J. Appl. Environ. Biol. Sci., 4(11S)213-217, 2015
© 2015, TextRoad Publication
Journal of Applied Environmental
and Biological Sciences
Corresponding Author: Vahid Hakimzadeh, Department of Food Science & Technology, Quchan branch, Islamic Azad
University, Quchan, Iran. Email: email@example.com
Carotenoids Extraction Optimization of Lutein-Based Banana Peel
, Vahid Hakimzadeh
, Mohammad Reza Abedi
Department of Food Science & Technology, Quchan branch, Islamic Azad University, Quchan, Iran
Department of Applied Chemistry, Quchan branch, Islamic Azad University, Quchan, Iran
Received: October 29, 2014
Accepted: December 31, 2014
Carotenoids are among pigments with antioxidant properties and healthy effects on human body. Lutein is one of the
most important carotenoids that is abundantly found in banana peel and belongs to xanthophyll’s family. The present
study aims to banana extract in order to use the applied properties of its carotenoids specially lutein. The banana
peels were dried in vacuum oven and changed to powder, three solvents of ethanol 96%, methanol 99.5% and ethyl
acetate 80% were used in 27 and 35 centigrade to extract its juice and study the amount of lutein. HPLC, UV-VIS
spectrophotometer and FRAP test were respectively used to determine the amount of lutein and the antioxidant
power of the extracts. The findings showed that considering the inhibition time of pure lutein with HPLC that took
about 2.311 minutes, the amount of lutein gained by ethyl acetate solvent was more as compared to other extracts
and its peak area was about 400.Similarly, through UV-VIS colorimetry and considering the amount of light
absorbance of pure lutein, the amount of light absorbance in ethyl acetate solvent in 27°C was more as compared to
other samples and its amount of absorbance was about 2.204.The antioxidant power of ethyl acetate in 27°C in
FRAP method showed the higher amount.
KEYWORDS: Antioxidant, Ethyl acetate, Colorimetry, Carotenoids, Lutein
Nowadays, the waste materials of food industries are of significant importance. The extractions of major and
useful compounds such as antioxidants, enzymes, antimicrobial compounds and the like from these materials
looking to be useless add to their values. One of these waste materials is banana peels with significant carotenoids
compounds including lutein.
Carotenoids including beta-carotene, alpha carotene and lycopene play a part in light absorption by the plants.
Beta- carotene and alpha- carotene are responsible for the orange color of carrot, lycopene is responsible for the red
color of tomato and Astaxanthin is responsible for the pink or red color in crabs and salmon fish .
Thanks to their wide distribution in animal and plant resources, and their antioxidant and bioactive properties,
Carotenoid shaves so many applications in coloring food materials. Among the carotenoids that have more usage as
strainers are beta- carotene, lutein, zeaxanthin and lycopene .
For the customer, the desired color in different edible products is a sign of suitable production process and
favorable quality of the product. That is why using synthesized or natural pigments in food industry has become
widespread. However, nowadays, the concern about using synthesized colors and the customers’ tendency to use
natural colored materials, has directed the attention toward using natural pigments .
Carotenoids as antioxidants have an effective role in preventing oxidation reactions.
In a large number of fruit wastes, the antioxidant properties are available because of the presence of
carotenoids. For instance, the remaining of star fruit, pressed grapes, citric fruit peels, pomegranate wastes and
banana peels are ranked as cheap sources of antioxidants [1, 2, 3, 4, 5].
The skin and pericarp layer of ripe tomato almost contain 80% of the total lycopene existing in ripe tomato .
The fruit peels are rich source of colored and antioxidant compounds .By extracting different isomers of
carotene and tocopherol from the peels and suitable processing, the risk of contamination caused by wastes removal
can be reduced and the productivity of production in agriculture and food sector can increase .
2. MATERIALS AND METHODS
Here we are studying some carotenoid compounds in lutein-based banana peels. In extracting carotenoid
extract from banana peels, the solvents ethanol 96%, methanol 99.5% and ethyl acetate 80% have been used, all the
three was made by Merck Company in Germany.
