Encapsulation of Anthocyanin and Flavonoid Watermelon Rind

(Citrullus lanatus) as a Natural Food Preservative

Lilis Kistriyani1, a *, Annisa Alvi Ramadhani1,b, Dika Puji Resphaty1,c
1
Departemen of Chemical Engineering, Universitas Islam Indonesia, Yogyakarta, Indonesia
a
lilis.kistriyani@uii.ac.id,bannisaalviramadhani@gmail.com,cdikapuji96@gmail.com

Keywords: encapsulation, watermelon, anthocyanin, flavonoids, phenolic

Abstract: Special Region of Yogyakarta is one of the regions that has high potential for natural
disasters. Food is the consumable material which is the most needed when natural disasters occur.
To maintain nutritious food that will be distributed to victims of natural disasters still appropriate to
eat, an alternative form of food preservative is needed. In this research, watermelon rind was chosen
as an ingredient to make natural preservatives because they contain flavonoids and anthocyanins.
This research aims were to determine the total content of anthocyanins, flavonoids and phenolics in
red watermelon rind and yellow watermelon rind, also to determine the effect of chitosan
composition to the ability of flavonoids and phenolics release in food. This research was carried out
by extraction and encapsulation methods. The analysis included the content of Total Anthocyanin
(TA), Total Flavonoids (TF) and Total Phenolic (TPC) in the supernatant. The other analysis was in
vitro tests that was done to know the ability of flavonoids and phenolics release in food. The total
anthocyanin content in red watermelon rind is 0.0334 mg L-1 while in yellow watermelon rind is
0.0668 mg L-1. The total content of flavonoids in red watermelon rind is 0.7369 g mL-1, while in
yellow watermelon rind is 0.3296 g mL-1. The total phenolic content of red watermelon rind is
0.3669 g mL-1, while in yellow watermelon rind is 0.2273 g mL-1. In red and yellow watermelon
rind, the highest release of flavonoid and phenolic levels occurred in variations of chitosan mass 0.4
grams. The highest flavonoid content released was 0.0638 g mL-1 on red watermelon rind and
0.0702 g mL-1 on yellow watermelon rind. The highest phenolic content released was 0.0321 g mL-1
on red watermelon rind and 0.0408 g mL-1 on yellow watermelon rind.

Introduction
Natural preservatives are an option to maintain nutrients in food. Watermelon rind is one of
the ingredients that can be used to make natural preservatives because they contain flavonoids and
anthocyanins [1]. Flavonoids and anthocyanins that have been extracted from watermelon rind will
be encapsulated with the intention for obtaining long-lasting preservative products.
Watermelon rind was chosen as the main ingredient in making natural food preservatives.
The reason for choosing was because it contained flavonoids and anthocyanins (Afrianti, 2010).
Flavonoids have high antioxidant activity [2]. Antioxidants protect tissues to against oxidative
damage from free radicals from processes in the body or outside and have a synergistic relationship
with vitamin C. While anthocyanins have the ability to capture free radicals and inhibit fat
peroxidation, the main cause of damage to cells associated with aging and degenerative diseases [3].
Not only anthocyanin compounds and flavonoids contained in watermelon rind but also phenolic
compounds that function as antioxidants are also found it. Its ability to form phenoxy radicals that
are stable in the oxidation process causes these compounds to be widely used as antioxidants.
Materials and method
A. Materials
The materials that were used in this research were watermelon rind, chitosan, Na (TPP) as
crosslinker, ethanol 96%, acetic acid, KCl Buffer pH 1.0, Sodium acetate buffer pH 4.5, HCl 37
%, aquadest.
B. Instruments
The main equipment that used in this research were homogenizer, rotary vacuum evaporator,
centrifuge, UV-VIS spectrophotometer and magnetic stirrer.
C. Method

