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Identification of Bioactive Compounds of Seaweed Sargassum sp. and Eucheuma cottonii Doty as a Raw Sunscreen Cream

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This study aimed at determining proximate value, vitamin E, antioxidant activity and active components extracts of Sargassum sp. and Eucheuma cottonii Doty . Extraction was done by maceration stratified method. The yield of Sargassum sp. used solvent n -hexane, ethyl acetate and methanol and amounted to 0.027 3 %, 0.133 3 %, 7.332 8 %. T he yield of E. cottonii used solvent n -hexane, ethyl acetate and methanol and amounted to 0.025 7 %, 0.078 8 %, 6.758 6 %. Proximate value of Sargassum sp. is a row of moisture, ash, fat, protein and crude fiber that amount ed to 82.26 %, 5.09 %, 1.26 %, 0.41 %, 0.43 %, respectively , and E. cottonii amounted to 77.27 %, 5.84 %, 2.39 %, 0.12 % and 0.67 % , respectively . Vitamin E value of Sargassum sp. was 165.19 μg mL –1 while vitamin E value of E. cottonii amounts to 160.01 μg mL –1 , using HPLC method. The antioxidant activity of Sargassum sp. and E. cottonii from the methanol extract were 57.050 μg mL –1 and 105.040 μg mL –1 . The active component s of Sargassum sp. and E. cottonii contained in the methanol extract were flavonoids, phenols hydroquinone and triterpenoid
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Research Article
Proceedings of the Pakistan Academy of Sciences: Pakistan Academy of Sciences
B. Life and Environmental Sciences 54 (4): 311–318 (2017)
Copyright © Pakistan Academy of Sciences
ISSN: 2518-4261 (print), ISSN 2518-427X (online)
Identification of Bioactive Compounds of Seaweed Sargassum sp.
and Eucheuma cottonii Doty as a Raw Sunscreen Cream
Nurjanah1, Mala Nurilmala1, Effionora Anwar2, Novi Luthfiyana1,
and Taufik Hidayat3*
1Department of Aquatic Product Technology, Faculty of Fisheries and Marine Science,
Bogor Agricultural University, Agatis street IPB Darmaga, Bogor-16680, Indonesia
2Faculty of Pharmacy University of Indonesia, Depok Campus 16424, Indonesia
3Department Fisheries, Faculty of Agriculture, Sultan Ageng Tirtayasa University, Serang Campus Jalan
Raya Jakarta KM. 04 Pakupatan, Kota Serang 42124, Banten, Indonesia
Abstract: This study aimed at determining proximate value, vitamin E, antioxidant activity and active
components extracts of Sargassum sp. and Eucheuma cottonii Doty. Extraction was done by maceration
stratified method. The yield of Sargassum sp. used solvent n-hexane, ethyl acetate and methanol and
amounted to 0.027 3 %, 0.133 3 %, 7.332 8 %. The yield of E. cottonii used solvent n-hexane, ethyl acetate
and methanol and amounted to 0.025 7 %, 0.078 8 %, 6.758 6 %. Proximate value of Sargassum sp. is a
row of moisture, ash, fat, protein and crude fiber that amounted to 82.26 %, 5.09 %, 1.26 %, 0.41 %, 0.43
%, respectively, and E. cottonii amounted to 77.27 %, 5.84 %, 2.39 %, 0.12 % and 0.67 %, respectively.
Vitamin E value of Sargassum sp. was 165.19 μg mL–1 while vitamin E value of E. cottonii amounts to
160.01 μg mL–1, using HPLC method. The antioxidant activity of Sargassum sp. and E. cottonii from the
methanol extract were 57.050 μg mL–1 and 105.040 μg mL–1. The active components of Sargassum sp. and
E. cottonii contained in the methanol extract were flavonoids, phenols hydroquinone and triterpenoids.
Keywords: Antioxidant, extraction, E. cottonii Doty, Sargassum sp., sunscreen
1. INTRODUCTION
Indonesia is widely known as an archipelago, of
which 2/3 of its territory is ocean and has the
longest coastline in the world which is ± 80 791.42
km. One potential commodity that grows and
thrives in the ocean is kelp [1]. Seaweed is one of
the commodities of pro and its production
increased by 20.9 % in 2013 to 7.5 × 106 t, and in
2014 the its production was 10 × 106 t. Seaweeds
are taxonomically classified as algae, pertaining to
four main classes, namely Rhodophyceae (red
algae), Chlorophyceae (green algae),
Chyanophyceae (blue green algae), and
Phaeophyceae (brown algae) [2].
