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Chapter II
REVIEW OF RELATED LITERATURE AND STUDIES
2.1 Natural colorants as a substitute for synthetic dyes
Synthetic dyes have been used for various applications for a long time.
These dyes are commonly added to food, cosmetics, textiles, and
pharmaceutical drugs. They provide bright colors to these materials,
enhancing their appearance and quality. Consumers, from individual
households to large industries, have widely utilized synthetic dyes because of
their low production costs, chemical stability, and high tinctorial strength
(Sigurdson et al., 2017). During the 20th century, several dyes, including
Yellow NO 6, Blue FCF, Blue NO 1, Blue NO 2, Red NO 2, and Red NO 4,
which have been used for different industrial applications, especially in food
and pharmaceutical manufacturing processes, were reportedly discontinued
due to their harmful effects on human health (Sharma et al., 2010; Nawaz et
al., 2020). The long-term use of these dyes was found to have caused
cancers in the skin, bladder, and liver due to their high azo-dye/heavy metal
compositions. Furthermore, the precursors used in the production of these
dyes, along with the wastes generated, have caused harmful environmental
effects, rendering them non-environmentally friendly and non-biodegradable
(Kumar et al., 2017; Nawaz et al., 2020).
Many studies have shown the toxicity of various azo-synthetic dyes,
particularly Red No. 2 (commonly known as amaranth), which caused cancer
in laboratory animals, especially mice, after long-term exposure. (Sharma et
al., 2010; Tsuda et al., 2001). The chemical structure of azo dyes, specifically
Amaranth as shown in Figure 1, and the mechanisms related to their
breakdown in the human body indicate potential risks to human health. Figure
1 shows the chemical structure of Amaranth, also called as Red No. 2.
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Figure 1. Chemical Structure of Amaranth (Red No. 2) (Sarıkaya et al., 2012).
The azo bond (-N=N-) in the chemical structure of amaranth (Figure 1)
links two aromatic rings. This bond is systematically absorbed in the body and
can be metabolized by azoreductase of intestinal microflora by liver cells and
skin surface bacteria. This metabolism produces aromatic amines, which can
pose significant health risks to humans (Nikfar & Jaberidoost, 2014). Thus,
natural colorants are more favored than synthetic dyes due to their lower
toxicity and benefits in health and environmental aspects. Natural colorants
are obtained from natural sources, ensuring that they have minimal impact on
the environment, making them more biodegradable than synthetic ones.
Significant efforts have been made to replace synthetic dyes with natural
alternatives. Some natural colorants, such as carmine, usually incorporated
into lipsticks, do not cause health problems, especially when ingested (Kumar
& Choudhury, 2018). Although they are more eco-friendly and safer, natural
colorants require higher costs due to the significant amounts of raw materials
needed for their production. The stability and quality of natural colorants may
not be consistent with those of synthetic dyes. Additionally, the availability of
raw materials for the production of natural colorants is limited due to the
seasonal growth of their plant sources (Kumar & Choudhury, 2018).
Nevertheless, the potential of natural colorants as substitutes for synthetic
dyes could significantly contribute to achieving a sustainable environment and
promoting health and wellness.
Natural colorants are organic compounds that are derived from
different naturally occurring sources, such as minerals (e.g., ochre, ferrous
sulfate, and clay); animals (e.g., some species of shellfish or mollusks);
insects (e.g., lac scale insects and cochineal beetles); and plants (e.g., saffron
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and indigo), without undergoing chemical modification or treatment (Nambela
et al., 2019). The natural coloring properties produced from these sources can
be classified into four main classifications, namely anthocyanins, carotenoids,
chlorophyll, and betalains. Anthocyanins display color hues of blue, red, or
purple. Carotenoids produce colors that range from red to yellow. Chlorophylls
give plants their green color, although they can also create a blue tone. Lastly,
betalains provide colors of yellow-orange, red-violet, and primarily pink (Vega
et al., 2023). These compounds not only possess colorant properties but also
exhibit beneficial bioactive effects for the human body when consumed
(Sigurdson et al., 2017).
Numerous studies have explored the use of natural colorants from
various sources in food, cosmetics, textile, and pharmaceutical products. For
example, betalains have been utilized to color dietary supplements.
