers. Many developments

are being made so that the food will have a better quality. Food technology is very

important in order to fulfill every progressive and dynamics changing demands of

consumers. Food technology is responsible to every parts of food development. It

concerns in many aspects, such as food safety, product development, food quality

management, food packaging, food nutrition, food chemistry, food waste management,

and food microbiology. In real life work, we as a food technologist will use the skills to

be applied in our work.

For that reason, Department of Food Technology, Soegijapranata Catholic University

(SCU) sets up a training program to let the student improve their skills and knowldges.

In this program, student is given an opportunity either to join a food industry company or

to take a part in-house training (research). Student that is stay in food industry company

will know the real bussiness practices and gains a big view about how actual food-related

research is executed in the industry. This experience will be helpfull once the student

graduates and goes into the working world. Student who chooses in-house training has to

go to the selected university to be their facilitator of research. The selected university for

this training is Food Science Department, Fu Jen Catholic University (FJU), Taiwan,

which is sophisticated in applied biotechnology and microbiology sectors. Through this

program, the student is given an opportunity to conduct their research abroad, to

experience cultural diversity. This program can held since there is a student exchange

mutual agreement between SCU and FJU.

The title of the research was “Anti-Inflammatory Effects of Strawberry Wine Extract on

LPS-Stimulated Raw 264.7 Macrophage Cells”. The advisor of this research was Dr.

2

Tsung-Yu Tsai, as the Assistant Professor of Food Science Department, Fu Jen Catholic

University, Taiwan. The mentor of this research was Jenny as the student of master degree

program of Food Science Department, Fu Jen Catholic University, Taiwan.

1.2. Purpose of Practical Training

a. To give experience about doing food science research with the new environtment.

b. To give an opportunity to adapt with new circumtances and society in another country

with their own culture.

c. To sharpen and broaden knowledge and experience that could not be learnt in the real

industry or scienctist world.

d. To meet new friends and build an international relationshiop network.

3

2. INSTITUTION PROFILE

2.1. Fu Jen Catholic University

Fu Jen Catholic University (FJU) is a famous private university in Taiwan that is found

in 1925 by the Benedictines of Saint Vincent Archabbey. In 1961, FJU was re-established

by Society of Jesus, Society Divine World, and Regional Bishop Conference. FJU is

located in New Taipei City which is really strategic and has an easy acces to the diversity

of cultural and social activities of the capital of Taiwan. FJU is moved by Christian

understanding and inspired by the high ideals of Confucian education, and is noted for

attracting foreign students from another country and also Indonesia. There are 21,671

undergraduate students, 3,891 graduated students (master program), more than 180,000

alumni that graduated and more than 39 alumni associations worldwide. FJU provides 11

colleges with 48 departments, 47 master program, 23 in-service master program, 11 Ph.D.

program, and School of Continuing Education. Seven goals of FJU are human dignity,

meaning of life, academic research, community awareness, dialogue with cultures,

religious cooperation and spirit of service.

2.2. Food Science Department

Department of Family Studies and Nutrition Sciences was established and grouped into

the Family Studies section and Nutrition section in 1963. Nutrition Sciences section was

combined with Food Science section as Department of Nutrition and Food Science in

1971. The Graduate Institute of Nutrition and Food Science was established in 1983, and

in 1995 the doctoral program was joined to the Institute. In 2006, Food Science became

an individual department and now they offers Bachelor degree program and master degree

program. The mission of Food Science Department is to promote the healthier, tastier and

safer for improving eating quality, human health and wellness. They have 11 lecturers

mastered in their own major of food science.

4

3. RESEARCH PROJECT

3.1. Background of Research

Oxidative stress has been implicated in human diseases conditions, such as cardiovascular

diseases, cancer, aging and neurodegenerative diseases (Bagchi et al., 2000). However,

the immune systems in human body is not enough for severe oxidative stress. Hence,

certain amounts of exogenous antioxidants and anti-inflammations are required to

maintain an adequate immune system in human body.

Strawberry (Fragaria ananassa) is widely cultivated in USA, Spain, Japan, Poland,

Korea and Russian Federation. Strawberry are not only available fresh, but also consumed

and processed into jams, juices, ice cream, confectionary and other concentration product.

The utilization of strawberry can be maximized when strawberry is processed into wine.