Brown algae is a photoprotective agent that is
capable of absorbing UV rays. Genetically, brown
algae is more frequently induced by sunrays so
that it is able to synthesize the compounds that
have a capability of absorbing UV rays. [3]. One
type of brown algae that has potential as raw
material for sunscreen cream is Sargassum sp.
Sargassum sp. has greater antioxidant activity
than other types of Caulerpa racemosa (Forsskal)
J. Agardh, Ulva lactuca Linn. and Gracilaria
tenuistipitata (C.F. Chang & B.M. Xia) with IC50
values of each (1.08 ± 0.83) µg mL–1, (15.05 ±
0.61) µg mL–1, (103.73 ± 0.59) µg mL–1, (24.22 ±
0.87) µg mL–1 [4]. Sargassum sp. contains
ascorbic acid, as well as phlorotannin compounds
that play a role in inhibiting the formation of
melanin. Phlorotannin is a polyphenolic
compound that is only found in brown algae, but is
not found in terrestrial plants, and is formed from
units of phloroglucinol (1,3,5-trihydroxybenzene)
[5]. Based on several studies that have been
————————————————
Received, February 2017; Accepted, November 2017
*Corresponding author: Tauk Hidayat; Email: besthd22@gmail.com
312 Nurjanah et al
carried out, it proved that phlorotannin has activity
Biological broad as anti-proliferative and
antioxidant [6], inhibitors of matrix
metalloproteinases [7], and an ability to absorb
UV radiation [8].
E. cottonii is red seaweed that can be
cultivated and consumed. Seaweed is growing
very fast and can be harvested every 45 d for
consumption. Seaweed cultivation is abundant in
Southeast Asia potentially in Africa and the
islands located in the Pacific region [9].
Manufacture of sunscreen formulations is also
needed in gelling agent and stabilizer.
Carrageenan has been widely used in food
industry, medicine, textiles, and cosmetics,
because it is as an emulsifier, thickener, stabilizer,
and gelling [10]. E. cottonii is a commercial
source of carrageenan which is a gelling agent and
stabilizer in food industry. E. cottonii contains
nutrients comprising proteins, lipids,
carbohydrates, vitamin C, α-tocopherol, and
minerals that can be used as a medium for the
growth of lactic acid bacteria [11]. Bioactive
components found in seaweed are very prospective
in cosmetics because they contain terpenoids,
carotenoids, and polysaccharides (fucoidan,
carrageenan, alginate, and jelly). This study aimed
at determining chemical characterization
proximate value, vitamin E and the antioxidant
activity as well as active components extracts of
Sargassum sp. and Eucheuma cottonii Doty. This
study aimed to determine proximate value, vitamin
E, antioxidant activity and active components in
extracts of Sargassum sp. and Eucheuma cottonii
Doty.
2. MATERIALS AND METHODS
2.1 Sampling
The sample used comprised of two types of natural
seaweeds, i.e., Sargassum sp. and E. cottonii.
Sargassum spp., obtained from the Kepulauan
Seribu beach. Samples were obtained in wet
conditions. The Sargassum sp. samples were
transported manually to the laboratory by using a
cool box and then were dried under the sun.
Samples were dried and then were cut into pieces
using scissors to simplify the process of
destruction. E. cottonii seaweed sample was
obtained from the beach Serang, Banten by
collecting the cultivation of local communities. E.
cottonii samples were obtained in the dry state to
avoid the risk of damage. The samples of E.
cottonii were washed first to remove dirt and salt
content. All the samples were identified prior to
enumeration or cutting.
