FUNCTIONAL COMPONENTS OF COOKED AND RAW BANANA (Musa sp. cv. saba) BUD1

Jastinne D. DecenaOctober 2010

1Scientific Report submitted in partial fulfillment of the requirements in HNF 152 (Food and Nutrition Research) under the supervision of Dr. Wilma A. Hurtada and Prof. Felecito M. Rodriguez, 1st semester, AY 2010-2011.

ACKNOWLEDGEMENT

As always, there are so many people to thank.

To Dr. Wilma Hurtada and Sir Chito Rodriguez, for their availability and efforts to share the knowledge that they have so that we, their students, will strive at all times to do our best. You are very good in what you are doing: molding students into the kind of future professionals that the university and the country will be proud of someday.

To Tita Tess of IPB as well as the other lab assistants, thank you for the countless times that you helped me during my chemical analysis in the laboratory. You have no idea how much it meant to me.

To my blocmates: Abby Pabro, Jo-anne Pascual, and Rachel Rodulfo who had been with me during this endeavor, your encouraging words helped me get through this. You are more than just blocmates to me.

To my friends: Angelica Cenizal, Anna Balmonte, Aina Tesorero, Ching Dee, Mouree Tumanguil, Maja Cantong, and more CCC friends, thanks for your prayers, words of encouragement. Ate Aina and Ate Ching, I really appreciate you trying to help me understand some of the points in my study that didn’t make sense to me. Haha! Ate Anna, your presence at that particular day lightened up my mood. God indeed knows how to make me smile. Maja, you are one of God’s evidences that He is at work. Anj, I wish you were tangibly with me while I was doing this, but I know that you’re with me in prayers. Thanks for listening, rebuking, and encouraging me.

To my family: Nanay, Tatay, and Sam. I’m always thankful to God that He gave me such wonderful family. Thank you for all the support, moral and financial. Haha. You are one of my inspirations in everything that I do. Nanay and Tatay, thank you for not giving up on me whenever I disappoint you. Sam, you are the greatest sister a person could have. I’m blessed to have you. I love you all.

To other people whom I failed to mention in this acknowledgement page, I equally thank you. You will always be part of this undertaking.

To my all-powerful, all-knowing, omnipresent Father, without You, even typing the front page of this manuscript will be impossible. Thank You for Your love and Your grace and Your faithfulness and for Your promise of wonderful plan for my life. I am eternally grateful!

ABSTRACT

The study focused on the determination of functional components of the different parts of

cooked and raw banana bud: the outer and inner bract and the male flower. Cooked banana bud

(outer and inner bract, flower) was subjected to 100o C water and cooked to edibility for

corresponding amount of time. It was found that there were significant variances in the amount

of functional components present in the cooked and raw samples. The results showed that certain

functional components such as the Antioxidative Activity, Tannin, Phenols, Flavonoid, and

Saponin of the samples decreased after cooking. In contrast, the Anthocyanidin content of the

samples increased after cooking. The Alkaloid content of the inner banana bract was lost after

cooking. Therefore it is concluded that cooking can alter the functional components of banana

bud. Moreover, it was found that the raw outer and inner bract contained high amount of

saponin; cooked outer bract, cooked flower and raw flower contained high amount of flavonoid;

and cooked inner bract contained high level of anthocyanidin.

TABLE OF CONTENTS

Acknowledgement ii

Abstract iii

Table of Contents iv

Introduction 1

Objectives of the Study 2

Significance of the Study 2

Limitations of the Study 3

Review of Related Literature 4

Banana Bud 4

Antioxidant 5

Phenols 7

Flavonoids 8

Anthocyanidin 9

Tannins 11

Alkaloids 13

Saponin 14

Methodology 16

Raw Material 16

Methods 16

Sample Extraction 16

Time and Place of Study 17

Determination of Total Phenolics 17

Total Flavonoid Assay 18

Total Anthocyanidin Assay 18

Total Tannins Assay 19

Antioxidative Activity 19

Alkaloids Assay 20

Saponin Content 20

Research Design 21

Statistical Analysis 21

Schematic Diagram 22

Results and Discussion 23

Summary and Conclusion 27

Recommendation 28

Literature Cited 29

Appendices 31

Appendix A. Raw Data/ Computed Data 31

Appendix B. Standard Curves 35

Appendix C. Statistical Analysis 37

INTRODUCTION

With the advancement of technology today comes the growing consciousness of people

for health. Scientists are yet to find cure for cancer and a variety of diseases. Several studies have

led to the discovery of certain substances which can inhibit the destructive effects of oxidation,

the antioxidants. The studies about antioxidants revealed that it is intimately related to the

prevention of cellular destruction (Parnes 2007). It has become a popular topic in health science.

Antioxidants are available from various plant sources. Interestingly, many of these plant

sources can be consumed by humans. One of these is the banana bud, more locally known as

“puso ng saging”. In many parts of Southeast Asia, it is eaten as a boiled vegetable after the

external, older, fibrous bracts have been removed. In the Philippines, banana bud is used as an

ingredient in a range of culinary dishes such as kare-kare, and laksa. (De Vera, 1992) Though

bananas are grown commercially for its fruits some of its parts, particularly the banana bud are

equally important. Its growing popularity demands that the banana bud’s functional components

be studied as well.

In this study, the buds of Musa sp. cv. saba were used. Of the 75 cultivars identified by

the National Plant Genetic Resources Laboratory, the bud of the Saba variety is the only one

found in the market and consumable. (Valmayor, et al., 1981 as cited by De Vera, 1992)

Objectives of the Study

The general objective of this study aimed to determine the effect of boiling on the

functional components of banana bud.