Recent study has shown that wine has a wide range of phytochemical such as sugars, ethyl

alcohol, tannins, aldehydes, esters, amino acids, minerals, vitamins, anthocyanin and

other compounds, including phenolic compounds. The phenolic compounds found in

strawberry wine are catechin, epicatechin, quercitin and ellagic acid. (Joshi et al., 2005;

Sharma, 2000). Phenolic compounds contained in strawberry wine may give a great

protection due to oxidative stress in human body.

The aim of this research are to know the optimum concentration of strawberry wine that

the cells still can survive and also can reduce oxidative stress in LPS-induced Raw 264.7

macrophage cell. LPS is used to stimulate inflammation condition in cells and produce

nitric oxide (NO). The NO production of cells is measured using NO production test and

the viability of cells is measured using MTT assay method.

5

3.2. Literature Review

3.2.1. Strawberry (Fragaria ananassa) Wine

Strawberry is a fruit from genus Fragaria that has red colour with unique shape and

flavour. Strawberry is a nutritious fruit because it has various nutrition and also rich in

vitamin C which is important for natural antioxidant, iron and other minerals. The

phenolic compounds of strawberry is located in major constituents of strawberry water.

Strawberry flavour is characterized as fruity, sweet and tart, and the aroma is mainly

determined by a complex eters, aldehydes, alcohols and sulfur compounds. Esters, like

ethyl and methyl ester, are important for imparting the fruity and floral notes to

strawberry. The composition of strawberry fruit in 100 gram can be seen in Table 1.

Table 1. Composition of Strawberry in 100 gram.

Constituents Average Range

Edible portion (%) 97

Water (%) 89.9-92.4

Total soluble solid (oB) 7-10.2

Total sugars (%) 3.3-9.1

Titratable acidity (%) 0.52-2.26

Organic acids (mg/100 g) 658-2,162

Total phenols (mg/L) 58-210

Protein (%) 0.23

Total anthocyanin (mg/L) 55-145

Minerals (mg/100 g) 157.8

Source: Sharma, 2000; Spayd & Morris, 1981.

Strawberry fruit are a very delicious fruit and ussualy used for appetizer and dessert.

Different utilization of strawberry are used to make jam, juices, puree, confectionary and

concentration, and other preserves food, including strawberry wine. Alcoholic beverages

are theoretically can be prepared from any fruit that have fermentable sugars and nutrient

required for fermentation. The sugars content in strawberry is high enough and can be

used as a raw material to make a wine (Table 1.) (Rana et al., 1986; Amerine et al., 1980).

6

A method for strawberry wine making has also been described (Joshi et al., 2006). To

make the wine, strawberry should be washed to remove dirts and soils, then crushed into

pulps. A palatable strawberry wine made from dilution of pulp with 50% water is

suggested available to reduce the acidity (Joshi et al., 2005). Thermovinification (heat

treatment) is done to improve colour extraction from weakly colour of strawberry. This

process can be done by heating the berries with 50% water at 60-65oC for 5-6 minutes

and raising the total suspended solid (TSS) after crushing to improve the sensory quality

of wine. The fermentation is done by using yeast (Saccharomyces cereviceae) for 4 days

then pressed the fermented wine to dryness (Sims & Bates, 1994).

During fermentation, the physicochemical characteristic of strawberry wine is improved.

It contains sugars, ethyl alcohol, higher alcohol, tannin, aldehydes, esters, amino acids,

minerals, vitamins, anthocyanin, minor constituent like methanol and a number of

flavouring compounds (Joshi et al., 2006; Sharma, 2000). Higher alcohol of strawberry

wine are formed due to amino acid biosynthesis from carbohydrates complex or directly

from existing amino acids by deamination and decarboxylation. Esters in wine are formed

as a result of esterification of alcohols with respective acids (Amerine et al., 1980). While

ageing/ maturation of strawberry wine, the phenolic compounds are formed as a result of

maceration of anthocyanins. The phenolic compounds in strawberry wine are catechin,

epicatechin, quercitin and ellagic acid (Pilando et al., 1985). Phenolic compounds is

important as antioxidant and usually used to reduce oxidative stress such as inflammation

in living organisms. Pasteurized and depectinized of strawberry wine is suggested since

pectin may cause a bad sensory and make the colour unstable. Depectinized can be done

by using 100 ppm of liquid pectic enzyme to breakdown pectin.

