SYNOPSIS

on
Conventional And Ultrasound Assisted Extraction of Flavonoid
and Total Phenolics Compounds from Pineapple Core
Presented By
SONALI SINGH
ID NO-22MTPFE006
[Link] (PROCESSING AND FOOD ENGINEERING)

2023

Adviser
Dr. DORCUS MASIH
(Assistant Professor)

DEPARTMENT OF PROCESSING AND FOOD ENGINEERING

VAUGH INSTITUTE OF AGRICULTURAL ENGINEERING AND TECHNOLOGY
SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND
SCIENCES
PRAYAGRAJ (U.P.) 211007
CONTENTS

1. INTRODUCTION
2. JUSTIFICATION
3. OBJECTIVES
4. REVIEW OF LITERATURE
5. MATERIALS & METHODS
6. REFERENCES
INTRODUCTION
The pineapple (Ananas comosus) is a tropical plant with an edible fruit; it is the most
economically significant plant in the family Bromeliaceae. The pineapple is indigenous to
South America, where it has been cultivated for many centuries. The introduction of the
pineapple to Europe in the 17th century made it a significant cultural icon of luxury. Since
the 1820s, pineapple has been commercially grown in greenhouses and many tropical
plantations.

The pineapple is one of the leading commercial fruit crops of the tropics. Pineapple is also a
commercially important fruit crop of India with around 90,000 ha area under this crop, 28.65
lakh tonnes annual production and 15.3 tonnes /ha productivity (Anonymous, 2021). It is one
of the choicest fruit all over the world because of its pleasant taste and flavour. It is also a
source of flavonoids, bromelain (proteolytic enzyme) & phenolic compounds, which have a
medicinal benefits, included anticancer, antioxidant, anti-inflammatory, and antiviral
properties. In addition to being eaten fresh, the fruit can also be canned and processed in to
different forms.

Phenolic compounds are secondary plant metabolites, which are important in determining the
sensory and nutritional quality of fruits, vegetables and other plants (Tomas- Barberan et al
2000, Lapornik et al 2005). These compounds possess an aromatic ring bearing one or more
hydroxyl groups and their structures may range from simple phenolic molecule to that of a
complex high molecular mass polymer (Balasundram et al 2006). These compounds are a
large group of bioactive chemicals having diverse biological functions. They may act as
phytoalexins (Popa et al 2008), antifeedants, attractants for pollinators and contributors to
plant pigmentation, antioxidants and protective against UV light (Naczk and Shahidi 2006).
These types of properties of phenolic compounds play an important role in plant growth and
reproduction (Popa et al 2002, Bravo 1998). In particular, natural phenols have been
reported to have excellent properties as food preservatives and play an important role in
protection against a number of pathological disturbances, such as atherosclerosis, brain
dysfunction and cancer (Gordon 1996, Valenzuela et al 1992). Phenolics are also capable of
scavenging free radicals, chelate metal complexes, activate antioxidant enzymes, reduce α-
tocopherol radicals and inhibit oxidases (Alia et al 2003, Amic et al 2003).
Flavonoids are the main class of phenolic compounds. More than 8000 polyphenolics
including over 4000 flavonoids have been identified and number is still growing. Flavonoids
are further classified as flavones, anthocyanins, isoflavones, flavonols and flavanols (Tsao
and Yang 2003). Flavonoids are low molecular weight compounds, consisting of fifteen
carbon atoms, arranged in C6-C3-C6 configuration. The structure has two aromatic rings A
and B joined by a 3-carbon bridge, usually in the form of a hetrocyclic ring C. The ring A is
derived from the acetate/malonate pathway, while ring B is derived from phenylalanine
through shikimate pathway (Merken and Beecher 2000).