The specific objectives were:

a. to measure the functional components such as total phenolic content, total flavonoid

content, total tannins, total anthocyanidin, alkaloid content, saponin content as well as

the antioxidative activity of cooked and raw banana bracts and flowers; and

b. To compare the above mentioned functional components of cooked and raw banana

bract and flowers.

Significance of the Study

Banana bud is quite a popular ingredient

in particular dishes served in the Philippines. Considered a vegetable, this part of the

banana plant also is a good source of certain health beneficial components. Its growing

popularity demands that its composition be studied as well. The study was conducted in order to

discover the effect of boiling in the functional components of the banana bud.

Scope and Limitation

The limitations of the study were as follows:

1. Only the bud of saba cultivar was used in the study and its three main parts (outer bract,

inner bract and flower) were used.

2. Cooked samples were prepared by boiling (100o C) at different times for each part,

without any other treatment.

REVIEW OF RELATED LITERATURE

Banana Bud

Banana (Musa sp.) is grown in all regions of the Philippines throughout the year. It was

described by Fray Blanco, the first botanist in the Philippines, as variety compressa. It is known

in Indonesia as Pisang Kepok, in Malaysia as Pisang Nipah, and in Thailand as Kluai Hin. Saba

is the type cultivar of edible and cultivated Musa balbisiana species (Valmayor et al. 2002).

The banana bud is a component in the inflorescence of the banana plant. The male and

female flowers of the plant are both present but come out of the plant separately. The female

flowers are the first to come out which then develops into fruits. The first 5-15 basal nodes or

hands produce female flowers and the upper digital nodes produce male flowers (Del Rosario

1990 as cited by Villa 1993).

Banana is grown commercially for its fruits. The various parts of the plant other than the

fruit are also used for food, packaging, and other purposes. Banana bud (bracts and flowers), one

of its parts, is eaten as boiled vegetable and is also used as an ingredient in an assortment of

cuisines (Villa 1993).

Banana bud or inflorescence is one of the most important parts of the banana plant. Aside

from its being a mere ingredient in vegetable preparations, it is also processed and exported as

canned banana bud (Evangelista et al. 1977 as cited by De Vera 1992).

Composition analysis shows that boiled banana bud of the saba variety contains 59.1%

moisture, 1.2% protein, 38.6% carbohydrates, 0.3% fiber, and a total of 143 calories (FNRI 1980

as cited by Villa 1993).

Figure 1. Banana bud of saba variety

Antioxidant

Antioxidants are compounds that protect cells against the damaging effects of reactive

oxygen species, such as singlet oxygen, superoxide, transition metals such as iron and copper,

nitric acid, and ozone (Malayba 2009). Antioxidants are nutrients, vitamins and minerals, as well

as enzymes, proteins in your body that assist in chemical reactions (Parnes 2007).

Oxidative stress occurs when the production of harmful molecules called free radicals is

beyond the protective capability of the antioxidant defenses (Parnes 2007). Free radicals are

atoms or groups of atoms with an unpaired electron and can be formed when oxygen interacts

with certain molecules. Examples of free radicals are the superoxide anion, hydroxyl radical.

Their chief danger comes from the damage they can do when they react with important cellular

components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs.

To prevent free radical damage the body has a defense system of antioxidant

(http://www.rice.edu).

Because they have one or more unpaired electrons, free radicals are highly unstable. They

scavenge the body to grab or donate electrons, thereby damaging cells, proteins, and DNA. The

same oxidative process also causes oils to become rancid, peeled apples to turn brown, and iron

to rust.

Antioxidants work in two ways. They can either be chain-breaking or preventive. Chain-

breaking occurs when a free radical releases or steals an electron, a second radical is formed.

This molecule then turns around and does the same thing to a third molecule, continuing to

generate more unstable products. The process continues until termination occurs. On the other

hand, antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase

prevent oxidation by reducing the rate of chain initiation. That is, by scavenging initiating

radicals, such antioxidants can thwart an oxidation chain from ever setting in motion. They can

also prevent oxidation by stabilizing transition metal radicals such as copper and iron. The

effectiveness of any given antioxidant in the body depends on which free radical is involved,

how and where it is generated, and where the target of damage is. Thus, while in one particular

system an antioxidant may protect against free radicals, in other systems it could have no effect

at all (Parnes 2007).

Phenols

Phenolic acids are plant metabolites widely spread throughout the plant kingdom. Recent

interest in phenolic acids originates from their potential protective role, through ingestion of

fruits and vegetables, against oxidative damage diseases. Phenolic compounds are essential for

the growth and reproduction of plants, and are produced as a response for defending injured

plants against pathogens. The importance of antioxidant activities of phenolic compounds and

their possible usage in processed foods as a natural antioxidant have reached a new high in recent

years (Sahelian 2009).

Phenolic acid compound can be found and is universally distributed in plants. Several

studies in chemistry, biology, agriculture and medicine are made in which these compounds have

been the subject. Phenolic acids form a diverse group that includes the widely distributed

hydroxybenzoic and hydroxycinnamic acids (Sahelian 2009).

The concentration of phenolics may be affected by some factors like cultivar, such that

different varieties and cultivars exhibit different phenolic acid and flavonal content. However,

the location where it is grown had no effect. In terms of maturity, young fruits have higher

phenolic contents compared to the mature ones (Oomah and Mazza 1996 as cited by Abalayan

2004).

Plant phenolic compounds are diverse in structure but are characterized by hydroxylated

aromatic rings. They are categorized as secondary metabolites, and their function in plants is

often poorly understood. Many plant phenolic compounds are polymerized into larger molecules

such as the proanthocyanidins (PA; condensed tannins) and lignins. Furthermore, phenolic acids

may occur in food plants as esters or glycosides conjugated with other natural compounds such

as flavonoids, alcohols, hydroxyfatty acids, sterols, and glucosides (Sahelian 2009).