Sheikhzadeh et al.,2015
2.1. Banana peels preparation
After providing banana with Dwarf Cavendish variety (preferably a little more yellow) and complete wash,
they were peeled and were put in vacuumed VO400 oven made in Germany in 40°C temperature for 14 hours to dry.
The dried banana peel was then powdered by mill (made in Germany) and was placed in a dry place.
2.2. Extraction with solvent
At first, 15 gr Banana peel was mixed with50mL of each solvent and the mixture was poured in Erlenmeyer
flask and was covered by an aluminum sheet. Then it was placed into shaker incubator (Fanavarn Sahand model
made in Iran) in 27 and 35°C for 150 rounds per minute for 12 hours and was then filtered by Whatman paper
number 2.It was finally put in rotary machine (LABOROTA 4003 – control) in 40°C with 70 rounds in suitable
vacuum so that the remained solvent is omitted and was finally filtered by Buchner funnel (filtration set made by
commonwealth market) and was moved to 100mLvolumetric flask. The flasks were covered by aluminum sheet in
5°C inside the fridge in order to keep away from sun.
2.3. High Performance Liquid Chromatography (HPLC) preparation
At first, a mixture of methanol (80%), distilled water (5%) and methyl-tert-butyl-alcohol (15%) was added in
solvent container (mobile phase) of HPLC (Agilent Technologies 1200 series) until the machine reaches its base
state .Then the extracts were injected and twenty minutes were given for each one until the peaks appear. This is
the time that after 4.1 minutes, the first and highest peak of extracts appears and the peak area was read.
2.4. UV-VIS Spectrophotometric Determination
At first, UV-VIS Spectrophotometer (Agilent Technologies Cary 8454 made in USA) in 245 nm wavelength
was calibrated by pure methanol. Then the pure lutein absorption with it was read .This method was used to measure
lutein-based colorimetry of the extracts .
2.5. Measuring antioxidant power of the extracts by Ferric reducing Antioxidant power (FRAP) method
FRAP solution was primarily prepared as follows: Buffer acetate, TPTZ reagent and 20 mL solution of iron
(III) chloride hexahydrate were mixed in 1:1:10 volume ratio and were kept in a dark place. Then spectrophotometer
was calibrated by TPTZ solution and the amount of absorption accompanied with iron power of inhibition against
control in 595 nm was read .
In the presence of antioxidants, the intensity of color increases. By measuring the color intensity, antioxidant
capacity can be gained.
The results of FRAP test is strictly related to the time of analysis .Therefore FRAP method will be appropriate
for comparative studies such as the impact of one treatment on antioxidant capacity.
3. FINDINGS AND DISCUSSION
3.1. Quality Identification of lutein
In HPLC, the peak of different extracts with 5µL in 446nm wavelength was compared with pure standard lutein
peak .The summary of which is shown in table 1.
Fig.1 Standard pure Lutein with HPLC
J. Appl. Environ. Biol. Sci., 4(11S)213-217, 2015
According to fig.1, the inhibition time of pure lutein with HPLC was about 2.311 minutes and lutein level below the
peak was read as 15614. (The time was constant as 4.1in all extracts)
Table1. The level below the peak of extracts with different solvents compared with the level below
peak in pure lutein
Type of solvent Time (min) Temperature (
C) Peak Area
Ethyl acetate 4.1 27 400
Ethyl acetate 4.1 35 394
Methanol 4.1 27 369
Methanol 4.1 35 342
Ethanol 4.1 27 201
Ethanol 4.1 35 152
Fig. 2 The graph of sample lutein of powdered banana peel with ethyl acetate 80% in 27°C
According to table 1 and fig.2 the amount of lutein in the extract gained from ethyl acetate was higher as
compared to other extracts and its level below peak was about 400.The temperature of extraction had no significant
effect in gaining effective carotenoids .In this way, ethyl acetate 80%with a lower density and temperature was more
appropriate as compared to other solvents.