Flavonoids are present in most of the plants and are responsible for color of flowers in plants.
Flavonoids represent a common constituent of the human diet. In some countries, the daily
dietary intake of mixed flavonoids is estimated to be in the range of 500-1000 mg. Now-a-
days flavonoids are becoming the subject of medicinal research. They have been reported to
possess many useful properties like anti-allergic (Toshio et al 2002), anti- inflammatory,
antimicrobial (Cottiglia et al 2001), anti-cancer activity (Androutsopoulos et al 2009),
oestrogenic activity, enzyme inhibition activity, antioxidant and antitumour activity. An
important effect of flavonoids is the scavenging of oxygen-derived free radicals (Asgary et al
1999). Flavonoids in red wine and in tea are also demonstrated to be effective antioxidants.
The flavonol glycoside rutin from buckwheat and rue, and the flavonone glycoside hesperidin
from citrus peels have been included in the dietary supplements as vitamin P.

These bioactive compounds are reported to have numerous medicinal and health promoting
effects. The inclusion of fresh green onions in the diet can influence the initiation and the
progression of carcinogenesis as it acts on the pathways implied in cell proliferation,
apoptosis and metastasis. Onion is main green food item which contains active components,
which possess antioxidant, cytotoxic and pro-apoptotic properties. Food-derived flavonoid
quercetin, can inhibit the growth of various cancer cells and is considered as a good candidate
for anticancer therapy. It behaves as an antioxidant and/or prooxidant as well as modulates
different intracellular signaling cascades that play a significant role in the body Bakht et al.
(2014). It showed anti-proliferation, cell cycle arrest, and induction of apoptosis of cancer
cells Zeng et al. (2013).

Quercetin and its derivatives are the major flavonoids of onion that shows chelating,
antioxidant Torel et al. (1986) and free radical scavenging activity Murota and Terao
(2003) that provides disease resistance in plants Trammell and Peterson (1976), prevents
coronary health diseases Hertog et al. (1992), inhibits cancer cell growth Chu et al. (2000),
diabetes, and antherosclerosis Cazzola et al. (2011, Suleria et al. (2015).

Phenolic compounds are secondary plant metabolites, which are important in determining the
sensory and nutritional quality of fruits, vegetables and other plants Tomas-Barberan et al.
(2000), Lapornik et al. (2005). Flavonoids are further classified as flavones, anthocyanins,
isoflavones, flavonols and flavanols Tsao and Yang (2003). Onion contains high levels of
antioxidant compounds (phenolics) with protective effects against different degenerative
pathologies Siddiq et al. (2013).

More rapid and automated methods have recently been used, e.g. supercritical fluid extraction
(SFE), pressurized liquid extraction (PLE) or microwave-assisted extraction (MAE). They are
advantageous compared to conventional methods because they can be carried out in the
absence of light and protected from oxygen, cope with the demand for a reduction in organic
solvent consumption and improve the extraction time due to the possibility of working at
elevated temperatures and/or pressures in inert atmospheres Sterbova et al. (2004).

Conventional liquid extraction methods, e.g. water bath or ultrasonication, with aqueous
methanol or acetonitrile have been widely used as simple and easy methods for analysis of
flavonoids. Extraction of flavonoid aglycones has most often been done by acid hydrolysis,
but harsh extraction conditions (e.g. acid or heating) should be avoided if intact flavonoid-
glycosides are desired Rijke et al. (2006).
 JUSTIFICATION

