Effect of the different drying process on the total Anthocyanin content of Violet

(viola odorata L.) flower

 ACKNOWLEDGEMENT

The researchers would like to express their profound gratitude for the persons
who always help them and guide them in making this research proposal possible.

The researchers want to thank Mr. Jerandel Gaspar for editing and teaching the
better way to present a research proposal. They also want to thank and glorify their
parents and friends for the light and courage to make this project possible. To all they
are giving so very thankful.

C.H.S.A.

H.C.F.

J.K.M.Q.
 INTRODUCTION

BACKGROUND OF THE STUDY

Today, interest in anthocyanin pigments has intensified because of their possible

health benefits as dietary antioxidants (Ronald E, Wrolstad, Ph. D.OSU, distinguished

profession of food science & technology) that may protect body systems (Marilyn

S,R.D.). The mission of antioxidants is to find out free radical inside the body and

(http://www.mangosteen-natural-remedies.com/how-do-antioxidant-work.htm) and

protect the cells against its effects

(http://www.nlm.nih.gov/medlineplus/antioxidant.html).

Free radicals are molecules that frequently damage the body and may play a role

in heart disease, cancer and other diseases (http://www.hlm.nih.gov/mrdline

plus/antioxidants.html). It is produced when the body breaks down food or by

environmental exposures like tobacco and radiation (http://www.mangosteen-natural-

remedies.com/how-antioxidants-work.html).

Violets have edible flowers and leaves (http://factoidz.com) that is considered to

be anthocyanins (A.Kariot Et aL, separation of micromolecules). Violets are made into

syrup used in alternative medicine mainly for respiratory ailmets asssociated with

congestion, coughing and soar throat. Violet flower are also edible and used as food

additives for instances in salad, made into jelly and candied for decoration

(www.googlescholar.com). They also contain antioxidants and have medicinal

properties (http://factoids.com/edible-flowers-violet-or-viola-history-culinary-uses-and-

nutrition).
 This study seeks to determine the level of anthocyanin content of Violets (Viola

odorata L.) in different drying process through extraction of pH-differential method by

Kong Et aL, In the year 2003.

STATEMENT OF THE PROBLEM

This study seeks to find out the effect of different drying process on the level of

anthocyanin content of Violets (Viola odorata L.) flower.

Specifically, this study seeks to find answers to the following questions:

1. How will the drying process affects the level of anthocyanin content of

Violets (Viola odorata L.) flower?

2. Which drying process can produce a highest level of anthocyanin?

HYPOTHESIS

Ho: There is no significant difference on the level of anthocyanin content that can

produce by the different drying processes.

 SIGNIFICANCE OF THE STUDY

This study will be conducted to find out the effects of the different drying

processes to the Violet and to determine which treatment has the highest possible

content of anthocyanin.

This study can also help the future researchers by giving them an idea and

overview about the athocyanin content of Violet.

SCOPES AND LIMITATIONS

This study is only limited on the measurement of anthocyanin content of violet

(viola odorata L.) in different drying process. This study will not use any other plant than

violets (viola odorata L.).

This project focuses on the highest content of anthocyanin in different drying

process. The analysis and evaluation will be conducted at chemistry Laboratory, Central

Luzon State University (CLSU).

REVIEW OF RELATED LITERATURE

A method based on liquid chromatography with diode array detection (HPLC-

DAP) coupled to an electrospray ionization (ESI) interface was developed for the

determination of constituents in the aqueous preparation of viola odorata L. flowering

tops. Characteristic constituents of violet flowers are considered to be the anthocyanins;

however, a detailed literature research showed that data concerning their chemical

content are scarce, studied by Anastacia Karioti Et aL.

According to the department of Pharmaceutical Science (college of pharmacy,

university of Kentucky, Levington, KY) their study is to investigate and compare the

composition, stability, antioxidant and anticancer properties and mechanisms of

anthocyanin- containing blackberry extracts (ACEs) from selected cultivars and using

different extraction methods. ACEs were analyzed for the total anthocyanin and

phenolic content, polymeric color and total antioxidant capacity (TAC). These data

suggest that anthocyanin and non-anthocyanin phenolics in ACEs act symergistically or

additively in producing anti-cancer effects.