Extraction of Anthocyanins and Flavonoids from Red Watermelon Rind

Fifty grams of fresh sample was inserted into the homogenizer at 4oC and then the cellular structure
was broken down using a high-speed homogeneous machine at 15,000 r min-1 for 3 minutes. The
paste formed was mixed with 175 mL 96% ethanol (ethanol: water = 80: 20) containing 0.1% HCl.
The sample solution was filtered using a separating funnel aided by filter paper and vacuum pump
until it was filtered completely. Then the solution and sediment were separated by using a 4000 rpm
centrifuge for 10 minutes. The solution was filtered again using a separating funnel aided by
Whattman paper and vacuum pump until it was filtered completely. 0.01 ml of 0.1N HCl was mixed
with 100 ml of aquadest, and 25 ml of the solution was taken and mixed with 100 ml of sample
solution. Solvents with extracts were separated using rotary vacuum evaporator 40 rpm, with a
temperature of 35˚C, for 120 minutes [4].

Process of Encapsulation of Anthocyanins and Flavonoids

Anthocyanins and flavonoids extract were dissolved in KCl buffer pH 1.0. Chitosan with variations
of 0.3 - 0.6% (c/v) was dissolved in acetic acid solution pH 2.6 (1.0% v/v). The crosslinker pH was
set to 2.6 using 0.1N HCl, while added 1 mL anthocyanin extract (pH 1.0) to 20 mL chitosan
solution pH 2.6 (0.1% b/v). At the same time, the solution was dropped into 20 mL NaTPP 0.5%
(b/v) solution pH 2.6 and stirred for 3 minutes. Then the encapsulation suspension was separated by
a centrifuge at 10,000 g for 10 minutes. The capsules and supernatants formed were separated to do
further analysis [5].

Analysis
1. Total Anthocyanin (TA) and Total Flavonoid (TF)
This analysis of anthocyanin was carried out by the pH difference method adopted from the
Wrolstad study et al. (2005) and Huang (2009). TA was carried out by dissolving 1 mL extract of
watermelon rind into 0.2 M KCl buffer pH 1.0 until the volume reaches 25 mL. Then, 1 mL of
anthocyanin extract and flavonoids were dissolved in 0.2 M sodium acetate buffer pH 4.5 to 25 mL
volume. The sample were analyzed using a UV-1800 Spectrophotometer (Shimadzu UV
Spectrophotometer) with a wavelength of 525 nm and 700 nm. Calculation of total anthocyanin
(TA) is carried out by the following formula: TA (mg / L) = [(OD525 – OD700) pH1.0– (OD525 –
OD700) pH4.5] x449.2x1000 / 26900xDF.

Where,
449.2 = molecular mass of cyanidin-3-glucoside
1000 = conversion factor from g to mg
26900 = molar absortivity
DF = dilution factor

Total flavonoids (TF) were measured by a spectrophotometric method that was carried out by Wang
et al. (2014). Routine was used as a standard compound in this analysis. The sample absorbance was
measured at a wavelength of 510 nm using Shimadzu UV Spectrophotometer. The amount of
flavonol (R) was calculated using a calibration curve. The levels of flavonoids (TF) were obtained
with the following calculations:

TF (g / mL) = R x DF / m
With,
DF = 2500
m = number of watermelon rind samples (grams)

Total Phenolic Content (TPC)

The total phenolic content (TPC) in the extract was analyzed using the Foin-Ciocalteu method [6].
Gallic acid was used as a standard solution. The extract sample was measured for its absorbance at a
wavelength of 750 nm using Shimadzu UV Spectrophotometer.

In vitro Release Test

In vitro release of anthocyanin and flavonoid compounds was carried out on the aquadest
release media. The media was chosen because the purpose of the encapsulation results would be
applied to food or drinks. The release test was carried out by taking 100 mg of encapsulation and
put in 50 mL of distilled water. The mixture were put into a cup and then stirred using a magnetic
stirrer at a speed of 120 rpm for 30 minutes at room temperature. The sample solution was analyzed
periodically every 5 minutes to determine the amount of flavonoids and phenolics released into the
media [5].

