ABSTRACT: every year(Li et al., 2006). citrus peels


Bioactive compounds presents in different
plants are most important due to their nutritional potentials which are
responsible for different properties like color, taste and flavors. Citrus
fruits are generally used for manufacturing citrus based drinks and juices in
the food industries. Deep eutectic solvents are alternative of ionic liquids
having more advantages like cost effective, easy preparation and environment
friendly. Solvents will be synthesized by the reaction of choline chloride
which act as a hydrogen bond acceptor and ethylene glycol having hydrogen bond
donor property. FT-IR spectroscopic method will be used for the
characterization of synthesizes solvents. For the extraction of bioactive
compounds from citrus fruits ultrasound and microwave extraction methods will
be used. High performance liquid chromatography (HPLC) will be used for
characterization and quantification of extracted bioactive compounds. For
higher extraction efficiency parameters of extraction will be optimized like
time, temperature, solid to solvent ratio. Total flavonoids and phenolic
contents and their antioxidant activities will be accessed by recognized
biological procedures. Response surface methodology will be used for optimized
study of process variables.

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Citrus fruits belong to the genus Citrus
whichcontains oranges, lemons, limes and grapefruits. Citrus fruits
make up the largest sector of the world’s fruit production, and their average
production are 100 million tons. About 34% of citrus fruits are made into
juices; therefore, largeamounts of residues are formed every year(Li
et al., 2006). citrus peels
contain more bioactive compounds, such as phenolic acids, flavonoids,
limonoids, and fibre than do juices(Bocco et al., 1998).

is a highly poly-embryonic, even surfaced and tight skinned fruit, which has
beenpart of human diet for ages due to its high nutritional and medicinal
values. Consumption of orange fruit generates a large amount of peels as waste,
which could be a potential cause of environmental pollution, if not handled
properly.Orange peel mostly consists of cellulosic fibres and pectin. Further,
it has been reported that orange peel extract comprises several bioactive
compounds, such as flavonoids and limonoids, which are known to act as
anti-cancer and anti-oxidant agents (Gacche et al., 2011). These are also
known to enhance vitamin absorption and possess a broad spectrum of medicinal
properties, including anti-inflammatory, anti-allergenic, anti-tumor and anti-microbial
activities(Di Mauro et al., 1999).

 Solvent extraction remains one of the
traditional basic ways of isolating bioactive compounds from plants. The
process is based on mixing the plant samples with a suitable solvent using any
of the extraction methods. There are several extraction techniques that have
been reported in the literature such as ultrasound-assisted extraction (UAE)(Xu
et al., 2013), pressurized
liquid extraction (PLE)(Stalikas, 2007), microwave-assisted extraction
(MAE)(Xiao et al., 2008), heat reflux
extraction (HRE)(Chen et al., 2007) and
headspace-solvent micro-extraction (HS-SME)(Theis et al., 2001).Traditional
solvents commonly used are mostly volatile organic compounds (VOCs). However,
there are environmental concerns due to their toxicity as well as low yield
resulting from the use of VOCs as an extraction media. Hence, such class of
solvents limits their application in areaswhere purity of products is vital
such as in pharmaceutical and food industry. This has necessitated the search
of sustainable and benign solvents as extractants. To this, deep eutectic
solvents have emerged as an alternative due to the aforementioned desirable
properties that qualify them as green solvent.DESs have a superior dissolution
and extraction ability(Zhang et al., 2012).

alternative of ionic solvents are deep eutectic solvents that were first
introduced by Abbott and his co-workers(Abbott et al., 2004), abbreviated as
DESs.. These solvents have many
advantages than others which are as biodegradability, low toxicity,
sustainability, low costs and preparation by simple way.DESs are synthesized
by mixing hydrogen bond acceptor (a salt) and a Hydrogen Bond Donor (HBD)
molecule resulting in a mixture. This mixture has melting point lower than the
melting point of respective individual component/compound.Mostly prepared deep
eutectic solvents are choline chloride (Ch-Cl) based due to their ease of
biodegradation, relatively high availability and very low toxicity (Singh et al., 2012; Radoševi? et al., 2015). DESs based on Ch-Cl had played
major role in many technologies.Most commonly DESs are prepared by mixing two
or three safe and cheap compounds. The melting point is usually lower than the
individual compound due to the formation of inter-molecular hydrogen bonding.