Carotenoids are usually applied in oil, butter, juices, cheese, baked goods,
confectioneries, and yogurt. Chlorophyll is added to bed sheets, chewing
gums, and toothpaste (Nabi et al., 2023). For decades, anthocyanins have
been used as a natural colorant in food and beverages. They are incorporated
into a variety of products, including confectioneries, fruit preserves, drinks,
and powdered foods. Anthocyanins are water-soluble flavonoids primarily
found in different parts of plants, such as fruits, leaves, roots, and flowers
(Heinonen et al., 2016). A previous study evaluated the potential benefits of a
skincare product, particularly a cream base made from bilberry anthocyanin
extracts. The results indicated that the cream formulation containing these
extracts demonstrated good antioxidant properties. However, the instability of
anthocyanins was evident as the color of the formulation changed from bright
pink to faded pink (Ștefănescu & Marian, 2023). Nonetheless, applying these
natural colorants to various products is becoming increasingly important, as
they provide not only aesthetic value and quality but also yield potential health
benefits.
2.2 Extraction and Encapsulation of anthocyanins
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Anthocyanins must first be extracted from plant sources prior to
encapsulation. The anthocyanins are water-soluble pigments that can be
extracted using acidified solvents, commonly in polar organic-water mixtures,
under mild conditions. A variety of organic solvents, including methanol,
acetone, ethanol, and acetonitrile, have been employed for anthocyanin
extraction, with methanol being the most commonly utilized (Puértolas et al.,
2013). These solvents work by breaking down the cell membranes of the plant
material, facilitating the efficient dissolution of anthocyanin pigments while
preserving their stability (Navas et al., 2012). Bridgers et al. (2010)
demonstrated that acidified methanol was the most effective solvent for
anthocyanin recovery. Similarly, Taghavi et al. (2023) found that methanol and
methanol-water mixtures outperformed other solvents in their study, achieving
the highest anthocyanin extraction efficiency.
While methanol and other organic solvents yield excellent results for
extraction, their prolonged use can lead to significant environmental concerns
and pose health hazards. In response, some researchers prefer water as an
alternative solvent due to its accessibility, safety, environmental friendliness,
and cost-effectiveness compared to organic solvents (Vuong et al., 2011).
Ekaputra and Pramitasari (2020) employed a water-based solvent system
acidified with citric acid for extracting anthocyanins from purple sweet potato.
The authors had successfully recovered significant amounts of anthocyanins
from the sweet potato.
The next step after extraction is encapsulation, which involves coating
the extracted compounds with an encapsulating agent. This method protects
the compounds from chemical and environmental degradation, ensuring the
stability of the bioactive components present (Peixoto et al., 2022). A suitable
encapsulating agent should meet several key criteria: it must possess high
stability, excellent water solubility, emulsifying properties, and effective drying
capabilities. Additionally, it should form a fine and dense network during the
drying process (Chranioti & Tzia, 2013). Maltodextrin is a common
encapsulating agent with high emulsifying properties. It is derived from
hydrolyzed starch from corn, rice, potato, or wheat and has a dextrose
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equivalence (DE) of less than 20 (Harshitha et al., 2023). In a review article by
Antares et al. (2024) about the use of maltodextrin in Indonesia, the authors
emphasized its encapsulation properties. The study highlighted that
maltodextrin is effective in enhancing the stability, solubility, and bioavailability
of active ingredients, nutrients, and flavors. The incorporation of maltodextrin
into pasteurized orange juice was found to significantly preserve its bioactive
compounds, including total phenols, total carotenoids (TC), ascorbic acid
(AA), and vitamin C, while also maintaining its antioxidant capacity (AC) (Arilla
et al., 2021). Ferrari et al. (2013) evaluated the storage stability of spray-dried
blackberry powder and found that incorporating maltodextrin significantly
enhanced stability. The addition of maltodextrin resulted in particles with an
extended half-life and a reduced anthocyanin degradation rate, making the
powder a suitable functional ingredient for various food applications.