3.2.2. Characteristics of Inflammation

Inflammation is a response to protect the cells from harm effects. Inflammation may

happen in every part of our cells, like blood vessels, immune cells and molecular

mediators. Inflammation has various functions to eliminate the initial cause of cell injury,

necrotic cells and tissues damage, and also to initiate tissue repair. Two kinds of

inflammations are acute and chronic (Table 2.). Acute inflammation can be achieved by

7

the rapid movement of plasma and leukocytes from blood into injured tissues as an initial

response of body to harmfull stimulus. Chronic inflammation is a prolonged

inflammation, which mean the harmfull stimuli leads to a progressive shift in the type of

cells present and can be characterized by simultaneous damage (Abbas & Lichtman,

2009).

Table 2. Characteristics of Acute and Chronic Inflammation.

Features Acute Inflammation Chronic Inflammation

Onset Fast: minutes or hours Slow: days

Cellular infiltrate Mainly neutrophilis Monocytes/ macrophages

Tissue injury Usually mild and self-

limited

Often severe and

progressive

Local and systemic signs Prominent Less prominent

Source: Cotran et al., 1998.

Inflammation can be defined by the sequential release of mediators such as pro-

inflammatory cytokines, NO and prostaglandin E2 (PGE2). Nitric oxide synthase (NOS)

will produce NO by converting L-arginine to L-citruline, accompanied by NO production

(Lin et al., 2003). High amount of NO produced by NOS indicates that the cells are in

diseases and inflammation. NO may be generated in excess during the host response

againts viral and bacterial infections, and also contribute to some pathogenesis by

promoting oxidative stress, tissue injury and cancer (Hanada & Yoshimura, 2002; Maeda

& Akaike, 1998).

3.2.3. Raw 264.7 Macrophage Cell

Raw 264.7 cell is a macrophage-like monoyte cells that can be found in mouse (Mus

musculus). This cells have function as defense and immunity mechanism of the living

host response to inflammatory effect like tumor. The protective function of this cells are

presented as innate immunity and inflammation, foreign antigen presentation and

scavenging dead cells (Lee et al., 2011). Macrophage cells can produce two groups of

protein mediators to inhibit inflammation, interleukin 1 (IL-1) and tumor necrosis factor

(TNF). Those two cytokines appear to play an important role in the immune processes

8

(Han et al., 2002). As a response of inflammation effects, this cells also will produce NO

and the production of NO will endows macrophage cell with cytotoxic activity againts

bacteria, viruses, fungi, protozoa and tumor cells. However, high concentration of NO in

living host will lead to neuropathogical diseases, rheumatoid arthritis and other disorders.

In other words, inflammation mechanism is just a response to a harmful thing but if the

healing of wound part of cell take time too long, it will cause many diseases.

To date, raw 264.7 macrophage cell are usually used to do cell culture with growth

conditions, protein expression and determination, RNAi, stable cell transfection and also

gene transfection (He et al., 2011). The thaw and subculture techniques are very important

things to do experiment using raw 264.7 macrophage cells. Pure raw cells should be

stored in liquid nitrogen vapour phase to deactivate the cells and prevent the growth of

cells. The thaw and subculture process should be done in laminar flow by using aseptic

methods.

3.2.4. Lipopolysaccharide (LPS)

Lipopolysaccharide (LPS) is a large molecule consisting of lipid and polysaccharide. LPS

can be found in the outer membrane of Gram-negative bacteria which outer and inner

core joined by a covalent bond (Hirai et al., 2014). Richard Friedrich Johannes Pfeiffer is

a scientist who discovered LPS in Escherichia coli and classified it as an endotoxin. LPS

called endotoxin because the toxin is kept within the bacteria outer membrane and will

only released to the environment when there is a destruction of bacteria membrane. LPS

plays a major role in bacteria because it contributes greatly on cell structure formation,

give protection from harmful chemical attack and also may stimulates strong response of

immune system (Rietschel et al., 1994).

LPS is a complex molecule which composed of three major parts, the parts are O

polysaccharide, core oligosaccharide and lipid A.

a. O polysaccharide (O antigen) comprises the outer domain of LPS molecule and

attached to the core oligosaccharide. O polysaccharide in the membrane will determine

whether the membrane is rough or smooth, depend on how many O polysaccharide

contains in Gram-negative bacteria. Bacteria with smooth LPS will have less

9

penetrable cell membranes to medicine like antibiotic because smooth LPS is less

hydrophobic (Rittig et al., 2003; Raetz & Whitfield, 2002).

b. Core oligosaccharide attached to O polysaccharide and contains simple sugar molecule

like heptose and 3-deoxy-D-mannooctulosonic acid and also contain other components

like amino acids, phosphate and ethanolamine (Hershberger & Binkley, 1968).

c. Lipid A is a phosphorylated glucosamine disaccharide attached with various fatty acids.