Flavonoids

The term “flavonoid” is generally used to describe a broad collection of natural products

that include a C6-C3-C6 carbon framework, or more specifically phenylbenzopyran functionality

(Grotewold 2007).

Flavonoids are compounds found in fruits, vegetables, and certain beverages that have

diverse beneficial, biochemical and antioxidant effects. The antioxidant activity of flavonoids

depends on their molecular structure, and structural characteristics of certain flavonoids found in

hops and beer confer surprisingly potent antioxidant activity exceeding that of red wine, tea, or

soy (Buhler and Miranda 2000).

Over 4,000 flavonoids have been identified, many of which occur in fruits, vegetables

and beverages. The flavonoids have aroused considerable interest recently because of their

potential beneficial effects on human health; they have been reported to have antiviral, anti-

allergic, antiplatelet, anti-inflammatory, antitumor and antioxidant activities (Buhler and

Miranda, 2000). 

Recent studies have demonstrated that flavonoids found in fruits and vegetables may also

act as antioxidants. Like alpha-tocopherol (vitamin E), flavonoids contain chemical structural

elements that may be responsible for their antioxidant activities. A recent study by Dr. van Acker

and his colleagues in the Netherlands suggests that flavonoids can replace vitamin E as chain-

breaking antioxidants in liver microsomal membranes. The contribution of flavonoids to the

antioxidant defense system may be substantial considering that the total daily intake of

flavonoids can range from 50 to 800 mg. This intake is high compared to the average daily intake

of other dietary antioxidants like vitamin C (70 mg), vitamin E (7-10 mg) or carotenoids (2-3

mg). Flavonoid intake depends upon the consumption of fruits, vegetables, and certain

beverages, such as red wine, tea, and beer. The high consumption of tea and wine may be most

influential on total flavonoid intake in certain groups of people (Buhler and Miranda 2000). 

The capacity of flavonoids to act as antioxidants depends upon their molecular structure.

The position of hydroxyl groups and other features in the chemical structure of flavonoids are

important for their antioxidant and free radical scavenging activities. Quercetin, the most

abundant dietary flavonol, is a potent antioxidant because it has all the right structural features

for free radical scavenging activity (Buhler and Miranda 2000). 

Anthocyanidin

Anthocyanidins are in a select group of flavonoids technically known as "flavonals,"

which provide crosslinks that connect and strengthen intertwined strands of collagen protein.

Being water soluble, they also scavenge free radicals they encounter in tissue fluids. This quality

makes anthocyanidins especially beneficial for athletes and others who exercise, because heavy

exercise generates large amounts of free radicals. Anthocyanidins are found in blue-purple

colored foods such as grapes and blueberries (http://www.altmeds.com).

There are six most prevalent anthocyanidins in nature namely: pelagonidin, cyanidin,

peonidin, delphinidin, maldivin, and petunidin. In nature, different anthocyanidins are commonly

bound to sugars such as glucose, galactose, rhamnose, xylose, or arabinose; the resulting

molecules are called anthocyanins (Knasmuller et al. 2009).

Plant foods have long been known to promote health and wellness. With over two

thousand known plant pigments presently identified, the chemicals that give foods their colors

may also translate into vibrant health. There are researches about the antioxidant properties of

anthocyanidin that shows that not only does it support the health of plants but can support the

health of humans as well (http://www.whfoods.com).

Among the most abundant commercial sources of anthocyanidins are berries; for

example, blueberries, blackberries, black currants, elderberries, grapes, and strawberries. The

anthocyanidin content of the berry increases with its maturation stage, and is highest in ripe

berries. In general, the darker the berry, the higher its anthocyanidin content; the higher its

antioxidant potential (Knasmuller et al. 2009).

As researchers confirm that metabolites of oxidation, known as free radicals, are at the

root of the progression of both chronic diseases such as arthritis, atherosclerosis, diabetes and

cancer and other signs of aging, such as the loss of skin elasticity and cognitive function,

antioxidants are gaining an ever more important place in health promotion. Among the

antioxidants, anthocyanidins have been found to have some unique features. They are able to

protect cells and tissues from free radical damage in both water-soluble and fat-soluble

environments. And, their free radical scavenging capabilities are thought to be more potent than

many of the currently well-known vitamin antioxidants (http://www.whfoods.com).

Much of the studies on anthocyanidins have focused on their ability to protect collagen

from the destructive effects of free radical damage. This protection of capillary integrity

manifests in the ability of anthocyanidins to reduce the fragility and permeability of these small

blood vessels. Anthocyanidins are therefore thought to be able to reduce edema as well as

vascular conditions such as varicose veins and hemorrhoids. Their ability to inhibit the

degradation of collagen found in blood vessels and cartilage has led researchers to propose that

anthocyanidins may play a role in the prevention of atherosclerosis and arthritis

(http://www.whfoods.com).

Tannins

Tannins are complex chemical compounds that are extensively distributed within the

plant kingdom. While tannins in some quantity may be found in most plant tissues, when there

are high concentrations, the tannins are usually localized to just a few plant parts, such as the

bark, roots, or leaves. They may be considered as those substances of vegetable origin which

may be found, as water-soluble bodies, in many plants, exhibiting certain chemical behavior,

possessing astringent properties (Grasser 2007).

It is difficult to give a concise and complete definition of the word “tannin”. The term

tannin, commonly used for the phenolics of cereals and legumes, has been questioned.