Result and discussion

3.1. Total Anthocyanin Content (TA)
Determination of total anthocyanin content in red watermelon and yellow watermelon rind were
carried out using UV-Vis spectrophotometry method. This analysis was carried out by the pH
difference method adopted from the research of [7]. By looking for absorbance at a wavelength of
525 nm and 700 nm, an anthocyanin level value was obtained by entering the absorbance value into
the total anthocyanin (TA) calculation formula. The total anthocyanin value of red watermelon rind
was 0.0334 mg L-1, while the total anthocyanin value of yellow watermelon rind was 0.0668 mg L-1.
From these results it could be seen that the total anthocyanin value of yellow watermelon rind was
bigger than red watermelon rind. The results of the analysis were not suitable with previous studies
conducted by [8] in the "Assessment of some Antinutrient Properties of the Watermelon (Citrullus
lanatus) Rind and Seed" where there was no anthocyanin content in watermelon rind.
Fig.1 showed the reaction that occurred during extraction. The reaction was between the
ethanol compound bond and HCl compound bond. The two compounds reacted with anthocyanin
functional groups caused the amount of anthocyanin could be taken more.

Fig.1. The Anthocyanin Functional Group

Total Flavonoids (TF)

Analysis of Total Flavonoids (TF) was measured by a spectrophotometric method that was
conducted by [5]. Routine was used as a standard compound in this analysis. The absorbance
samples were measured at 510 nm wavelength, then the red watermelon absorbance value was
0.381 while the yellow watermelon absorbance value was 0.170, the absorbance value was used to
find the total flavonoid content value. By using the standard flavonoid curve, the total flavonoids
 content of red watermelon rind was 0.7369 mg L-1, while the total flavonoids content of yellow
watermelon rind was 0.3296 mg L-1.
Based on the results of research conducted by [9] in the "Effect of Extraction Solvents and
Drying Conditions on Total Phenolic Content and Antioxidant Properties of Watermelon Rind
Powder", the flavonoid content in yellow watermelon rind was higher than red watermelon rind.
While the results of this analysis show different things where the sample on yellow watermelon rind
has a lower flavonoid content than the sample on red watermelon rind. This difference could be
caused by errors in the extraction process originating from internal and external factors such as
inaccuracy in all measurements taken and external factors such as decreased of tool performance.

3.3. Total Phenolic Content (TPC)

Determination of the total phenolic content of red watermelon and yellow watermelon rind
was carried out using the UV-Vis spectrophotometry method. The extract sample was measured for
its absorbance at a wavelength of 750 nm, then the absorbance value of red watermelon was 0.184
while the absorbance result of yellow watermelon was 0.114. The absorbance value was used to find
the value of the total phenolic content. Using a standard phenolic curve, the total phenolic content of
red watermelon rind was 0.3669 mg L-1, while the total phenolic value of yellow watermelon rind
was 0.2273 mg L-1.
Based on the results of research conducted by [9] in the "Effect of Extraction Solvents and
Drying Conditions on Total Phenolic Content and Antioxidant Properties of Watermelon Rind
Powder", the phenolic content of red watermelon rind was higher than yellow watermelon rind. This
was appropriate with the result from this experiment that the phenolic content of red watermelons
was higher than yellow watermelons.