Since this part attach with lipid, then the characteristic is hydrophobic. Lipid A is

responsible to the toxicity level of Gram-negative bacteria and the toxicity level is

depend on how much lipid A is contained on bacteria. Once the bacteria cells is

destroyed by immune system, lipid A will be released to the environment and cause

diseases like diarrhea, fever and also fatal endotoxic shock (Tzeng et al., 2002).

The injection of LPS which contain lipid A in mammalian cells may cause uncontrolled

immune systems and will stimulate to produce inflammatory mediators (Kilar et al.,

2013). The mediators of inflammation system is Toll-like receptor 4, which is responsible

to activate the immune cell system. Damage in endothelial layer of blood vessel caused

by Toll-like receptor 4 can lead to capillary leak syndrome, dilation of blood vessels and

decrease in cardiac functions. The high concentration of LPS injected to mammalian cells

may cause further effects like disseminated intravascular coagulation (DIC) with loss of

organs function such as kidneys, adrenal glands, lungs and heart (Stephens et al., 2007).

3.2.5. Trolox

Trolox (C14H18O4) is an analog of vitamin E. International Union or Pure and Applied

Chemistry (IUPAC) name of trolox is 6-hydroxy-2,5,7,8-tetramethylchroman-2-

carboxylic acid and the molar mass is 250.29 g/mol. It is a water-soluble substance and

have an antioxidant-like function vitamin E. Trolox can be found in many fruits with high

vitamin E, such as berries and tomatoes. In many research, trolox usually used for

biological and biochemical applications to reduce damage and oxidative stress. The

effectivity of trolox as antioxidant and anti-inflammatory effect can be measured using

trolox equivalent antioxidant capacity (TEAC) assay. The units of this measurement is

trolox equivalents (TE) (Berg et al., 1999).

10

3.2.6. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) Assay

MTT assay is a test based on the conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5

diphenyl tetrazolium bromide) into formazan crystals by living cell. Mitochondiral

activity is used to determines the formazan crystal formation in living cells. The number

of the cell populations are related to the number of cell viability (Van Meerloo et al.,

2011). Cell viability assay developed for a 96-wells format that suitable for high

throughput screening. Viable cells can convert MTT reagent into purple colored formazan

product that can be detected by using 570 nm wavelengths (Marshall et al., 1995). A dead

cell will lose the ability, thus unable to convert MTT into formazan. Formazan can be

absorbed and precipitated by the cells surfaces and the color of the cells will change into

dark purple. Formazan should be soluble when recording the absorbance using

absorbance-reader equipment (Tada et al., (1986). There are various liquid to soluble the

formazan such as dimethylformamide, SDS, acidified isoporopanol, dimethyl sulfoxide

(DMSO) and also combination of detergent and organic solvent. DMSO is the one of the

most common solvent that used to dissolve hydrophobic substances, such as formazan

crystals, for in vivo and in vitro purposes (Bartsch, et al., 1976).

3.2.7. Nitric Oxide (NO) Production Test

Nitric Oxide (NO) plays a major roles in plants, bacteria and even in mammalian life

(Moncada et al., 1991). Multiple physiological and pathophysiological function of NO

are achieved by using various classes of nitric oxide synthase (NOS). The excess or lack

of NO production in cells can cause nitrosative stress, leading damages of protein (DNA)

and cell injury and death (Murphy, 1999). Wagner and coworkers established that the

excess amount of NO in cells indicates that inflammation have occured in these cells

(Wagner et al., 1984). This shown that NO is involved in many of human diseases and

need to be regulated because it plays multiple role in living organisms (Lum et al., 2002).