Originally, this word was coined by Seguin to describe the substances present in vegetable

extracts which are responsible for converting animal skins into stable product leather. The

phenomenon is called tanning process, and the substance responsible to convert skin into leather

are called tannins. In plant extracts, these substances are polyphenols of varying molecular size

and complexity (Grasser 2007).

Tannins’ molecular weight is between 500 and 3000. They are usually complex

polyphenol mixtures and are difficult to separate because they do not readily crystallize. Tannins

form colloidal solutions in water; precipitate protein solutions such as gelatin as well as alkaloids

in solution produce a deep red color when mixed with potassium ferricyanide and ammonia, and

form green-black or dark blue soluble compounds with ferric salts. Tannins are also known to

absorb oxygen while in strong alkaline solutions (Grasser 2007).

Tannins are sometimes called plant polyphenols, a name which originally included

polyphenolic compounds of various types including flavonoids, lignans, etc. At the same time,

some polyphenols which contain a non-tannin polyphenol part, such as flavan-flavonol

condensates, are classified as tannins (Okuda 1999).

Alkaloids

The term “alkaloid” essentially means a substance with an alkali-like character. So far,

there are an enormous number of alkaloids that are already isolated from plant and animal

organisms, fungi, or natural folk medicines (Hesse 2002).

Though, chemistry has provided a definition of alkaloids in purely chemical terms.

Chemists stress that alkaloids are any group of complex heterocyclic nitrogen compounds, which

have strong physiological activity, are often toxic, and retain their own basic chemical properties

(Aniszewski 2007).

The pharmaceutical drug industry has succeeded in the use of natural plant alkaloids for

the development of anti-malarian agents, anticancer agents and agents promoting blood

circulation in the brain. Moreover, they have been and continue to be the object of human

interest concerning new possibilities for their safe utilization and ensuing health benefits

(Aniszewski 2007).

Alakloids are generally classified by their common molecular precursors, based on the

biological pathway used to construct the molecule. From a structural point of view, alkaloids are

divided according to their shapes and origins. There are three main types of alkaloids namely:

true alkaloids, protoalkaloids and pseudoalkaloids. True alkaloids and protoalkaloids are derived

from amino acids while the pseudoalkaloids are not (Aniszewski 2007).

Alkaloids are most commonly found in higher plants and are abundant. At least 25% of

higher plants contain these molecules. Some plant families are especially rich in alkaloids such

as the Dogbane botanical family (Apocynaceae), the Logan botanical family (Loganiaceae), the

Poppy botanical family (Papaveraceae), the Citrus botanical family (Rutaceae), the Nightshade

botanical family (Solanaceae), the Coca botanical family (Erythroxylaceae), the Borage botanical

family (Boraginaceae), and the Legume botanical family (Fabaceae) (Aniszewski 2007).

Saponin

Saponins are glycosides of triterpenes, steroids or steroidal alkaloids. They can be found

in plants and marine organisms. Very diverse biological activities are attributed to saponins and

they play important roles in food, animal feedstuffs, and pharmaceutical properties (Oleszek and

Marston, 2000). The name saponin was derived from the Caryophyllaceae plant Saponaria

officinalis (soapwort), the root of which was used as soap since time immemorial (Jia, et al

2002).

Saponins occur in plants which are used as human food. Among them these foods are:

soybeans, chick peas, peanuts, mungbeans, broad beans, kidney beans, lentils, garden peas,

spinach, oats, asparagus, fenugreek, garlic, sugar beets, potatoes, green peppers, tomatoes,

onions, cassava, yams, and legumous forage spices. Though, the contents may depend on factors

such as the cultivar, the age, the physiological state and the geographical location of the plant

(Hostettmann and Marston 1995).

Usually highly polar compounds, saponins occur as complex mixtures and their

separation into individual components is a very tedious task. They act as emulsifiers, stabilizing

the oil or water interface. Saponins have a high capacity for dissolving monoglycerides. Based

on these activities, it can be speculated that dietary saponins could improve fat emulsification

and digestion. However, the opposite appears to be true, with several studies finding that dietary

saponin reduces fat digestibility. The major effect of saponin on lipid digestibility appears to be

exerted through effects on bile acids. Saponin form micelles, electrically charged molecules,

with bile acids, reducing availability of bile acids for formation of micelles with fatty acids. The

bioavailability of vitamin A and E may also be reduced by saponins, probably because of

sequestration of bile acids (Oleszek and Marston 2000).

Also, saponins increase the effectiveness of oral vaccines by altering the permeability of

the intestinal mucosa. It was determined that some saponins increase the permeability of

intestinal mucosal cells, facilitating the uptake of substances to which the gut would normally be

impermeable. It was proposed that saponins react with cholesterol in the membranes of the

microvilli, causing structural lesions (Oleszek and Marston 2000).

MATERIALS AND METHODS

Raw Materials

Banana buds were acquired from a local wet market in Cabuyao, Laguna. It was fresh

and free from blemishes. It was taken to a plant taxonomist, Dr. William Gruezo, for

identification.

Sample Preparation and Extraction

The banana bud’s outer bract, inner bracts, and the male flowers were subjected to a

treatment. Each of the samples was cooked in boiling water (100oC). Each treatment was cooked

at 5, 15, and 20 minutes, respectively.

For two hours, two hundred milligrams of each sample was extracted with 10ml of 50%

methanol at room temperature with constant agitation by a vortex mixer. The mixture was

centrifuged at 10,000g for 15 minutes, and the supernatant was decanted into 50 ml volumetric

flask. The residue was re-extracted with the same solvent and the supernatants were combined

and diluted to 50 ml with 50% methanol. Then, the samples were filtered and further diluted.