3.4. In Vitro Test Analysis for Flavonoids and Phenolic Compounds

This analysis was done to determine how much flavonoid and phenolic compounds could be
released. The analysis was carried out using a UV-Vis spectrophotometer. The absorbance results of
spectrophotometric analysis then calculated by means of a numerical approach and the simulation
shown in the graph.The results of analysis of flavonoids and phenolics content which could be
released into aquadest from encapsulants with different variations of chitosan mass on red
watermelon rind and yellow watermelon rind showed almost the same data. The level of flavonoid
and phenolic showed a slightly decrease but it has no significant effect. By using a calibration curve
on the total flavonoid and total phenolic content analysis, a comparison of flavonoids and phenolic
in various of chitosan mass in red watermelon rind and yellow watermelon rind was showed with in
Fig.2, Fig.3, Fig.4 and Fig.5

0.0600 0.0600
phenolic contents (g/mL)

phenolic contents (g/mL)

0.0500 0.0500
0.0400 0.0400
0.0300 0.0300
0.0200 0.0200
0.0100 0.0100
0.0000 0.0000
0 10 20 30 40 0 10 20 30 40
time (minutes) time (minutes)
chitosan 0.3 g chitosan 0.4 g chitosan 0.3 g chitosan 0.4 g
chitosan 0.5 g chitosan 0.6 g chitosan 0.5 g chitosan 0.6 g

Fig.2. Phenolic Release in Red Watermelon Rind Fig.3. Phenolic Release in Yellow Watermelon Rind
 0.0800 0.1000
flavonoid contents (g/mL)

flavonoid contents (g/mL)

0.0600 0.0800

0.0600
0.0400
0.0400
0.0200
0.0200
0.0000 0.0000
0 10 20 30 40 0 10 20 30 40
time (minutes) time (minutes)
chitosan 0.3 g chitosan 0.4 g chitosan 0.3 g chitosan 0.4 g
chitosan 0.5 g chitosan 0.6 g chitosan 0.5 g chitosan 0.6 g

Fig.4. Flavonoid Release in Red Watermelon Rind Fig.5. Flavonoid Release in Yellow Watermelon Rind

The release curve of phenolic showed that the more time added, the more flavonoid and
phenolic content released into the distilled water solution. In the release process also obtained the
mass transfer from the capsule to the aquadest solution.
Encapsulation with mass of chitosan 0.4 grams on in red and yellow watermelon rind
showed the highest levels of flavonoids that could be released to aquadest. This condition also
occured in the phenolic release. Encapsulation with variations chitosan 0.4 grams in red and yellow
watermelon rind showed the highest phenolic content that could be released to aquadest. This was
shown in Fig.6 and Fig.7.

0.0800 0.0450
flavonoid content (g/mL)

0.0700 0.0400
phenolic content (g/mL)

0.0600 0.0350
0.0500 0.0300
0.0400 0.0250
0.0300 0.0200
0.0150
0.0200
0.0100
0.0100
0.0050
0.0000
0.0000
0 0.2 0.4 0.6 0.8
0 0.2 0.4 0.6 0.8
chitosan mass (g)
chitosan mass (g)
red watermelon rind yellow watermelon rind
red watermelon rind yellow watermelon rind

Fig.6. Flavonoid Release in Various Chitosan Mass Fig.7. Phenolic Release in Various Chitosan Mass

For red watermelon rind, the average value of flavonoid released into the aquadest solution
at variation of 0.3 gram mass chitosan was 0.0467 g mL-1, at 0.4 gram chitosan mass was 0.0638 g
mL-1, at 0.5 gram chitosan mass was 0.0277 g mL-1 and at 0.6 gram chitosan mass was 0.0544 g
mL-1. For yellow watermelon rind, the average value of flavonoid released into the aquadest
solution in the variation of chitosan 0.3 gram mass was 0.0445 g mL-1, at 0.4 gram chitosan mass
was 0.0702 g mL-1, at 0.5 gram chitosan mass was 0.5474 g mL-1 and at 0.6 gram chitosan mass was
0.0355 g mL-1.
For red watermelon rind, the average phenolic value released into the aquadest solution in
the variation of 0.3 gram chitosan mass was 0.0275 g mL-1, at 0.4 gram chitosan mass was 0.0321 g
mL-1, at 0.5 gram chitosan mass was 0.0162 g mL-1 and at 0.6 gram chitosan mass was 0.0278 g
mL-1. For yellow watermelon rind, the average value of flavonoid released into the aquadest
solution at a variation of 0.3 gram chitosan mass was 0.0205 g mL-1, at 0.4 gram chitosan mass was
0.0408 g mL-1, at 0.5 gram chitosan mass was 0.501 g mL-1 and at 0.6 gram chitosan mass was
0.0291 g mL-1.
The average release rate of phenolic for red and yellow watermelon rind in each chitosan
variation showed a strong increase in 0.3 gram chitosan mass to 0.4 gram chitosan mass. The
increase was due to the driving force. For yellow watermelon rind, the curve tends to decrease. This
happened because of the strong bond between Na(TPP) and chitosan, which caused the flavonoid
and phenolic components released slowly into the aquadest solution.