, The Violet plant was collected during mid-morning during summer and winter

and it was extracted with dicloromethane or methanol at room temperature 22ᵒc, the

extract was partitioned with methanol and water and the aqueous layer was

concentrated on a rotary evaporator prior of freezedrying. (“A novel suite of cyclotides

from viola odorata”, 2006)

 The extract of 4 medicinal plants including Violets were investigated their

antioxidant potency employing six various established in vitro system. (M.

Ebrahimzadeh Et aL.,)

According to Ronald E, Wrolstad, Ph. D.OSU, the interest in anthocyanin

pigments has intensified because of their possible health benefits as dietary

antioxidants. Solutions of these compounds are colorless or pale-yellow. Other phenolic

compounds that comprise part of our diet include phenolic acids and their esters. The

term polyphenolics is increasingly used to describe phenolic-based compounds having

similar solubility properties.

MH-Gordon said, together with (www.nlm.nih.gov/medline

plus/antioxidants.html), antioxidants are substances that may protect your cells against

the effects of free radicals (molecules that produced when your body breaks down food)

and the natural antioxidants have been interested for many years because they have

the ability to retard the off-flavors in foods.

Most Violets contain anthocyanin and have medicinal properties. Violets have a

delicate, sweet and sometimes peppery flavor. Although they may be an underrated

food resource, identifying them in the wild requires knowledge because there are some

flowers that resemble Violets, such as spring larkspur and monkshood. From

(http://factoidz.com/edible-flowers-violet-orviola-history-culinary-uses-and-nutrition)

Jennifer Ferniza (2009) said, fruits rich in anthocyanin may provide health

benefits including promoting healthy blood sugar, reduced oxidation, anti-aging benefits,

joint health and protection against certain types of toxins.

 Anthocyanins are common in plant foods with bright, distinctive colors, such as

red cherries, orange carrots and purple blueberries (for instance). The most common

antioxidants are vitamins A, C, and E, beta carotene, selenium and lycopene.

(http://www.fitday.com/fitness-articles/nutrition/vitamins-minerals/what-are-antioxidants-

where-can-i-find-them.htm)

Juane Dong Et al studied the effects of microwave drying, a newly emerging

drying method, on the contents of functional constituents contained in the flower from

the aspects of microwave output power and time of treatment. The results indicated that

microwave drying method could maximally maintain the functional constituents (Juane

Dong Et al., Effects of microwave drying on the contents of functional constituents of

Eucommia ulmoides flower tea, date received (February 20, 2011), date revised (March

22, 2011).

Flowers from several common Mediterranean shrubs, such as those from the

Cytus genus and Genista genus have long been used for medicinal purposes and

seasoning in the north-eastern Portugese redion. Despite, the shade drying traditionally

used to process these plants, freeze drying is claimed to better preserve the quality of

medicinal plants. This process could be applied in scale-up treatments of the studied

plants for cosmetic or pharmaceutical applications (Jose pineal et al., Influence of the

drying method in the antioxidant potential and chemical composition of four shrubby

flowering plants from the tribe Genisteae, Date received (June 16, 2011).
 Fresh flowers, leaves,stems, and roots of Echinacea pupurea were

subjected to vacuum freeze- drying, cool wind-drying (30 oC) and hot air- drying (40, 55,

or 70oC), and then stored under different environmental conditions. The cichoric acid

was the main phenolic compound detected in dried E. pupurea materials, followed by

caftaric acid. The bioactive constituent contents in different plant parts were in

descending order: flowers>leaves>stems>roots>. Both caffeic acids derivatives and

total phenolic contents were affected by drying method and storage condition. Cool-

wind dried materials retained more bioactive constituents content (>85%) compared to

vacuum freeze-dried materials. The packing material also affected the storability of E.