This was used for total phenolic, total flavonoid, total tannins, anthocyanidin content and

antioxidative content assay.

Time and Place of Study

Samples were prepared and extracted at the Bio-Assay Laboratory, Institute of Human

Nutrition and Food while the chemical assays were done in the Analytical Services Laboratory,

Institute of Plant Breeding, University of the Philippines, Los Baños from August 4 to September

8, 2010.

Determination of Total Phenolics

Total phenolics was determined colorimetrically by using the method described by

Velioglu et al. (1998) as cited by Rodriguez and Mendoza (19910 with slight modifications. Two

hundred microliter (200 µl) of extract was mixed with 1.5 ml of Folin-Ciocalteau reagent which

was previously diluted 10-fold with distilled water. It was allowed to stand at room temperature

for 5 minutes. A 1.5 ml sodium bicarbonate solution (60 g/L) was added to the mixture and

incubated further at room temperature for 90 minutes. Absorbance was measured at 725µm using

Cecil UV Visible spectrophotometer. Total phenolics was quantified by calibration curve

obtained from measuring the absorbance of known concentrations of catechins standard which is

25-150µg/ml of 50% methanol. They were expressed as grams catechin equivalents per gram of

dry weight.

Total Flavonoid Assay

The flavonoid content was measured using the colorimetric assay developed by Zhishen

et al (1999) as cited by Rodriguez and Mendoza, 1991. One ml of correctly diluted sample was

added to a 10 ml volumetric flask containing 4 ml of distilled water. At time 0 (zero), 0.3 ml of

5% NaNO2 was added to each volumetric flask. Then, after 5 minutes, 0.3 ml of 10% AlCl 3 was

added. After 1 minute, 2 ml of 1 M NaOH was added. Each reaction flask was immediately

diluted and mixed with 2.4 ml distilled water. Absorbances of the mixtures upon the

development of pink color were determined at 510 µm relative to a prepared blank. The total

flavonoid contents of the samples were expressed in milligrams gallic acid equivalent per gram

of sample.

Total Anthocyanidins Assay

The colorimetric assay for anthocyanidins that was used in the experiment is the one by

Sun et al (1998) as cited by Rodriguez and Mendoza, 1991, with slight modifications. One ml of

sample, 2.5 ml of 1% (weight/volume) vanillin in methanol, and 2.5 ml of 9.0 N HCl in methanol

were mixed together in a test tube and were incubated for 20 minutes at room temperature. After

incubation, the absorbances were recorded at 500µm. A calibration curve using various

concentration of catechin were used to calculate the amount of anthocyanidins in the samples.

Total Tannins Assay

The analysis of total tannins was conducted by using the modified Prussian Blue

Reduction Method. An aliquot, 0.10 ml of the methanol extract was placed in a 17 by 100 ml

tube. Exactly 3 ml of distilled water was added and then shaken. One ml of 0.008 M potassium

ferricyanide were added and then followed by 1.00 ml of 0.30 M ferric chloride solution. The

mixture was shaken thoroughly and allowed to stand for 15 minutes at room temperature. The

absorbance reading was taken at 710µm using a Cecil UV-Vis Spectrophotometer. A standard

curve was prepared using catechin to compute for the amount of tannins in the sample.

Antioxidative Acitivy

For the antioxidative activity, an aliquot of 2.9 ml of 10-4 DPPH solution in methanol

were added to a 25 µL of the extract and 75 µL of water. The mixture was shaken in a vortex

mixer and allowed to stand in the dark for 30 minutes. The absorbance is measured at 517 nm. A

reagent blank and a positive control using different concentrations of butylated hydroxtoluene

(BHT) are also prepare as in sample.

Antioxidative activity was measured from the difference between the absorbance of the

blank minus the absorbance divided by the absorbance of the blank multiplied by 100. This is

expressed as percent lipid peroxidation.

Percent Lipid Peroxidation = Absorbance blank – Absorbancex 100

Absorbance blank

The higher the percentage, the lower is the antioxidative activity.

Total Alkaloids Assay

The alkaloid content of the samples was analyzed using the qualitative determination of

alkaloids. One gram of each sample was extracted using 10.0ml absolute methanol overnight.

The mixture was placed in 40oC for 4 hours. It was filtered and then, using an oven dehydrator

volume was reduced to about 5ml. Ten milliliters of 1% HCl was added, followed by the

addition of concentrated ammonia. The mixture was placed on a separatory funnel and then

extracted 3 times with 10 ml chloroform each time. The chloroform extract was passed in sodium

sulfate to remove excess water. It was dried completely, and then re-extracted with 5ml

chloroform. Alkaloid was extracted using 5.0 ml 1% HCl. An aliquot was tested for precipitation

with Bismuth Nitrate.

Saponin Content

The saponin content of the samples was analyzed using the qualitative determination of

saponins. One gram of the fresh sample was finely cut and added with 10.0 ml of 80% ethanol. It

was macerated until it became homogenous. It was filtered using Whatman coarse filter paper

and the extract was placed in a screw-capped vial. The supernate was passed through PVPP

columns prepared overnight. The first volumes of eluents that were turbid were removed. One

ml of aliquot was placed in the test tube and was pre-treated with 0.5ml glacial acetic acid and

then mixed. Three ml of Lieberman-Buchard reagent was placed in the mixture and then heated

in a 90-100oC water bath for 30 minutes. It was then cooled to room temperature and the

absorbance was read at 450 nm using Cecil UV-Vis Spectrophotometer. The unit used is

milligram saponin.

Research Design

This was an experimental study, a true experiment. The whole banana bud, inner bracts

and male flowers were the independent variables in this study while the antioxidative activity,

total phenolic content, total tannins, total flavonoids, total anthocyanidins, alkaloid and saponin

content were the dependent variables. The data gathered for this study underwent t-test to see

their relationship.