Conclusion
Based on the results of research conducted, it can be concluded that:
1. The total anthocyanin content in red watermelon rind was 0.0334 mg L-1 and in yellow
watermelon rind was 0.0668 mg L-1. The total content of flavonoids in red watermelon rind was
0.7369 g mL-1 and in yellow watermelon rind was 0.3296 g mL-1. The total phenolic content of red
watermelon rind was 0.3669 g mL-1, and the yellow watermelon rind was 0.2273 g mL-1.
2. In red and yellow watermelon rind, the most release of flavonoid and phenolic levels observed in
variations of chitosan mass 0.4 gram. The highest flavonoid content released was 0.0638 g mL-1 in
red watermelon rind and 0.0702 g mL-1 in yellow watermelon rind. The most released phenolic
content was 0.0321 g mL-1 in red watermelon rind and 0.0408 g mL-1 in yellow watermelon rind.

References
[1] L.H. Afrianti, Kind of Fruits for Healthy, Alfabeta, Bandung, 2010.
[2] C.F. Zuhra, J.B. Tarigan and H.Sitohang, Activity of Antioxidant Flavonoid Compound
From Katuk Leaves (Sauropus androgonus (L) Merr.), J. Biologi Sumatera, 3(1)(2008) : 7-
10.
[3] B. A. Cevallos-Casals and L. Cisneros-Zevallos, Stability of anthocyanin-based aqueous
extracts of Andean purple corn and red-fleshed sweet potato compared to synthetic and
natural colourants, J. Food Chemistry, 86 (2004) : 69-77.
[4] Z. Shi-lin, D. Peng, X.U.Yu-chao, and W. Jian-jun, Quantification And Analysis of
Anthocyanin and Flavonoids Compositions, and Antioxidant Activities In Onions With
Three Different Colors, 15(9) (2016) : 2175–2181.
[5] L.J. Wang, S. Su, J.Wu, Variation of anthocyanins and flavonols in Vaccinium uliginosum
berry in Lesser Khingan Mountains and its antioxydant activity. J. Food Chemistry,
160(2014) : 357-364.
[6] A. Mansouri, G. Embarek, E. Kokkalou, P. Kefalas, Phenolic Prole and Antioxidant
Activity of Algerian Ripe Date Palm Fruit (Phoenix dactylifera). J. Food Chemistry,
89(2005) : 411-420.
[7] R. E. Wrolstad, R. W. Drust, and J. Lee, Tracking Color and Pigment Changes In
Anthocyanin Product, J. Trends in Food Science & Technology,16(9)(2005): 423-428.
[8] C. E. Anthony, Assessment of some Anti-nutrient Properties of the Watermelon (Citrullus
lanatus) Rind and Seed. Research Journal of Environmental Sciences 9 (5) (2015): 225-232.
[9] L.H.Ho, N.F. Ramli, T.C. Tan, N. Muhamad and M.N. Haron, Effect of Extraction Solvents
and Drying Conditions on Total Phenolic Content and Antioxidant Properties of Watermelon
Rind Powder, J. Sains Malaysiana. 47(1)(2018): 99–107.