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Functional Components of Cooked and Raw Banana Blossom (Decena, J.)
Transcript of Functional Components of Cooked and Raw Banana Blossom (Decena, J.)
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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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