The spray-drying technique is widely employed for drying heat-
sensitive food ingredients because it facilitates rapid solvent evaporation from
droplets, preserving the quality and stability of the ingredients (Muhamad et
al., 2017). The spray-drying process consists of three main stages: first, the
liquid feed is atomized into fine droplets within a spray chamber. Next, these
droplets are mixed with a heated gas stream, which rapidly evaporates the
solvent. Finally, the dried product is separated from the air stream and
collected (Liu et al., 2015). The spray-drying technique is favored over other
drying methods for several reasons, including its ability to produce high-
quality products, its low operational costs, compact equipment size, rapid
drying process, high solubility of the resulting capsules, and the capacity for
continuous operation (Mahdavi et al., 2014). However, this technique has
certain limitations that must be considered. These include its unsuitability for
temperature-sensitive ingredients, challenges in controlling particle size, and
the production of non-uniform microcapsules (Mahdavi et al., 2014).
2.3 Yautia as a natural food colorant
Xanthosoma sagittifolium (L.) Schott (X. sagittifolium), widely known as
cocoyam and locally referred to as “yautia,” “istaring,” “takudo,” “karlang,”
“butig,” and “lutya” across various regions of the Philippines, is a member of
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the Araceae family (Pardales, 1997). It is widely grown in tropical and
subtropical regions and is valued for its edible root and tuber crops. (Rojas-
Sandoval & Acevedo-Rodríguez, 2022). Red cocoyam is widely regarded as a
more affordable source of carbohydrates compared to other tuber crops. It
has the capacity to fulfill the body’s essential nutrient needs, as it is abundant
in minerals, proteins, carbohydrates, and vitamins (Mba & Agu, 2021).
Nishanthini and Mohan (2012) analyzed the total phenolic and flavonoid
content, as well as the antioxidant activity, of methanol extracts from the corm
of X. sagittifolium. They found that the extract of corm contained 0.32 g/100
g⁻¹ of gallic acid equivalents and 0.26 g/100 g⁻¹ of flavonoids. The extract
also exhibited strong antioxidant activity, with 83.27 ± 0.08% superoxide
radical scavenging, 78.22 ± 0.56% DPPH inhibition, 76.11 ± 0.07% ABTS
radical cation scavenging, and 69.11 ± 0.21% hydroxyl radical scavenging.
Amah et al. (2018) investigated the proximate composition of yautia (X.
sagittifolium) and reported the following values: moisture content (65.57%),
ash content (3.48 g/100g), crude fiber (1.93 g/100g), fat (0.40 g/100g), crude
protein (4.72 g/100g), and carbohydrates (20.92 g/100g). These findings
suggest that red cocoyam could serve as an affordable source of nutrients to
promote good health. However, its potential as a natural colorant remains
unexplored.
2.4 Purple Yam as a natural food colorant
Dioscorea alata, also known as the ‘purple yam’, is a species of yam
that is one of the most extensively cultivated in Asia. It is mostly grown in
tropical and subtropical regions and is characterized by its purple vibrant color
and viscosity (Moriya et al., 2015). Based on the statistics reported by BAS
(Bureau of Agricultural Statistics) (2006), as cited in Cornago et al. (2011),
among the approximately 600 species of yam, D. alata is one of the most
widely cultivated species in the Philippines, with an annual production of
26,464 metric tons recorded from 2000-2005. Previous studies have identified
purple yam as a source of natural anthocyanins, which are beneficial as
antioxidants and natural food colorants. Beyond its antioxidant capacity,
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purple yam also offers health benefits attributed to its rich nutritional profile. It
contains carbohydrates (17.10–29.37%), protein (1.29–3.00%), fat (0.29%),
fiber (6.70–11.62%), and ash (0.85–1.44%), with a high moisture content
ranging from 65.47% to 82.46%. Additionally, it is a good source of minerals,
including potassium (224.54–483.21 mg/100 g), calcium (15.63–61.97 mg/100
g), magnesium (16.75–43.06 mg/100 g), iron (1.40–13.40 mg/100 g), zinc
(0.43–2.83 mg/100 g), and phosphorus (329.37–699.91 mg/100 g). These
health-contributing attributes make purple yam a valuable functional food with
diverse applications in health and nutrition (Tamaroh & Sudrajat, 2021).