Statistical Analysis

Analyses were done in triplicate. Analysis of variance (ANOVA) was applied to the data

using T-test (Least Square Difference) to test any significant differences between raw and

cooked samples.

Schematic Diagram

Research Proposal

Bought fresh sample

Prepared methanolic extract

Antioxidative Activity Assay

Phenolics Assay

Tannins Assay

Anthocyanidin Assay

Flavonoid Assay

Alkaloid Assay

Saponin Assay

Raw data computed

Statistical Analysis

Research paper done

Identification of the sample by a

taxonomist

RESULTS AND DISCUSSIONS

The samples were subjected to treatments: raw and cooked. Table 1 shows the

comparison between the two. All samples of cooked in contrast to raw are significantly different.

Table 1. Functional Components of Cooked and Raw SamplesSample TPC Flavonoid Tannin Anthocyanidin AOA SaponinRaw 87.2489 a 407.238 a 117.4033 a 37.236 b 38.4600 b 491.070 aBoiled 40.7900 b 172.571 b 56.6367 b 98.061 a 69.4833 a 123.408 bPr > F 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001LSD 0.8262 15.832 2.1557 4.6965 1.176 7.4615NOTE: Means with the same letter are not significantly different at 5% level using LSD.

Table 2 shows the comparison of the different parts of the banana bud and its functional

components. Almost all parts of the banana bud showed significant differences in their functional

components except for the saponin content of inner bract and flower.

Table 2. Functional Components of the Different Parts of the Banana BudSample TPC Flavonoid Tannin Anthocyanidin AOA Saponin

Outer bract

92.0800 c 155.048 c 113.333 a 50.467 c 79.3567 a 210.508 b

Inner bract

65.4833 b 287.618 b 90.000 b 94.827 a 46.1283 b 358.737 a

Flower 92.0800 a 427.047 a 57.727 c 57.467 b 36.4300 c 352.472 aPr > F 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001LSD 1.0118 19.38 2.6402 5.752 1.4404 9.1385 NOTE: Means with the same letter are not significantly different at 5% level using LSD.

Table 3 shows the measurement of functional components of cooked and raw parts of the

banana bud. The antioxidative activity (AOA) of the cooked samples decreased in relation to the

raw samples. This decrease may be due to possible disruption of AOA of the sample which is

caused by the onset of destruction of cell wall due to the heat applied (Guerrero 2009). Also,

since the sample was submerged to water while being cooked, this might have caused the

antioxidant content of the samples to denaturize, thus, decrease in AOA (Amin and Lee 2005 as

cited by Guerrero 2009).

Table 3. Measurement of the Functional Components of the samples.

Sample

Antioxidative Activity

(% Lipid Peroxidation)

Phenols(mg

catechins/ 100g)

Flavonoid(ml gallic acid/ 100

g)

Anthocyanidin(mg catechins/

100g)

Tannins(mg

catechins/ 100g)

Saponin(mg

saponin)

Alkaloids(+, -)

Raw Outer Bract

75.89 32.10 182.86 28.01 165.23 386.22 (+)

Cooked Outer Bract

82.83 36.89 127.24 72.92 61.43 34.80 (-)

Raw Inner Bract

15.20 104.82 470.09 60.34 119.38 627.00 (+)

Cooked Inner Bract

77.06 26.14 105.14 129.31 60.6290.47

(+)

Raw Male

Flower24.29 124.82 568.76 23.35 67.60 459.99 (+)

Cooked Male

Flower48.57 59.34 285.33 91.96 47.85 244.96 (-)

Only the cooked outer bract of the banana bud indicated an increase in phenolic content

while the rest of the cooked samples decreased. The increase in phenols may have been due to

the breakage of supramolecular structures, releasing the phenolic sugar glycosidic bounds, which

react better with the Folin-Ciocalteau reagent (Bunea et al. 2008 as cited by Jimenez-Monreal et

al. 2009). Heat treatment may also reduce the activity of phenylalanine ammonia lyase (PAL),

the enzyme responsible for the biological production of polyphenols. It is possible that while the

activity of PAL was subdued, the phenolics that are already present were more efficiently

released from the cells or from being bound to other molecules. The decrease in phenols,

however, can be accounted to its dissolution to water while cooking through thermal degradation

or inactivation of antioxidant enzymes (Recuenco 2007). The use of hot water results to greater

amount of water-soluble functional components lost due to leaching (Morris et al. 2004 as cited

by Recuenco 2007).

The relationship between the phenols and flavonoids may be considered in view of the

fact that flavonoid is a part of a major group of phenols (Guerrero 2009). Therefore, any increase

or decrease of flavonoid is directly related to any increase or decrease of phenols.

Tannin is known as the contributor to the astringent taste of certain crops. It is indicated

in Table 1 that the samples decreased their tannin content after cooking. There are two main

categories of tannin: the hydrolysable tannin and the condensed tannin (Cannas 2008). Loss of

tannin can be accounted to its being hydrolysable. This kind of tannin can be hydrolyzed by mild

acids or mild bases to yield carbohydrate and phenolic. It can also be hydrolyzed by using hot

water or even enzymes, particularly tannase (Cannas 2008). In this case, it was the hot water.

Anthocyanidin, on the other hand, being condensed tannin (proanthocyanidin), increased

in levels after cooking. Since they are linked by carbon-carbon bond, they are not susceptible to

cleavage by hydrolysis (Cannas 2008). When applied with heat, molecules produce stronger

radical-scavenging antioxidants by thermal chemical reaction (Jimenez-Monreal 2009).