2.2 Extraction [12]
The method of extraction was done in some stages
using the solvent n-hexane, ethyl acetate and
methanol. E. cottonii and Sargassum sp. were
chopped each as much as 100 g and then inserted
into Erlenmeyer and the solvent was added with a
ratio (1:5 (w/v). Samples were soaked with solvent
n-hexane, then covered with cotton, and wrapped
in aluminum foil. Samples macerated and mixed
using a shaker at a speed of 150 rpm (1 rpm = 1/60
Hz) for 3 d.
The obtained extract solution was filtered
with Whatman filter paper number 42 to separate
the filtrate and the residue. Residues were soaked
again with 500 mL of solvent ethyl acetate and
stirred for three days using a shaker with a speed
of 150 rpm. The filtrate and the residue were
filtered using filter paper, and then the same
process using methanol. The filtrate was
evaporated by rotary evaporator at 40 °C.
2.3 Yield Analysis [13]
Yield was calculated as a percentage of the weight
of seaweed extract that had been obtained from the
weight of the initial sample.
2.4 Proximate Analysis [14]
Proximate analysis is a method of chemical
analysis to identify the nutritional content of the
sample. Proximate analysis was conducted on the
levels of ash, fat, protein and crude fiber.
2.5 The Content Analysis of Vitamin E [15]
Qualitative method used for analyzing vitamin E
compounds in E. cottonii and Sargassum sp.
seaweed is a comparison between the retention
time (RT) and vitamin E with standard samples.
Bioactive Compounds of Seaweed as a Raw Sunscreen Cream 313
Quantification method used for quantitative
analysis in this study is an external standard.
2.6 Antioxidant Activity Analysis [16]
Testing the antioxidant activity uses DPPH
method (method of reduction of free radicals).
Test activities include the manufacture stock
DPPH antioxidant, vitamin C stock, stock
samples, blank, and testing the activity using
DPPH method. The antioxidant activity was
calculated based on the linear regression equation
and expressed in IC50 (mg L–1). Ascorbic acid is
used as a benchmark commercial antioxidant
compound at a concentration of 0.1 mg L–1 to 0.5
mg L–1.
2.7 Phytochemical Analysis [17]
Phytochemical analysis was performed to
determine the bioactive components contained in
the extracts of seaweed Sargassum sp. and E.
cottonii. Phytochemical composition comprised of
steroids / triterpenoids, flavonoids, phenols
hydroquinone.
2.8 Steroids / Triterpenoids
Samples were dissolved in 2 mL of chloroform in
a test tube. As many as 10 drops of acetic
anhydride and sulfuric acid as much as three drops
added to the mixture. Positive test results of
samples containing steroids and triterpenoids was
the formation of a solution of the red for the first
time and then changed to blue and green.
2.9 Flavonoids
A mixture between 0.1 mg of magnesium powder
and 0.4 ml of amyl alcohol (a mixture of
hydrochloric acid 37 % and 95 % ethanol by
volume each) were added to the sample and 4 ml
of alcohol, and then the mixture was shaken.
Positive test results of samples containing
flavonoids was the formation of red, yellow or
orange in the lining of amyl alcohol.
2.10 Phenol Hydroquinone
The sample, which is 1 g, was extracted with 20
mL of 70 % ethanol. The resulting solution was
taken as 1 mL then two drops of 5 % FeCl3
solution were added. Positive test results of
samples containing phenol compounds were
shown the formation of green or blue green
solution.
The experimental data were analyzed by using
statistic descriptive with Ms Excel.
3. RESULTS AND DISCUSSION
3.1 Yield of Sargassum sp. and E. cottonii
Extraction is the separation of one or more
ingredients of a solid or a liquid with the aid of
solvents. Separation occurs on the basis of
different solubility of the components in the
mixture [18]. Extraction with organic solvents can
be done by percolation, maceration and
soxhletation [19].
Maceration is a crude drug extraction process
by using a solvent with some time shaking or
stirring at room temperature (room temperature).