major task of current research is the extraction of bioactive compounds from
the orange peels using choline chloride based deep eutectic solvents due to
their low cost and no toxicity effects. FT-IR spectroscopic method will be used for the characterization of
synthesizes solvents.High performance liquid chromatography (HPLC) will be used
for characterization and quantification of extracted bioactive compounds.


eutectic solvents:

Another type of
solvent with similar physical properties and phase behavior to ILs are deep
eutectic solvents (DES). These solvents are mixtures of compounds that have a
much lower melting point than that of any of its individual components, mainly
due to the generation of intermolecular hydrogen bonds.
The principle of creating ILs and DES was demonstrated for mixtures of
quaternary ammonium salts (Abbott et al., 2003)with a range of amides and
carboxylic acids and later extended to choline chloride with alcohols (Gorke et al., 2008) and urea with sugars or organic
acids (Gore et al., 2011).

features of these DES make that they have an advantage over ILs because they
are easier to prepare with high purity at low cost. Higher melting points of
many DES, however, can hamper their application as a green solvent at room
temperature. Compared to the broad applications of ILs (Tang et al., 2012), the application of DES has
been so far limited to organic reactions (Ilgen and König, 2009), organic extractions (Abbott et al., 2009), electrochemistry (Nkuku and LeSuer, 2007) and enzyme reactions carried
out at 60 ?C. Moreover, the synthetic ILs
suffer from high toxicity of some of the ingredients (Docherty and Kulpa Jr, 2005) which is hampering their use
in pharmaceutical and food related product.

DESs can be synthesized easily by mixing a salt and
HBD at room temperature or elevated temperature (Abbott et al., 2004). Two methods were used for preparing deep
eutectic solvents (DES): a vacuum evaporating and a heating method.The
two-component mixture with calculated amounts of water was placed in a bottle
with a stirring bar and cap and heated in a water bath below 50 ?C
with agitation till a clear liquid was formed (about 30–90 min). The viscous
liquids were tested on a 1H NMR spectrometer at40°C(Dai et al., 2013).


Bioactive compounds are extra-nutritional constituents that are found in
small quantities in foods providing health benefits beyond the basic
nutritional value of the product. These compounds vary widely in chemical
structure and function and are grouped accordingly. Some examples of bioactive compounds are carotenoids, flavonoids, carnitine, choline,
coenzyme Q, dithiolthiones, phytosterols, phytoestrogens, glucosinolates,
polyphenols, and taurine. Since vitamins and minerals elicit pharmacological
effects, they can be categorized as bioactive compounds as
well. Most of the bioactive compounds have
antioxidant, anticarcinogenic, antiinflammatory, and antimicrobial properties(Hamzal?o?lu
and Gökmen, 2015). 

Bioactive compounds in orange

peels contain more bioactive compounds such as phenolic acids, flavonoids,
limonoids, and fibre(Gorinstein et al., 2001). Among the
well-known citrus bioactive compounds, flavonoids, especially the citrus unique
polymethoxy flavones and flavanone glycosides, attract considerable attention
for their significant biological activities (Tripoli et al., 2007). The most
extensively studied actions proved are their anti-inflammatory and anti-cancer
activities.Hesperidin is a predominant flavourless flavonoid present in the
peel and membranous parts of orange, which shows such bioactivity(Bilbao et al., 2007). Rutin is
another major flavonoid that can chelate heavy metals like iron and also
enhances vitamin absorption (Tundis et al., 2014). Limonoids are
organic compounds commonly found in the peel of citrus fruits, exhibit several
beneficial health effects (Bicu and Mustata, 2013). Therefore, the isolation of a
fibrous assembly from orange peel extract, preserving such bioactive compounds,
will have great potential for various healthcare applications.