purpurea materials sealed in polyethylene terephthalate/aluminum foil/polyethylene or

nylon/ polyethylene bags and stored under 10-20 oC and 40-60% relative humidity

without light conditions retained the highest content of bioactive content of bioactive

compounds. From Sheng-Dun Lin Et. aL, Department of food science and technology.
 METHODOLOGY

Gathering of
Materials

Preparation of
Treatments

Drying
Process

Extraction

Determination of
Anthocyanin

Statistical
Tools
Gathering of Materials

The flower to be used, Violet (Viola odorata L.), will be gathered at Fort

Magsaysay, Nueva Ecija. At least 2 kilograms of Violet flowers will be gathered, ¼ for

each treatment. The Extraction will be conducted at Central Luzon State University

(CLSU).

Preparation of treatments

Violets will be washed with tap (running) water to remove unnecessary particles.

Then, it will undergo three different drying processes such as: (a) microwave drying; (b)

air drying; and (c) Freeze drying.

Drying Process

The treatment A will be heated for 47 minutes at 40 oc (Hyun-ock Kim et al.).

Treatment B will be stored at room temperature for 4 days (Hyun-Ock Kim). Lastly,

treatment C will be kept inside the freezer, -5 oc (Kazuko Kaneko et al) for 4 days (H

Scherm).

Extraction

After going through the drying process, each treatment will be extracted using the

pH- differential method studied by Kong Et.aL (2003). 1.00g of Bolatinao flour will be

placed in a 50ml centrifuge tube and 20 ml of each solvent will be added. The mixture

will be shaken for 1 hour and centrifuged at 2000. 1 rpm for 10 minutes. Anthocyanin

extracts with intense violet color will be selected for pH- differential analysis.
Determination of Anthocyanin

The absorbance in each buffer will be taken at 510, 520, 530 and 700 nm using

spectrophotometer (Fuleki and Francis, 1968).

Statistical Tools

The final absorbance will be calculated using the formula:

A= (A530nm-A700nm) pH 1.0- (A530nm-A700nm) pH 4.5.

BIBLIOGRAPHICAL REFERENCES

 Ronald E, Wrolstad, Ph. D.OSU, distinguished profession of food science &

technology

 Marilyn S.R.D (2001)

 http://www.mangosteen-natural-remedies.com/how-do-antioxidant-work.htm

 Antioxidants, Retrieved January 19, 2012 from

http://www.nih.gov/medlineplus/antioxidant.html.

 http://factoidz.com

 www.googlescholar.com

 Anastasia Et aL. Separation of Science macromolecules. 1399-1570

 Department of Pharmaceutical Sciences, College of Pharmacy, University of

Kentucky, Lexington , (May 13, 2008) retrieved January 8, 2011

 Jennifer Ferniza, Phoenix Diets Examiner August 24, 2009

 Spices and Medicinal Plants. Journal of herbs. 79(3) Retrieved 2011

 Fuleki and Francis, 1968

 Kong Et.aL(2003)

 “A novel suite of cyclotides from viola odorata”, (2006)

 Hyun-Ock Kim, Retention of Caffeic Acid Derivatives in Dried Echinacea

purpurea, J. Agric. Food Chem., 2000, 48 (9), pp 4182–4186 (August 8, 2000)

 Kazuko Kaneko et al, FREEZE-DRYING PROCESS FOR PRODUCING A DRY

PLANT (August 21, 1973)

 H Scherm Et al. Biological control of infection of blueberry flowers caused by

Monilinia vaccinii-corymbosi, (September 30,2002)
 Effects of microwave drying on the contents of functional constituents of
Eucommia ulmoides flower tea, date received (February 20, 2011), date revised
(March 22, 2011)

 Sheng-Dun Lin Et. aL, Effect of drying and storage conditions on caffeic acid
dertivativesand total phenolics of Echinacea Pupurea grown in Taiwan.
Received April 22, 2010. Revised June 24, 2010. Accepted September 1, 2010