The measurement of NO in living organism is not a simple way because NO is rapidly

oxidized to its derivatives, such as nitrite and/or nitrate oxygen (Feldmen, 1993). There

11

are three ways to measure NO level, using fast-response amperometric electrode sensors,

in-vivo trapping techniques and estimation of determining the concentrations of nitrite

and nitrate concentration (Kleschyov & Munzel, 2002; Kojima et al., 1998; Moshage,

1995) The determination of nitrite and nitrate concentration in sample can be done by

using Griess assay. Griess assay was first described in 1879 and used widely in analysis

of various samples including urine, salive and cell culture media. In this method, nitrite

is treated with diazotizing reagent, such as sulfanialmide (SA) in acidic media to form a

transient diazonium salt. N-naphthyl-ethylenediamine (NED) is added and react with the

sample to form a stable azo compound. The purple color of the sample can be used to

measure nitrite concentration by using absorbance-reader equipment at 540 nm (Green et

al., 1982).

12

4. RESEARCH METHODOLOGY

4.1. Time and Place of Practical Training

The practical training is conducted in Food Science Department, Fu Jen Catholic

University, Xinzhuang District, New Taipei City, Taiwan (Figure 1.). This activity take

place between January 12th to March 12th 2015.

4.2. Strawberry Wine Extraction

a. Materials

Materials and tools for the extraction was strawberry wine (11% of alcohol), aquadest,

condensator, freezer, sonicator, centrifuge, centrifuge tube, freeze dryer and electric

balance.

b. Methods

Strawberry wine with 11% of alcohol was condensed at 40±3oC. Put sample into tube and

freeze at -20oC for 24 hours. The water content of sample was removed by using freeze

dryer for 3 days. After removal of water content, the powder can be collected. One gram

of powder was extracted with 2.5 ml aquadest and then homogenized it using sonication

for 30 minutes. The sample was centrifuged at 9,449 rpm at 4oC for 20 minutes. The

supernatant was removed and put into tube, then freeze it at -20oC for 24 hours. After

freezing, the sample was freeze dried for 72 hours and then the stock of sample at

concentration 1,000 mg/ml is prepared. The sample was stored at -20oC (Fuhrman et al.,

2001).

13

4.3. Raw 264.7 Macrophage Cell Preparation

a. Materials

Materials and tools for this preparation was raw cell 264.7 (bought from Food Industry

Research and Development Institute, Bioresource Collection and Research Center,

Hsinchu, Taiwan), PBS, TryplE, DMEM medium with 5% of FBS, alcohol, laminar flow,

micropipette, tips, volume pipette, auto pipette, dish, centrifuge, tissue paper, high

pressure suction unit, incubator and falcon tube.

b. Methods

Laminar air flow was wiped with 75% of ethanol and UV light was turned on for 15

minutes before use. Medium was warmed using waterbath until the temperature reached

37 oC. B16 cells were taken out from the liquid nitrogen tank and warmed using waterbath

until the temperature reached 37 oC. Cells were added by 5.5 ml medium and centrifuged

at 1000 rpm for 3 minutes. Dish was prepared and added by 7 ml medium. After

centrifuged, medium was removed using high pressure and the cells were diluted using 1

ml medium. The cells were added to dish plate and incubated at 37 oC for 24 hours. The

medium was removed after incubated and washed using 2 ml phosphate buffer saline

(PBS). PBS was removed and then 7 ml medium were added to the dish plate. The cells

were incubated at 37oC for 24 hours (Hirai et al., 2014).

4.4.Raw 264.7 Macrophage Cell Counting and Seeding

a. Materials

Materials and tools for this preparation was raw cell 264.7, PBS, TryplE, DMEM medium

with 5% of FBS, alcohol, laminar flow, 96-well plates, 8-channel micropipette,

micropipette, tips, volume pipette, auto pipette, dish, centrifuge, high pressure suction

unit, tissue paper, incubator and falcon tube.

14

b. Methods

Cells were taken out from incubator and removed the medium. Cells were washed using

2 ml PBS and removed the PBS. 1.5 ml TryplE were added to the dish and removed it.

The cells were incubated at 37oC for 3 minutes. After 3 minutes , the dish was knocked

and then the cells were collected using 7 ml medium. The solution was put into falcon

tube and then centrifuged at 1,000 rpm for 3 minutes. The supernatant (medium) was

removed and the cells were collected using 10 ml medium. Three micro tubes were

prepared and 900 μl medium were added to second micro tube. One ml of cells were taken

and put into first micro tube. First micro tube 100 μl were added to the second micro tube

(10 fold dilution). Second micro tube 20 μl was taken and put into the third micro tube.