 The pineapple [Ananas comosus (L.) Merr.] is one of the leading commercial fruit
crops of the tropics. Pineapple is also a commercially important fruit crop of India
with around 90,000 ha area under this crop, 28.65 lakh tonnes annual production and
15.3 tonnes /ha productivity (Anonymous, 2021). It is one of the choicest fruit all over
the world because of its pleasant taste and flavour.
 It is also a source of flavonoid, bromelain (proteolytic enzyme) & phenolic
compounds. which have a medicinal benefits, included anticancer, antioxidant, anti-
inflammatory, and antiviral properties. In addition to being eaten fresh, the fruit can
also be canned and processed in to different forms.
 Nowadays, the common synthetic antioxidants occupied the most market. Long-term
animal experiments had proved that excessive use of oxidants could cause irreversible
damage to the body. Therefore, people began to pursue green and healthy natural
antioxidants, such as flavonoids, alkaloids, and unsaturated hydrocarbons.
 The UAE extraction protocol was used to increase bioactive compounds extraction
efficiency by breaking the plant cell wall integrity. Considering the situation, the
objective of this study was to comparing and maximizing the efficiency of extracting
the flavonoids and total phenolic compounds from pine apple core through
conventional and ultrasound-assisted extraction.
 Conventional extraction has certain limitations in terms of time, energy, and solvent
requirements. Ultrasound assisted extraction (UAE) can extract bioactive components
in very less time, B at low temperature, with lesser energy and solvent requirement.
OBJECTIVES

The present study will undertake with the following objectives are:

 To determine the flavonoids and total phenolic compounds from pineapple through
conventional and ultrasound assisted extraction.
 To compare the yield of flavonoids and total phenolic compounds.
 To assist the antioxidants activity of extracted bio-active compounds of pineapple core.
REVIEW OF LITERATURE

Britsch et al. (1981) Flavonols are the class of flavonoids having antioxidant activity. The
synthesis of flavonol depends on the enzyme flavonol synthase (FLS). It was first
characterised as 2- oxoglutarate dependent dioxygenase from irradiated parsely cells in in-
vitro studies.

A diet rich in fruit and vegetables is associated with a reduced risk of developing
chronic diseases such as cancer (Block et al 1992, Fresco et al 2006, Rajamanickam and
Agarwal 2008), cardiovascular disease (Bazzano et al 2003, Hu 2009), and age-related
neurodegenerative diseases (Ramassamy 2006, Shukitt-Hale et al 2008). In fruits, the
components involved in the prevention of degenerative diseases include soluble and
insoluble dietary fibre, vitamin C, vitamin E, folate, carotenoids, selenium and phenolic
compounds (Abdel-Aal and Akhtar 2006, Szajdek and Borowska 2008).

Harborne et al. (1999) Phenolic compounds are naturally occurring secondary metabolites
present in plants. Secondary metabolites are the compounds that do not participate in
essential processes but play an important role in the defence mechanisms. These are mainly
divided into two groupsphenolic acids and polyphenols. These naturally occurring
compounds are linked with one or more phenolic groups, carbohydrates (mono- and
polysaccharides) or methyl and ethyl esters.

Fiehn, (2002) Extraction of phytochemicals requires the homogenization of material (tissue)

with suitable solvent. For extraction of metabolites, several methods are used like manual
grinding in pestle mortar with liquid nitrogen, electric drills connected with pestle mortar
and ultra turrax devices. The most simple and commonly used method is to shake tissue with
suitable solvent(s) at specific (high/low) temperature.
Bahorun et al. (2004) Onion (Allium cepa L.) is good source of flavonoids, which are mainly
concentratedin edible part of onion i.e. the scales of bulb. Apart from quercetin, anthocaynins
also contribute a remarkable portion of flavonoids. Anthocyanin is responsible for skin colour
of onion. Red onions are the main source of anthocyanins. The major known anthocyanins in
red onions are cyanidins and their glycosides which are synthesised from extremely unstable
leuco-anthocyanidins by the action of anthocyanidine synthase (ANS).

Mogren (2006) Two main flavonoid subgroups are found in onion; flavonols such as
quercetin, which are responsible for the yellow and brown skins of many varieties, and
anthocyanins, which impart the red/purple colour to some varieties. The S-alk(en)yl-L-
cysteine sulphoxide (ACSOs) are the flavour precursors, which when cleaved by the enzyme
alliinase generate the characteristic odour and taste of onion.