The procedure is done by soaking bulbs in a
suitable solvent in a sealed container. Occasional
or constant stirring can increase the speed of
reaction [20]. Maceration has several advantages,
such as the amount of organic solvent used is not
too much and the extraction temperature used
below the boiling point of the solvent so that the
degradation of oil components due to heat can be
avoided [19].
Selection of solvents and extraction methods
will affect the results of the content of secondary
metabolites that can be extracted. Selection of
solvent extraction generally uses principles where
the non-polar compounds will dissolve in non-
polar solvents, while the polar compounds will
dissolve in polar solvents [21]. The extraction is
done in some stages. The organic solvents used in
this study are n-hexane, ethyl acetate and
methanol. Evaporation process is used to separate
the solvent from the extract. The temperature used
is 40 °C to 50 °C to prevent damage of the active
components contained in the extract.
Yield is an important parameter to determine
the economic value and effectiveness of a material
or product. Yield is the percentage share of the
raw materials that can be utilized. The yield of
314 Nurjanah et al
Sargassum sp. using solvent n-hexane, ethyl
acetate and methanol amounted to 0.027 3 %,
0.133 3 %, 7.332 8 % respectively. The yield of E.
cottonii using solvent n-hexane, ethyl acetate and
methanol amounted to 0.025 7 %, 0.078 8 %,
6.758 6 % respectively. The extracts of Sargassum
sp. and E. cottonii using the methanol produces the
greatest yield compared with the solvent n-hexane
and ethyl acetate. Putri et al. [22], states that
methanol can dissolve almost all organic
compounds that exist in the sample, both polar and
non-polar compounds. Harborne [23] reported the
differences of the yield of extract depends on the
natural conditions of the sample, the extraction
method, the particle size of the sample, the
conditions and the time of extraction, as well as a
comparison sample with solvent. Setha et al. [24]
states that methanol produce extracts with
antioxidant potential is better than other organic
solvents, which means that methanol is able to
attract the active components in the Sargassum sp.
and E. cottonii optimally. The extracts of
Sargassum sp. generated in this study is brownish
green. Limantara & Heriyanto [25], reported that
the color of Sargassum sp. is caused by three main
pigments such as chlorophyll (the green pigment
bluish), carotenoids (red pigment), and
fucoxanthin (brown pigment).
3.2 Chemical Composition of Sargassum sp.
and E. cottonii
The test results of proximate seaweed Sargassum
sp. and E. cottonii from Kepulauan Seribu are
presented in Table 1.
Table 1. Chemical composition of Sargassum sp. and
E. cottonii from Kepulauan Seribu.
Chemical
component
Sargassum sp.
(%)
E. cottonii
(%)
Water
82.26
77.27
Ash
5.09
5.84
Protein
1.26
2.39
Fat 0.41 0.12
Crude Fiber
0.43
0.67
Being marine in nature seaweeds contain a
large amount of water. In a fresh condition, they
have 75 % to 85 % water and 15 % to 25 %
organic components and minerals [26]. States that
the drying process is uneven and fluctuating
temperature changes affect the water content. The
longer the drying time is done, the water content
of a substance contained in the lower [27].
The ash content value of Sargassum sp. and E.
cottonii is 5.09 % and 5.84 %. The resulting ash
content values still meet the standards of ash
content in brown seaweed which is about 36 %.
Winarno [28] states that the ash is inorganic
substances waste products of combustion of an
organic material. Ash has to do with mineral
materials. High and low ash content contained in a
material can be attributed to the amount of mineral
elements [29], while the mineral content of
seaweed can be affected by processing [30]. In
addition, the level of each mineral component is
determined by species, physiological factors,
geographical conditions and frequencies, as well
as the types of methods used in the mineralization
process.
The protein content Sargassum sp. and E.
cottonii in this study was obtained by 1.26 % and
2.39 %. The protein content Sargassum sp. and E.
cottonii is lower when compared to the Burtin
research [31], Brown seaweed that contains
proteins of 3 % to 9 % of the weight of the wet,
while the red and green seaweed containing
protein at 6 % to 20 % of wet weight.
Environmental conditions, such as
temperature, salinity, water transparency for
synthesis of NFE and the nutrient uptake are the
factors that can affect the crude fibre levels [32].