Twenty μl of trypan blue stain (0.4%) were added to third micro tube (2 fold dilution).

Sample 20 μl was injected to cell counting plate using micro pipette. The cell was counted

in 5 squares of two sides of cell counting plate using microscope. The number of cells

and preparation proportion was calculated. The medium and cells were mixed using low

speed vortex.

𝑦

𝑎 × 𝑏× 𝑐 × 𝑑 ÷ 𝑒 × 𝑓 = 𝑥 × 𝑔

Preparation proportion = 1 ml medium with cells ∶ x − 1 ml medium without cells

Known:

y = cell number d = 2 fold dilution g = cell seeding number

a = two side of counting plate e = 10μl of sample

b = number of square f = Coefficient

c = 10 fold dilution x = Total medium (ml)

The 96 well-plate and 8-channel pipette were prepared. The solution of cells were put

into well and then 200 μl of cells solution were added to the 96 well-plate using 8 channel-

pipette carefully. The cells were incubated in 37oC for 24 hours.

15

4.5.Sample Preparation and Injection

a. Materials

Materials and tools for this preparation was strawberry wine extract, LPS, trolox, DMEM

medium with 5% of FBS, tip, micropipette, alcohol, laminar flow, 96-well plates,

micropipette, tips, volume pipette, auto pipette, dish, tissue paper, high pressure suction

unit, incubator, vortex and falcon tube.

b. Methods

b.1. Control, LPS and T100 Sample Preparation

Control was made by using 200 μl medium. LPS was made by mixing 1 ml medium with

1μl LPS. T100 was made by mixing 100 μl of trolox with 900 μl of medium, then mix 100

μl of solution with 900 μl medium.

b.2. Strawberry Wine Extract Sample Preparation

There are 5 variable of strawberry wine extract sample used in this experiment; 25, 50,

75, 100 and 250 μg/ml. Ten μl of strawberry extract was added to 990 μl of medium

(hundred-fold dilution). A 100 μl amount of hundred-fold dilution was added to 900 μl

medium (become thousand-fold dilution). Concentration at 25, 50, 75, 100 and 250 μg/ml

was made by mixing 25, 50, 75, 100 and 250 μl of thousand fold dilution with 975, 950,

925, 900 and 750 μl medium. One μl of LPS was added to T100, concentration 25, 50, 75,

100 and 250 μg/ml. All variable sample was homogenized using vortex.

b.3. Sample Injection

Medium were removed from the 96-well plates using high pressure suction unit. The 96-

well plates were divided into 3 repeats. A 200 μl amount of different concentration were

16

added to the 96-well plates carefully and then incubated at 37oC for 24 hours (Hirai et al.,

2014).

4.6. MTT Assay

a. Materials

Materials and methods for this test was MTT reagent, DMEM medium with 0% FBS, tip,

micropipette, alcohol, laminar flow, 96-well plates, micropipette, tips, volume pipette,

auto pipette, dish, tissue paper, high pressure suction unit, incubator, vortex and falcon

tube.

b. Methods

b.1. MTT Reagent Preparation

A 2.5 ml amount of MTT reagent were mixed using 22.5 ml of DMEM medium without

FBS and vortex.

b.2. MTT Test

The medium in 96-well plates was removed using high pressure suction unit. The solution

was put in the sample cage and 200 μl of solution was added to 96-well plates using 8

channels pipettes. 96 well-plates was incubated at 37oC for 1 hour, then the solution was

removed using high pressure suction unit. A 200 μl amount of DMSO were added to the

96-well plates to dissolve the purple crystals. Absorbance value of all plates were

analyzed using ELISA-reader at 550 nm (Sun et al., 2014; Kim et al., 2014).

17

4.7. Nitric Oxide Production Test

a. Materials

Materials and methods for this test was Griess reagent, sulfanilamide, phosphoric acid,

aquadest, naphthylethylenediamine dihydrochloride, NaNO2, DMEM medium without

FBS, tip, micropipette, alcohol, laminar flow, 96-well plates, micropipette, tips, volume

pipette, auto pipette, dish, tissue paper, high pressure suction unit, incubator, vortex and

falcon tube.

b. Methods

b.1. Griess Reagent Preparation

Griess reagent was made by mixing 3 ml of 0.1% naphthylethylenediamine

dihydrochloride with 3 ml of 1% sulfanilamide with 5% of phosphoric acid.