Medicinal importance:

the traditional Chinese medicine, chen-pi, the dried mature fruit peels of
Citrus reticulata and Citrus sinensis and theirvarieties, have been widely used
for centuries as remedies to treat indigestion and to improve inflammatory
syndromes of the respiratory tract, such as bronchitis and asthma(Ou, 1999). sweet orange (Citrus sinensis)
peel is concerned, it is found to have a good total radical antioxidative
potential and can be used as antioxidants in food and medicinal preparations(Anagnostopoulou et al., 2006).    

of bioactive compounds:

It is necessary
to build up a standard and integrated approach to screen out these compounds
carrying human health benefits. It is only possible
to conduct further separation, identification, and characterization of
bioactive compounds followed by an appropriate extraction process. Different
extraction techniques should be used in diverse conditions for understanding
the extraction selectivity fromvarious natural sources. Different techniques many
of them remain almost same through hundreds of years; can also be used to
extract bioactive compounds. All these techniques have some common objectives,
(a) to extract targeted bioactive compounds from complex plant sample, (b) to
increase selectivity of analytical methods (c) to increase sensitivity of
bioassay by increasing the concentrationof targeted compounds, (d) to convert
the bioactive compounds into a more suitable form for detection and separation and
(e) to provide a strong and reproducible method that is independent of
variations in the sample matrix (Smith, 2003). 

assisted extraction (MAE):

microwave-assisted extraction is also considered as a novel method for
extracting soluble products into a fluid from a wide range of materials using
microwave energy (Paré et al.,
1994). The extraction mechanism of
microwaveassisted extraction is supposed to involve three sequential steps described by(Alupului et al.,
2012). First, separation of solutes
from active sites of sample matrix under increased temperature and pressure; second,
diffusion of solvent across sample matrix; third, release of solutes from
sample matrix to solvent. Several advantagesof MAE have been described by (Cravotto et al.,
2008)such as quicker heating for
the extraction of bioactive substances from plant materials; reduced thermal
gradients; reduced equipment size and increased extract yield. MAE can extract
bioactive compounds more rapidly and a better recovery is possible than
conventional extraction processes.


extraction (UAE):

UAE is seemed to be an effective extraction technique for
bioactivecompound extraction from herbal plants(Rostagno et al., 2003). UAE have also been incorporated along with various
classical techniques as they are reported to enhance the efficiency of a
conventional system. In a solvent extraction unit, an ultrasound device is
placed in an appropriate position to enhance the extraction efficiency (Vinatoru et al.,
1998). The advantages of UAE include reduction in extraction time,
energy and use of solvent. Ultrasound energy for extraction also facilitates
more effective mixing, faster energy transfer, reduced thermal gradients and
extraction temperature, selective extraction, reduced equipment size, faster
response to process extraction control, quick start-up, increased production
and eliminates process steps (Chemat et al.,


Maceration is a
popular and inexpensive method of extraction which used to get essential oils
and for the extraction of bioactive compounds from different plants. For
extraction on small scale this extraction methods used which generally involves
several steps. First step is grinding in which plant materials convert into
small particle in order to enhance the surface area and hence proper mixing of
material with solvent take place. Secondly,suitable solvent selected and added
in closed container. In the third step, liquid is
strained off but the marc which is the solid residue of this extraction process
is pressed to recover large amount of occluded solutions. The obtained strained
and the press out liquid are mixed and separated from impurities by filtration.
Occasional shaking in maceration facilitate extraction by two ways; (a)
increase diffusion, (b) remove concentrated solution from the sample surface
for bringing new solvent to the menstruumfor more extraction yield (Azmir et al., 2013).


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