The variations in crude fibre of seaweeds can
occur due to differences in growth stages and
photosynthetic activity among seaweed species,
and season brought about by changing
environmental parameters that influence
photosynthesis and uptake of nutrients [33].
The crude fat of seaweed was less than 5%
reported on crude fat of seaweeds in other works
[34]. Dharmananda [35], reported that seaweed
generally contains fat by 1 % to 5 % of the dry
weight. Seaweed contains very little fat.
Bioactive Compounds of Seaweed as a Raw Sunscreen Cream 315
3.3 Vitamin E of Sargassum sp. and E. cottonii
Vitamins based on their solubility properties are
divided into fat soluble and water soluble. Vitamin
E is a fat soluble vitamin in seaweed which
contains many antioxidant activity [36].
Antioxidants are chemical compounds that can
donate one or more electrons to free radicals, so
that the free radicals can be suppressed [37].
Vitamin E (α-tocopherol) is widely used as an
antioxidant in cosmetic preparations for
preventing the aging process, maintenance and
protection of normal biological processes such as
anti-inflammatory. Vitamin E is an essential
nutrient that functions as an antioxidant in the
human body [38]. The results of the assay of
vitamin E Sargassum sp. and E. cottonii use HPLC
system. Levels of vitamin E obtained from
samples of Sargassum sp. amounted to 165.19 mg
L–1 and E. cottonii at 160.01 mg L–1.
3.4 Antioxidant Activity of Sargassum sp. and
E. cottonii
Antioxidants are defined as compounds that could
delay, slow down and prevent the oxidation of
lipids. In a special sense, antioxidants are
substances that can delay or prevent the
occurrence of free radical reactions in the
oxidation of lipids [39]. Free radicals can be
defined as a molecule or compound in a free state
that has one or more unpaired electrons free.
Electrons of unpaired free radicals are very easy to
attract electrons from other molecules so that they
become more reactive radicals. Therefore, highly
reactive, free radicals are very easy to attack
healthy cells in the body [40].
The different types of seaweed give
differences in the content and type of the
polyphenol compound. The differences of its
compounds will affect its ability to reduce free
radicals. The value of antioxidant activity
Sargassum sp. and E. cottonii from the methanol
extract is 57.050 mg mL1 and 105.040 mg mL–1
respectively. The results showed that the
antioxidant value of the Sargassum sp. and E.
cottonii sample is very strong. Molyneux [41]
states that a compound called active as an
antioxidant when the IC50 value is less than 200
mg mL–1. When the IC50 values obtained ranged
from 200 mg mL–1 to 1000 mg mL–1, the substance
is less active but still has potential as antioxidants.
Prior & Cao [42] suggest the lower the IC50 value,
the higher the antioxidant activity of antioxidants.
This was due to the use of lower concentrations
can already inhibit DPPH by 50 %.
3.5 Bioactive Components of Sargassum sp. and
E. cottonii Extracts
Sargassum sp. contains fucoidan and phenolic
components. Type of phenolic components that
are commonly found in brown seaweed is
phlorotanin ranging from 0.74 % to 5.06 % [43].
Sargassum sp. contains ascorbic acid, as well as
phlorotannin compounds that play a role in
inhibiting the formation of melanin [5].
The extracts of Sargassum sp. and E. cottonii
phytochemical were analyzed to determine the
active component compound that acts as a good
sunscreen to protect the skin. Phytochemical
analysis undertaken includes flavonoids, phenols
hydroquinone and triterpenoids. Phytochemical
analysis results of extract Sargassum sp. and E.
cottonii are presented in Table 2.
Table 2. Phytochemical analysis of extracts of
Sargassum sp. and E. cottonii.
Compounds Sargassum sp. E. cottonii
Flavonoid +++ +
phenol hydroquinone ++ +
triterpenoids ++ +
Description:
(+) = weak
(++) = strong
(++++) = very strong
Based on qualitative phytochemical analysis,
it can be shown that the extracts of Sargassum sp.
and E. cottonii contain the active components of
flavonoids, phenols hydroquinone and
triterpenoids which allegedly acted as a potential
agent for raw materials sunscreen cream.