b.2. NO Standard Curve Preparation

Sample for standard curve of NO test was made by mixing 69 gram of NaNO2 with 1 ml

aquadest, then 10 μl of the solution was added to 10 ml aquadest (1,000 μM). There were

8 different concentration of NaNO2; 0, 10, 20, 30, 40, 50, 60 and 80 μM. Concentration

at 0 μM was made by using 1,000 μl of aquadest. Concentration at 10, 20, 30, 40, 50, 60

and 80 μM was made by mixing 1,000 μM NaNO2 with 990, 980, 970, 960, 950, 940 and

920 μl aquadest. Standard curve of NO test was made by mixing 100μl of any different

concentration of NaNO2 with 100 μl Griess reagent in 3 repeats. The NO value was

analyzed using ELISA-reader at 550 nm (Hirai et al., 2014; Kim et al., 2014).

b.3. NO Production Test

After standard curve of NO was done, the determination of NO value in strawberry wine

can be implemented. A 100 μl amount of medium in 96-well plates were taken and put

18

into another 96-well plates. A 100 μl amount of Griess reagent was added to 96-well

plates and then the NO value of strawberry was analyzed using ELISA-reader at 550 nm

(Hirai et al., 2014; Kim et al., 2014).

4.8.Statistical Analysis

Statistical analyses were performed with SPSS program (SPSS version 10.0) using two-

way repeated ANOVA. Differences in the means were determined using LSD and Duncan

multiple range test (Hirai et al., 2014).

19

5. RESULT AND DISCUSSION

Anti-inflammatory effect of strawberry wine extract on LPS-induced RAW 264.7

macrophage cells were analyzed. Strawberry wine extract was made by using water

extraction method. LPS was injected to RAW 264.7 macrophage cells to stimulate the

inflammation condition of the cells. To know the anti-inflammatory effect of strawberry

wine extract, different concentrations of strawberry wine extract was injected to the cells

and trolox was used as the positive control. Anti-inflammatory effect was measured by

NO production test using ELISA reader. Cell viability was measured using MTT survival

test. The effect of strawberry wine extract to morphology cells were analyzed using

electronic microscope connected to personal computer (PC).

5.1.Cell Viability Test

Figure 1. Effect of strawberry wine water extract on the cell viability in LPS-stimulated

RAW 264.7 cells. Normal cells as control (C). LPS at 100 ng/mL (LPS). Trolox

at 100 μg/mL (T100). Strawberry wine extract at 25-250 μg/mL (25, 50, 75,

100, 250).

20

Cell viability test is used to know wether the sample is suitable for the cells or not. To

analyze the cell viability, MTT assay is used as a method using ELISA reader. Viable

cells in the bottom of the plate have the skill to absorb and convert MTT reagent into

formazan crystals which have purple color, but death cells are not. Formazan is dissolved

in DMSO solvent and change the color of solution into purple that can be detected by

using 550 nm wavelength of ELISA reader. Amount number of viable cell will determine

the color of solution and also the absorbance of ELISA reader (Marshall et al., 1995; Tada

et al., 1986; Bartsch et al., 1976).

Based on Figure 1. we can see that cell viability of LPS-stimulated RAW 264.7 cells were

depend on extract concentration. LPS, T100, 25, 50 and 75 μg/mL were not significantly

different with control. This means the addition of LPS and T100 were not hurt the cells,

thus the number of the cells in wells could be mantained. The same thing happened in the

concentration of 25, 50 and 75 μg/mL because it proved that the cell viability at these

concentrations are not significantly different from the control. This means that these

concentrations does not lead to cell death. However, cell viability at 100 and 250 μg/mL

of extract were decreased signicantly. This was happen because the high concentration of

extract could kill the cells and resulted in low cell viability. Strawberry wine extract

contains alcohol. The higher concentration of sample was added, the higher alcohol will

be. Alcohol is a damaging agent for all cells, from the liver to nerveous system (Blasiak

et al., 2000; Luo & Miller, 1997). High concentration of alcohol, especially ethanol, may

lead to necrotic cell death, mediated by a high production of reactive oxygen species

(ROS) (Peri et al., 2005).

5.2.Cell Morphology

Cell morphology can be used to evaluate the effect of strawberry wine water extract as

anti-inflammatory in LPS-induced RAW 264.7 cells (Figure 2.). This observation can be

carried out using microscope computer connected.