The saponin content of the cooked samples also decreased. It could be attributed to its

high polarity, thus, solubility in water. Presence of alkaloid, however, was found in all of the

samples except from cooked outer bract and cooked male flower. The decrease is most likely

physical than chemical (Recuenco 2007).

Figure 2. Comparison Functional Components of Cooked and Raw Banana Bud

Results indicated that the functional component highly abundant in the banana bud

sample were saponins, flavonoids and anthocyanidins (Figure 2). Saponin level is most elevated

on the raw outer bract and raw inner bract; flavonoids are most abundant in cooked outer bract,

raw male flower and cooked male flower; and the cooked inner bract is high in anthocyanidin.

SUMMARY AND CONCLUSION

With the rise of many lifestyle diseases as well as the growing consciousness of people

for their health come various discoveries about health benefits from plant sources. These

discoveries had led to various studies of certain phytochemicals and their properties. Plants,

being its source are placed under the healthiness spotlight.

The banana bud of saba cultivar was used in this study. It has three main parts: the outer

fibrous bract, the inner bract and the male flower. These samples were subjected to two

treatments: raw and cooked (boiled) to see the change in its functional component contents.

Results showed that applying heat has effect on the functional components of the samples. Most

of the functional components such as the total phenolic content, flavonoid content, tannins,

saponins, as well as the antioxidative activity of the cooked sample decreased in comparison to

the raw samples. On the other hand, anthocyanidin content increased due to its properties as

condensed tannin. The loss of alkaloid, however, can be attributed to physical loss rather than

chemical.

Particular parts of the buds presented large quantities of functional components. Among

these were saponins, flavonoids, and anthocyanidin. The raw inner and outer bract of the banana

bud exhibited generous quantity of saponins. The cooked outer bract and flower as well as the

raw flower presented high amounts of flavonoids and cooked inner bract was high in

anthocyanidin content. These components are also known to contribute astringency to a number

of crops.

RECOMMENDATIONS

The most usual mode of cooking prepared for banana bud is boiling, though it is possible

to try a different cooking method for it. Studies conducted showed that among the cooking

methods, boiling can lead to great loss of functional components since most of these components

are soluble to water.

Banana bud is native in the Philippines and comes in abundance and could also be a good

source of profit for the country. Modern technology has allowed it to be processed and exported

as canned banana buds. And with the continuing advancement in technology, new food product

could be developed from it.

Aside from profit, a growing interest for health improvement is a major concern. An

upsurge in research of edible plant parts has lead to the discovery of antioxidant-rich plant

extract as dietary supplement. Being rich in flavonoid that is diversely beneficial to human

health, it is not impossible that food supplement be developed out of it. Also, as mentioned

earlier, studies were conducted about the properties of saponins and it has shown that it could

increase the effectiveness of oral medicines. The study of the saponin content as well as the other

antioxidants can be an important basis for the pharmacological extraction.

LITERATURE CITED

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CRUZADA MAG. 2007. Functional Components of Cucumber (Cucumis sativus). BS Thesis, University of the Philippines, Los Baños.

DE VERA MCA. 1992. Reduction of Polyphenols in Banana Buds (Musa paradisiaca va Sapientum cv. lakatan). BS Thesis, University of the Philippines, Los Baños.

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GUERRERO SY. 2009. Functional Component of Alibangbang (Bauhinia malabarica Roxb.), Yampong (Ipomoea congesta), Lupo-lupo (Spenochlea zeylanica), and Takip-kuhol (Centella asiatica) Leaves. BS Thesis, University of the Philippines Los Baños.

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http://www.rice.edu cited June 28, 2010

http://www.altmeds.com/anthocyanidins cited July 1, 2010

http:www.whfoods.com/genpage cited July 1, 2010

APPENDICES

Appendix A

Raw and Computed Data

Table 1. Antioxidative activity of each sample

 AOA (% LIPID PEROXIDATION)          

  RAW     COOKED      1 2 3 1 2 3outer bract 77.155 75.304 75.201 83.311 82.861 82.306inner bract 15.595 15.655 14.343 77.565 77.733 75.871Flower 22.845 23.887 26.139 49.795 48.853 47.051

Table 2. Phenolic content of each samplePhenols abs x=y/m x/wt. spl % phenols mg/100 g

Raw outer bract 0.249 6.552632 0.000328 0.032763 32.76316 0.24 6.315789 0.000316 0.031579 31.57895 0.243 6.394737 0.00032 0.031974 31.97368

Raw inner bract 0.793 20.86842 0.001043 0.104342 104.3421 0.797 20.97368 0.001049 0.104868 104.8684 0.8 21.05263 0.001053 0.105263 105.2632

Raw flower 0.953 25.07895 0.001254 0.125395 125.3947 0.959 25.23684 0.001262 0.126184 126.1842 0.934 24.57895 0.001229 0.122895 122.8947

Cooked outer bract 0.28 7.368421 0.000368 0.036842 36.84211 0.282 7.421053 0.000371 0.037105 37.10526 0.279 7.342105 0.000367 0.036711 36.71053

Cooked inner bract 0.194 5.105263 0.000255 0.025526 25.52632 0.2 5.263158 0.000263 0.026316 26.31579 0.202 5.315789 0.000266 0.026579 26.57895

Cooked flower 0.45 11.84211 0.000592 0.059211 59.21053 0.451 11.86842 0.000593 0.059342 59.34211 0.452 11.89474 0.000595 0.059474 59.47368