Flavonoids are generally found in all parts of
the plant, including the fruit, pollen and roots in
316 Nurjanah et al
the form of glycosides. Flavonoids are classified
into flavones, flavonols, flavanones, flavanols,
isoflavones, kalkon, dihidrokalkone, aurone,
anthocyanidins, catechins and flavan-3,4-diol [44].
Flavonoids are good at reducing compounds,
inhibiting many oxidation reactions, enzymatic
and non-enzymatic [45] Flavonoids, one of
polyphenols, have a big role in the activity of
tyrosinase because they contain phenol group and
ring pyren. The structure of flavonoids in principle
is suitable as substrates and able to compete so
that it can be inhibiting tyrosinase [8].
Phenolic component is an aromatic structure
that binds to one or more hydroxyl groups which
are some may be substituted with a methyl group
or a glycosyl. Free phenolic compounds are
usually are found in wood tissue, while the
phenolic compounds are in another place usually
in the form of glycosides [23]. Phenolic
compounds are involved in electron transport in
photosynthesis and in the regulation of certain
enzymes. These compounds also have anti-
inflammatory activity, as it can inhibit
prostaglandin synthesis [46]. Kim et al. [7]
reported the chemical structure of phenolic
components has similarities with tyrosinase
substrate so that the phenolic components are
potential as competitive inhibitors of the tyrosine-
tyrosinase reaction.
Triterpenoids are natural compounds formed
by the process of biosynthesis and distributed
widely in the world of plants and animals.
Terpenoids structure is built by isoprene molecules
with terpenoid skeleton which is formed of two or
more units of isoprene (C5) [44]. Terpenoids
consisting of several kinds of compounds are
components of essential oils, diterpenoid,
giberaline, triterpenoidem, sterid and carotenoids
[47]. Three steroids isolated from Trifolium
balansae were reported to have activity of
tyrosinase inhibitor which isolates both have IC50
value of 2.39 μM and [48].
4. CONCLUSIONS
The yields of Sargassum sp. and E. cottonii,
generated by methanol, amounted to 7.332 8 %
and 6.758 6 %. The proximate value of Sargassum
sp. comprsing of moisture, ash, fat, protein and
crude fiber amounted to 82.26 %, 5.09 %, 1.26 %,
0.41 %, 0.43 %, respectively, and proximate E.
cottonii amounted to 77.27 %, 5.84 %, 2.39 %,
0.12 % and 0.67 %, respectively. The
concentration of vitamin E determined in
Sargassum sp., using HPLC, was 165.19 mg L–1
and E. cottonii at 160.01 mg L–1. The antioxidant
activity of Sargassum sp. and E. cottonii from the
methanol extract was 57.05 mg L–1 and 105 mg L1,
04 mg L–1. The active components of Sargassum
sp. and E. cottoni contained in methanol extracts
were flavonoids, phenols hydroquinone and
triterpenoids compounds which can potentially be
used as raw material for sunscreen cream.
5. ACKNOWLEDGEMENTS
This work was financially supported by the
KEMENRISTEK DIKTI no. 083/SP2H/PL/
Dit.Litabmas/II/2015.
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... Keberadaan senyawa phlorotannin yang turut berperan dalam melindungi kerusakan kulit terhadap radikal bebas yang disebabkan oleh paparan sinar UV sehingga menghambat pembentukan melanin (Svobodová et al. 2003). Nurjanah et al. (2015) melaporkan nilai IC 50 pada ekstrak metanol Sargassum sp. sebesar 57,05 ppm dan jumlah ketersediaan vitamin E sebesar 165,19 ppm. ...
... Kandungan vitamin E yang terdapat pada E. cottonii 0,23 mg/kg (berat kering) dan S. plagyophyllum 363,86 mg/kg (berat kering). Nurjanah et al. (2015) melaporkan kandungan vitamin E yang terdapat pada E. cottonii segar 160,01 mg/kg dan Sargassum sp. segar 165,19 mg/kg. ...
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