According to our findings, Vernalis W and Jacobaea studied the amount of lutein on two species of Senecio by
HPLC in 2009 with a similar laboratorial method .Their findings also showed that the best solvent was ethyl acetate
and the peak area was read as 444 .
Mahagamasekara in 2010 also studied the amount of lutein on 13 species of vegetables with green color in Sri
Lanka by HPLC .The most compound was related to lutein and by baking some percent of lutein in the laboratorial
level, the level of lutein decreased .The findings also indicated that lutein is more hydrophilic [16,17].
2.3. Lutein colorimetry with UV-VIS spectrophotometer
After pure lutein is poured into cell, the amount of pure lutein absorbance in 245nm was read as 1.347.
Then Absorbance of samples was read in 245nm. Thus it was revealed that the amount of light absorbance in
ethyl acetate solvent extract in 27°C was higher as compared to other extracts and the amount of its absorption was
Sheikhzadeh et al.,2015
Fig.3: Lutein absorbance in UV-VIS Spectrophotometer
According to these findings, Andrea investigated the different methods of lutein preparation of supercritical
fluid. Primarily the lutein absorbance was done using UV-VIS spectrophotometer and as the time passed,
absorbance increased .Finally, the esters released from free lutein created a reversible system with free fatty acids in
CALB system .
Similarly, Vernalis Waldst ., Kit and Jacobaea studied the amount of lutein on two species of Senecio by UV-
VIS spectrophotometer with similar laboratorial method, their findings also indicated that the amount of absorbance
read for lutein was higher as compared to other compounds and the amount of extract absorbance was read in 450
3.3. Determining the antioxidant power of the extracts
In determining the antioxidant power of the extracts, the light absorbance of the sample has a direct
relationship with the power of reduction. This means that lighter absorbance means more reducing property. This
test was used to determine the antioxidant power of the extracts. The antioxidant power of ethyl acetate extract in
27°C in FRAP method also showed the highest degree.
Ethyl acetate and ethanol had the highest and lowest antioxidant power in FRAP method. The extraction
conditions which determine the maximum antioxidant power has indicated that the number of stages of extraction,
temperature and time are among the effective factors in connection with antioxidant power of banana peel .
The powerful antioxidant property of ethyl acetate can be related to lutein existing in it .As lutein has
antioxidant and reducing property and play a part in eye health as well as preventing cardio-vascular diseases,
cerebral infraction and lung cancer.
Studies showed that antioxidant power of ethyl acetate was more than ethanol. Methanol 99.5% and ethanol
96% have the highest efficiency among common solvents for extracting herbal materials for polar and non-polar
compounds .Therefore, any measuring with methanol extract can cause extraction of polar and non-polar
compounds resulting to higher antioxidant power. The ferric reducing antioxidant power is a test which directly
measures the antioxidants or reducers in the sample and has a direct relationship with their antioxidant density .
In these methods the extracts that have a high ferric reducing antioxidant activity can easily neutralize free radicals
existing in the body.
In the test for measuring the reducing power of antioxidant activities made based on light absorbance, an
increase in light absorbance of the reaction indicated an increase in antioxidant activity as light absorbance was used
to intensify the reducing property.
Generally, the reducing power of ethyl acetate was gained as higher than methanol and methanol higher than
J. Appl. Environ. Biol. Sci., 4(11S)213-217, 2015
Extraction of banana peel was done by three solvents (ethanol, ethyl acetate and methanol) and HPLC method
was used to determine the amount of lutein and the best solvent was ethyl acetate. Peak area was about 400.UV-VIS
spectrophotometer was used for lutein colorimetry. The best amount of light absorbance was for ethyl acetate that
was read as 2.204. FRAP test was done to measure antioxidant power of the extracts .Ethyl acetate extract showed
the most antioxidant power and the property of reduction.
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