Table 3. Flavonoid content of each sampleFlavonoids Abs x=y/m x/wt. spl %flavo mg/ 100g

           raw outer bract 0.164 937.1429 0.018743 1.874286 1874.286  0.158 902.8571 0.018057 1.805714 1805.714  0.158 902.8571 0.018057 1.805714 1805.714raw inner bract 0.396 2262.857 0.045257 4.525714 4525.714  0.393 2245.714 0.044914 4.491429 4491.429  0.445 2542.857 0.050857 5.085714 5085.714raw flower 0.505 2885.714 0.057714 5.771429 5771.429  0.503 2874.286 0.057486 5.748571 5748.571  0.485 2771.429 0.055429 5.542857 5542.857cooked outer bract 0.108 617.1429 0.012343 1.234286 1234.286  0.112 640 0.0128 1.28 1280  0.114 651.4286 0.013029 1.302857 1302.857cooked inner bract 0.099 565.7143 0.011314 1.131429 1131.429  0.09 514.2857 0.010286 1.028571 1028.571  0.087 497.1429 0.009943 0.994286 994.2857cooked flower 0.25 1428.571 0.028571 2.857143 2857.143  0.257 1468.571 0.029371 2.937143 2937.143  0.242 1382.857 0.027657 2.765714 2765.714

Table 4. Tannin content of each sampletannins abs x=y/m x/wt. spl % tannin mg/ 100g           raw outer bract 0.869 16.24299 0.001624299 0.162429907 162.4299  0.888 16.59813 0.001659813 0.165981308 165.9813  0.895 16.72897 0.001672897 0.16728972 167.2897raw inner bract 0.624 11.66355 0.001166355 0.116635514 116.6355  0.638 11.92523 0.001192523 0.119252336 119.2523  0.654 12.2243 0.00122243 0.122242991 122.243raw flower 0.354 6.616822 0.000661682 0.066168224 66.16822  0.365 6.82243 0.000682243 0.068224299 68.2243  0.366 6.841121 0.000684112 0.068411215 68.41121cooked outer bract 0.321 6 0.0006 0.06 60  0.328 6.130841 0.000613084 0.061308411 61.30841  0.337 6.299065 0.000629907 0.062990654 62.99065cooked inner bract 0.319 5.962617 0.000596262 0.059626168 59.62617  0.32 5.981308 0.000598131 0.059813084 59.81308  0.334 6.242991 0.000624299 0.062429907 62.42991cooked flower 0.244 4.560748 0.000456075 0.045607477 45.60748  0.254 4.747664 0.000474766 0.047476636 47.47664  0.27 5.046729 0.000504673 0.05046729 50.46729

Table 5. Anthocyanidin content of each sampleanthocyanidin abs x=y/m x/wt. spl % anthocyanidin mg/ 100graw outer bract 0.021 4.525862 0.000226 0.02262931 22.62931

0.029 6.25 0.000313 0.03125 31.25 0.028 6.034483 0.000302 0.030172414 30.17241raw inner bract 0.058 12.5 0.000625 0.0625 62.5 0.048 10.34483 0.000517 0.051724138 51.72414 0.062 13.36207 0.000668 0.066810345 66.81034raw flower 0.025 5.387931 0.000269 0.026939655 26.93966 0.019 4.094828 0.000205 0.020474138 20.47414 0.021 4.525862 0.000226 0.02262931 22.62931cooked outer bract 0.07 15.08621 0.000754 0.075431034 75.43103 0.068 14.65517 0.000733 0.073275862 73.27586 0.065 14.00862 0.0007 0.070043103 70.0431cooked inner bract 0.116 25 0.00125 0.125 125 0.124 26.72414 0.001336 0.13362069 133.6207 0.12 25.86207 0.001293 0.129310345 129.3103cooked flower 0.088 18.96552 0.000948 0.094827586 94.82759 0.084 18.10345 0.000905 0.090517241 90.51724 0.084 18.10345 0.000905 0.090517241 90.51724

Table 6. Alkaloid content of bract and flower

Banana BudAlkaloids

raw cookedouter bract (+) (+)inner bract (+) (-)

Flower (+) (+)

Table 7. Saponin content of each samplessaponins abs x=y/m x/wt. spl %saponin mg/ 100g           raw outer bract 0.185 0.386221 3.86221E-06 0.000386 0.386221294  0.188 0.392484 3.92484E-06 0.000392 0.392484342

  0.182 0.379958 3.79958E-06 0.00038 0.379958246raw inner bract 0.302 0.63048 6.3048E-06 0.00063 0.630480167  0.299 0.624217 6.24217E-06 0.000624 0.624217119  0.3 0.626305 6.26305E-06 0.000626 0.626304802raw flower 0.212 0.442589 4.42589E-06 0.000443 0.442588727  0.225 0.469729 4.69729E-06 0.00047 0.469728601

  0.224 0.467641 4.67641E-06 0.000468 0.467640919

cooked outer bract 0.016 0.033403 3.34029E-07 3.34E-05 0.033402923

  0.015 0.031315 3.13152E-07 3.13E-05 0.03131524

  0.019 0.039666 3.9666E-07 3.97E-05 0.039665971

cooked inner bract 0.044 0.091858 9.1858E-07 9.19E-05 0.091858038

  0.041 0.085595 8.5595E-07 8.56E-05 0.08559499

  0.045 0.093946 9.39457E-07 9.39E-05 0.09394572

cooked flower 0.117 0.244259 2.44259E-06 0.000244 0.244258873

  0.118 0.246347 2.46347E-06 0.000246 0.246346555

  0.117 0.244259 2.44259E-06 0.000244 0.244258873

Appendix B

Standard Curve

Figure 1. Standard Curve for Phenol

Figure 2. Standard Curve for Flavonoid

Figure 3. Standard Curve for Tannin

Figure 4. Standard Curve for Anthocyanidin