21

Figure 2. Raw 264.7 Macrophage Cell Morphology. Normal cells as control (C). LPS at

100 ng/mL (LPS). Trolox at 100 μg/mL (T100). Strawberry wine extract at 25-

250 μg/mL (25, 50, 75, 100, 250)

The control cells had round shape with low degree of spreading (Figure 2.C.). Cell with

100 ng/mL of LPS significantly changed, the shape was not round anymore (Figure

3.T100.). LPS induced cell spreading and also pseudopodia formation (Figure 2.LPS.).

This means that LPS may cause the cells into inflammation state and NO will be produced

as a response (Harmey et al., 2002). When the strawberry wine water extract was added

with various concentration into LPS-induced RAW 264.7 cells, it can prevent the cell

from spreading and also the pseudopodia formation.

5.3.NO Production Test

In order to assess the anti-inflammatory activities of strawberry wine water extract, RAW

264.7 cells were stimulated with LPS in the presence or absence of the extract and the

NO production contained in medium was measured. The inhibition of NO production was

observed in cells with addition of the strawberry wine water extract (25-250 μg/mL).

C LPS T100 25

50 75 100 250

22

Figure 3. Effect of strawberry wine water extract on the NO production in LPS-stimulated

RAW 264.7 cells. Normal cells as control (C). LPS at 100 ng/mL (LPS). Trolox

at 100 μg/mL (T100). Strawberry wine extract at 25-250 μg/mL (25, 50, 75,

100, 250).

Based on Figure 3., the NO production in LPS-stimulated RAW 264.7 cells were different

from each other depends on the treatment. Control cell had the lowest NO production,

whereas LPS had the highest NO production. LPS, known as lipopolysaccharide, is a cell

wall component of gram-negative bacteria that has inflammatory stimulus potential which

may cause cell to produce NO (Harmey et al., 2002). The addition of strawberry water

extract on different concentration may reduce the NO production compared with LPS

treatment. The extract at 25 and 50 μg/mL were not significant different in preventing

NO production in cells compared with LPS. But NO production at concentration 50

μg/mL was lower than 25 μg/mL. The extract at 75, 100 and 250 μg/mL were significant

different in preventing NO production in cells compared with LPS. However, the NO

production level was not significant between 75 μg/mL against 100 μg/mL, 75 μg/mL

againts 250 μg/mL and 100 μg/mL againts 250 μg/mL, but the higher the concentration

of extract was added to the cells, the NO production level was lower.

23

T100 is positive control and contains trolox which is a water soluble substances and

analog of vitamin E used to reduce oxidative stress or damage in cells (Moulin et al.,

1998). T100 should has the lowest NO production among the other LPS-induced cells

because it may inhibit the production of NO in LPS-induced cells. NO production in cells

with 25 and 50 μg/mL of extract were significant different compared to T100, however,

at concentration 75-250 μg/mL were not. NO production at concentration 100 and 250

µg/mL of strawberry wine water extract were lower than T100 respectively. It means the

inhibition level of NO production in cell with 100 and 250 µg/mL was higher than in

T100. This is because the number of the cell in each well is not the same. Some of the

cells in concentration 100 and 250 µg/mL cannot survive and may influence the NO

production level.

NO production at concentration 25, 50 and 75 µg/mL decreases with the increasing of

strawberry wine water extract concentration. The extract may reduce the level of NO

production because strawberry contains so many phenolic compounds, such as

anthocyanins, ellagic acid, ellagic acid glycosides and ellagitannins (Clifford, 2004).

Those phenolic compounds can inhibit NO production, thus strawberry wine may reduce

inflammation proccess in cells. The extract at 75 µg/mL showed significantly higher anti-

inflammatory activities than those other concentrations (25, 50, 100 and 250 µg/mL)

compared with the cell viability (MTT test).

24

6. CONCLUSION AND SUGGESTION

6.1.Conclusion

The optimum concentration of strawberry wine that the cells still can survive and also can

reduce oxidative stress in LPS-induced Raw 264.7 macrophage cell is 75 µg/mL.

6.2.Suggestion

For future research, the measurement of phenolic content in strawberry wine and in vivo

research is needed for comparison reason.

25

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29

8. APPENDICES

Appendices 1. Research Schedule

December January February March April Note

4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Week(s)

Searching

references.

Practice cell sub

culture,

strawberry wine

concentration.

Experiment

execution.

Report making