Moriya et al. (2015) isolated new pigments found in purple yams from
the Philippines and evaluated their antioxidant activities. The new acylated
anthocyanin compounds found from the methanol extracts of purple yam were
cyanidin (alatanin D, alatanin E, and alatanin G) and peonidin (alatanin F)
compounds. Alatanin D, alatanin E, and alatanin G were elucidated to be 3-O-
(6-O-(6-O-(E)-sinapoyl-β-D-glucopyranosyl)-β-D-glucopyranosyl)-7-O-(6-O-
(E)-sinapoyl-β-D-glucopyranosyl) cyanidin, 3-O-(6-O-(3-O-(β-D-glucopyran-
osyl)-6-O-(E)-sinapoyl-D-glucopyra-nosyl)-β-D-glucopyranosyl)-cyanidin, and
3-O-(6-O-(6-O-(E)-feruloyl-β-D-glucopyranosyl)-β-D-glucopyranosyl)cyanidin,
respectively. Alatanin F was elucidated to be 3-O-(6-O-(6-O-(E)-sinapoyl-β-D-
glucopyranosyl)-β-D-glycopyranosyl)peonidin. The peonidin-type anthocyanin
(alatanin F) was found to exhibit less antioxidant activity than the cyanidin-
type anthocyanins (alatanin D, alatanin E, and alatanin G) (Moriya et al.,
2015). Thus, purple yam can be extracted as a natural colorant due to its
remarkable anthocyanin properties and antioxidant activity.
2.5 Antioxidant properties of anthocyanins
Antioxidants are known to reduce oxidative stress caused by free
radicals, protecting cells from damage. Free radicals are molecules that
contain an unpaired electron in the atomic orbital. They are highly reactive
and unstable. Free electrons can either donate to or accept from other
molecules, functioning as oxidizing or reducing agents (Cheeseman & Slater,
1993). Free radicals and oxidants can have both harmful and beneficial
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effects. They typically originate from normal cell metabolism or external
sources like pollution, radiation, medications, cigarettes, and various other
sources. The accumulation of free radicals inside a body generates a
phenomenon known as oxidative stress, which involves the development of
chronic diseases such as cancer, neurodegenerative and cardiovascular
diseases, and autoimmune disorders (Pham-Huy et al., 2008).
Antioxidants are compounds that neutralize the damaging effects of
free radicals. The free radical scavenging property of the antioxidants is linked
to their ability to delay or inhibit cellular damage. Some antioxidants are
naturally produced by the body during normal metabolism. However,
micronutrient antioxidants like vitamin C, vitamin E, and β-carotene cannot be
synthesized internally and must be obtained through dietary sources (Lobo et
al., 2010). When oxidation occurs, oxygen will be converted to several
reactive species, such as superoxide anion (O2•−), hydroxyl radical (•OH),
lipid peroxyl radical (LOO•), alkoxyl radical (LO•), ferryl and perferryl species
[Oxy-Fe(IV)•+], and many more (Jiang & Xiong, 2016).
In addition to their color attributes, anthocyanins are found to have
potential health benefits due to their significant antioxidant properties.
Anthocyanin, as part of the flavonoid family, exhibits potent antioxidant
properties and effectively neutralizes free radicals, showcasing its significant
free radical scavenging capabilities. Ke et al. (2019) investigated the total
anthocyanin content and cyanidin-3-O-glucoside (C-3-G) levels in commonly
consumed pigmented plants. The researchers examined how these
compounds contributed to the antioxidant activities of the plants. The study
revealed that most berry extracts demonstrated higher antioxidant activities
compared to vegetable extracts. The authors also indicated that the
antioxidant capacity of berries is influenced by their color, with darker-colored
berries displaying the highest antioxidant potential. Feng et al. (2016)
discovered that the antioxidant activities in blackberries were much higher
than in red berries. The findings from earlier studies suggest that the deeper
the color, the greater the antioxidant capacity.
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2.6 Total Monomeric Anthocyanin
Anthocyanins are pigments responsible for the purple, red, and blue
hues in plants and vegetables. The structural variations of six (6) common
anthocyanidin molecules—cyanidin, pelargonidin, delphinidin, peonidin,
petunidin, and malvidin—depend on the glycosidic substitutions at the 3 and 5
positions, as illustrated in Figure 2. Additional variations arise through the
acylation of sugar groups with organic acids (Lee et al., 2005). Figure 2
illustrates the basic structure of anthocyanin pigment.