Santas et al. (2008) studied the effect of extraction solvents (water, methanol, ethanol and
acetone) at various concentrations on yield of polyphenols and antioxidant activity of two
Spanish onion varieties: white onion and calcot de valls and reported that ethanol (75%),
methanol (100%) and methanol (75%) were the most efficient solvents for extraction of
phenolic compounds from Spanish onion varieties and regardless of the solvent used, the
calcot de valls variety contained lower concentrations of phenolic compounds than Spanish
white onions. A good correlation was found between phenol content and antioxidant capacity
and extracts with higher amounts of phenolic compounds showed higher TEAC and FRAP
values.

Owen et al. (2008) showed that among six AtFLS isoforms only AtFLS1 encodes
catalytically active enzyme for the production of flavonols. They reported that AtFLS
sequences expressed as tissue specific and cell type specific promoters. Similarly, cDNA was
isolated from Scutellaria baicalensis (SbFLS) encoding FLS enzyme, which shows structural
similarity with AtFLS. SbFLS was highly expressed in roots and flowers, among all plant
parts.

Santas and Almajano (2010) assayed the antioxidant and antimicrobial activity of ethyl
acetate and water sub fractions of methanolic extracts of three Spanish onion varieties against
some food spoiler microorganisms and reported that among the onion extract tested, only
ethyl acetate sub fraction showed anti-microbial inhibition. The study conducted in Spain,
showed that onions are a rich source of flavonoids with good antioxidant activity and
reported that onion extracts inhibited the growth of some important bacteria commonly
associated with food deterioration.

Lu et al. (2011) determined total phenolic content (TPC) and total antioxidant capacity
(TAC) of four onion varieties (red, yellow, white and sweet) using Fourier transform infrared
(FT-IR) spectroscopy (4000-400 /cm) and obtained a correlation of r > 0.95 between FT-IR
predicted and reference value (by folin-Ciocalteu 2,2- diphenyl- pierylhdrozyl (DPPH),
Trolox equivalent antioxidant capacity (TEAC) and Ferric reducing antioxidant power
(FRAP) assay. They concluded that use of mid infrared spectroscopy to predict the total
antioxidant capacity of vegetables provide a rapid and precise alternative to traditional
chemistry analysis.

Eliassen et al. (2012) Plant phenolic compounds have an important role in reduction of
reactive oxygen species that are responsible for lipid peroxidation both in plants and animals.
Consumption of good quality fruits and vegetables reduces the risk of developing chronic
diseases (cancer, cataract, cardiovascular disease, neurological disease) and ageing.

Garg et al. (2012) carried out a comparative analysis of total content of phenols, carotenes,
tannins, flavonoids and the antioxidant activities of methanol and aqueous extracts of the
leaves of herbs lemon grass, mint, coriander & curry leaves and reported that presence of
greater amount of phenolic compounds leads to a more powerful radical scavenging effect as
was shown by methanolic extract of the leaves when compared to the aqueous extracts.

Issa et al. (2013) A study conducted on total polyphenols, flavonoids content, kaempferol
concentration and antioxidant activity of two onions Syrian (spring and white) showed that
the concentration of polyphenols in spring onion is higher than white onions, with values
ranged from 1665.7±13.6 to 2288.9±12.5 mg Gallic acid equivalents/100g dry weight and
279.9±2.6 to 538.5±3.2 mg GAE/100g DW.
[Link] AND METHODS
The experimental work “Conventional and Ultrasound-assisted Extraction of Flavonoids And
Total Phenolics Compounds From pineapple core” will be carried out in the research lab of
“The Department of Processing and Food Engineering, Vaugh Institute of Agricultural
Engineering and Technology” SHUATS, PRAYAGRAJ.