Figure 2. Basic structure of anthocyanin pigment (Lee et al., 2005).
The pH differential method has been used to determine the total
monomeric anthocyanin content by observing the structural change of the
anthocyanin chromophore between pH 1.0 and pH 4.5. The pigments of
monomer anthocyanins change color irreversibly due to pH changes. At pH 1,
anthocyanins predominantly exist in their colored flavylium cation (oxonium)
form. However, as the pH increases to 4.5, their structure shifts to a colorless
semi-ketone form (Figure 3) (Teng et al., 2020). The difference in absorbance
of anthocyanins at 520 nm is directly proportional to their pigment
concentration (Lee et al., 2005). However, this method is limited to detecting
monomeric anthocyanins only. Polymerized anthocyanins resist color changes
with pH variations because they absorb light at both pH 1.0 and pH 4.5, as
noted by Teng et al. (2020). The structural forms of anthocyanin at pH 1.0 and
pH 4.5 are shown in Figure 3.
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Figure 3. Predominant anthocyanin structural forms at different pH levels (pH
1.0 and pH 4.5) (Teng et al., 2020).
2.7 Total Antioxidant Capacity
The quantitative determination of the antioxidant capacity of the
extracts is conducted through phosphomolybdenum assay. This method relies
on the reduction of molybdenum(VI) (Mo6+) to molybdenum(V) (Mo5+) by the
analyte in the sample, leading to the formation of a green phosphate Mo 5+
complex under acidic pH conditions (Prieto et al., 1999). This method was
originally developed by Prieto et al. (1999) to quantitatively determine the
vitamin E in seeds.
According to Bibi Sadeer et al. (2020), approximately 90 minutes is
enough for the complete formation of the greenish-blue phosphomolybdenum
complex, marking the endpoint of the reaction. This coloration arises from the
reduction of ammonium molybdate to an oxide called the Keggin ion
[H3PO4(MoO3)12] under acidic conditions. The Keggin ion is then reduced into
[H4PMo8VIMo4VO40]3− in the presence of an antioxidant. The
phosphomolybdenum complex can be produced at room temperature, but the
rate of reaction is very slow. The reaction rate can be enhanced by elevating
the temperature, increasing surface area, raising the concentration of a
reactant, or introducing a catalyst to expedite the reaction. Prieto et al. (1999)
further demonstrated that the reaction is temperature-dependent. It was
observed that raising the temperature to 95℃ had led to an acceleration of
the reaction rate, which consequently resulted in an increased yield of
greenish-blue phosphomolybdenum complex. The absorbance of the
greenish-blue complex can be measured at a wavelength of 695 nm, as
shown in Figure 4.
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Figure 4. Reaction mechanism of phosphomolybdenum (Bibi Sadeer et al.,
2020).
2.8 Total Phenolic Content
In determining the total phenolic content of plant and food samples,
Folin–Ciocalteu (F–C) assay is commonly employed. This assay is
characterized by its high sensitivity and capability to quantify a broad
spectrum of phenolic compounds. Several studies have utilized this method to
analyze the complex phenolic compounds found in vegetables, fruits, and
other foods. It is also widely used to identify and quantify TPC in plants to
determine their antioxidant activity (Pérez et al., 2023).
The Folin-Ciocalteu (F-C) method involves an electron-transfer
reaction, where the F-C reagent acts as the oxidizing agent and antioxidant
species serves as the electron donor, as seen in Figure 5. The chemical
structure of the F-C reagent is unknown, but it is composed of a complex
mixture of phosphomolybdic and phosphotungstic acids. Throughout the
assay, this mixture undergoes reduction, resulting in the formation of a blue
chromophore that can be measured at its maximum absorbance of 765 nm
(Figure 5). When antioxidants from a sample react with the F-C reagent, it
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results in the reduction of the anionic derivatives of phosphotungstic and
phosphomolybdic acids, leading to a change in color from yellow to blue. This
color change is directly correlated with the reducing capacity of the phenolic
compounds (Pérez et al., 2023).
Figure 5. General reaction mechanism in Folin–Ciocalteu (F–C) assay
(Pérez et al., 2023).