3. Procurement of raw material

3.1 Pineapple
Pineapple will obtained from the local market of Prayagraj.

3.2 List of chemicals

Ethanol
Sodium acetate
Folin-Ciocalteu reagent
DPPH (2,2- diphenyl picryl hydrazyl)

3.3 List of equipment

Tray dryer
Centrifuge
pH Meter
Hot Air Oven
Water Bath
Weighing balance
Mixer Grinder
Freezer
Rotary evaporator
Spectrophotometer

3.4 Experimental Plan

Variable/Parameter Levels Description

Raw Material 1 Pineapple core

Solvents 1 Ethanol+Water (50%/50%)

Processing 2 1. Conventional extraction

(50°,60°,70° for 60,120,180 Minutes)
2. Ultrasound–assisted extraction
(400 ,500,600 for 30,40,50 Minutes)
Physio-chemical 3 1. Determination of Flavonoids
2. Determination of Total phenolic compounds
analysis
3. Determination of Antioxidant activity

Packaging 1 LDPE
Storage condition 1 Refrigerator temp (5 ± 2)

Treatment 9

Replications 3 R1, R2, R3

Samples 27
3.5 Formulation Table
3.5.1 Formulation table of conventional extraction

Sample SSR (Ratio) Temperature Time Solvent

T1 20:1 50°C 60 min
T2 20:1 50°C 120 min
T3 20:1 50°C 180 min
T4 20:1 60°C 60 min
T5 20:1 60°C 120 min Ethanol+Water
T6 20:1 60°C 180 min (50%)
T7 20:1 70°C 60 min
T8 20:1 70°C 120 min
T9 20:1 70°C 180 min

3.5.2 Formulation table of Ultrasound-assisted extraction

Sample Duty cycle(%) Temperature Time Solvent
T1
T2
T3
T4
T5
T6
T7
T8
T9

3.6.1 Preparation of pineapple core Powder

Selection of pineapple
↓
Peeling
↓
Washing
↓
Cutting
↓
Coring Solid waste (core)

Tray drying 60° for 8 hour

↓
Grinding
(To Make Powder)
↓
Packaging
(LDPE)
↓
Storage
(Refrigerator Temp. 5±2)
 Fig. 1 Flow Chart for Preparation of pineapple core Powder

3.6.2 Conventional extraction (Soxhlet) and Ultrasound- Assisted Extraction of

flavonoids and total phenolic compounds from pineapple Core
Pineapple core Powder
↓
Extraction methods

Ultrasound-Assisted Extraction Conventional Extraction (Soxhlet)

200 W

Extraction Conditions

SSR Ratio : SSR Ratio : 20 : 1

Time (minute) : 30,40,50 Time (minute) : 60,120,180
Temperature (0C) : 40,50,60 Temperature (0C) : 50,60,70
Duty Cycle (%) : 60,80,100

Aliquot Extract Aliquot Extract

Analytical method
 1. Total Phenolic content
2. Total Flavonoids content
3. Antioxidant Activity

Fig. 2 Flow Chart for Conventional Extraction (Soxhlet Extraction) & ultrasound-
Assisted Extraction

4 OBSERVATIONS TO BE RECODED
4.1 Flavonoids
The flavonoids will be calculated using the method given by Benitez et al. (2012).
4.2 Total phenolic compounds
The phenolic compounds will be calculated using the method given by Turturica et al.
(2018).
4.3 DPPH Antioxidant activity
Antioxidant activity will be calculated using the method given by Abdou et al. (2011).
%RSA= 1- Sample absorbance × 100
Blank absorbance

4.4 Flavonoids extraction yield

Flavonoids extraction yield will be calculated using the formula given by Kharasani et al.
(2015).
Y = 100 C x V
m

4.5 Total Phenolic Compound extraction yield

Total Phenolic Compound extraction yield will be calculated using the formula given by
Folin Ciocalteu’s method (2015).
YTPC % = CTPC x V
m
Statistical Analysis
The experiment will be conducted by adopting completely randomized design the data
recorded during the course of investigation were statistically analyzed by the analysis of
variance – Two way classification or single factor ANOVA .
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