2.9 Stability of Anthocyanins
The usage of anthocyanin as a natural colorant in food and cosmetic
industries has been widely studied. However, it was found that this compound
is unstable when exposed to different factors including pH, temperature, light,
and oxygen (Muhamad et al., 2017). Anthocyanins exist in various structural
forms that are highly dependent on the pH of their environment. This
dependency significantly influences the coloration of anthocyanins in fruits
and plants (Andrés-Bello et al., 2013).
Due to their ionic molecular structure, anthocyanins' color is
significantly affected by the pH of the solution, making them most stable and
prevalent in acidic conditions (Turturică et al., 2015). Accordingly, certain
anthocyanins appear red in acidic conditions, shift to purple at neutral pH, and
transition to blue as the pH increases further. The red pigments found in
anthocyanins primarily exist as flavylium cations, which are more stable in
lower pH conditions (Bakowska-Barczak, 2005). The presence of flavylium
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cations makes anthocyanins highly water-soluble. However, when the water
concentration decreases, the deprotonation of the flavylium cation is favored,
leading to reduced color stability (Coutinho et al., 2014).
Vayupharp and Laksanalamai (2015) evaluated the stability of
anthocyanin extracted from purple corn cob in terms of different pH ranges.
The researchers found that the anthocyanin extracts from corn cob yield
colors of dark red, orange-red, light purple, and dark blue at pH 1, 4, 7, and 9,
respectively. Cai et al. (2023) highlighted that anthocyanins exhibit the
highest stability at lower pH levels but become less stable in neutral or basic
conditions. The authors noted that at pH 1, anthocyanins exist as flavin
cations, which produce a red color. At pH values between 2 and 4, quinodal
blue species are more prominent. As the pH increases to a range of 4 to 6,
the hydrolysis of the flavin cation at carbon 2 occurs due to a nucleophilic
attack by water, resulting in a colorless carbinol pseudoradical and a pale
yellow chalcone. As pH continues to increase to around 8 to 10, further
deprotonation takes place, shifting the color towards green as the chalcone
and quinone become ionized. When the pH is increased to 12 and above, the
dianion chalcone becomes the primary compound, imparting a yellow color to
the solution (Cai et al., 2023).
The stability of anthocyanins is also susceptible to temperature
changes. Broto et al. (2018) studied the stability of anthocyanin extracts from
the lether fruit of palm’s waste to varying temperatures (50 ℃, 60 ℃, 70 ℃,
80 ℃, 90 ℃, and 100 ℃). Researchers noted that the anthocyanin solution
lost its color due to high temperatures breaking down the anthocyanin's
aglycone form into chalcone. At even higher temperatures, it transformed into
a brown alpha-ketone. The study indicated that the color stability of
anthocyanin extracts from palm’s fruit peel diminished with increasing
temperatures. The authors determined that 50°C was the optimal temperature
for maintaining the stability of anthocyanin color (Broto et al., 2018).
One factor that can affect the stability of anthocyanins is light exposure.
The aromatic acyl group in anthocyanin molecules enhances their stability in
light conditions compared to their unacylated forms. These acyl groups act as
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a protective mechanism against photodegradation by absorbing photons.
However, the number of acyl groups in anthocyanins does not always
demonstrate a direct positive correlation with their stability. In acidic media,
anthocyanins exhibit greater stability under light. Moreover, the light stability of
anthocyanins is influenced by additives in the solution, such as ascorbic acid,
which enhances light stability by scavenging peroxide radicals formed during
light-induced stress. Dark storage conditions help maintain the stability of
anthocyanin molecules, unlike light exposure, which often leads to their
degradation (Li et al., 2023).
The stability of anthocyanin molecules is also influenced by exposure
to oxygen. Research indicates that anthocyanins are less stable in oxygen-
rich environments compared to storage under vacuum or inert gases like
nitrogen and argon. Additionally, previous studies have highlighted the
noticeable degradation of anthocyanins in wine when exposed to elevated
oxygen levels. Zhou et al. (2017) investigated the effects of various
processing and storage conditions on anthocyanin extracts from blueberry
peel. Their findings revealed that the degradation of anthocyanin extracts
occurred more rapidly under aerobic conditions compared to anaerobic
conditions. Therefore, careful control of oxygen levels during storage and
packaging is crucial to prevent future degradation of anthocyanins (Xue et al.,
2024).