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Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
1878-0296 © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of scientific committee of the ICTCRED 2014
doi: 10.1016/j.proenv.2015.01.042
Available online at www.sciencedirect.com
ScienceDirect
International Conference on Tropical and Coastal Region Eco-Development 2014(ICTCRED
2014)
Comparative Study of Bioactive Substances Extracted from Fresh
and Dried Spirulina sp.
Tri Winarni Agustini a*, Meiny Suzeryb, Danny Sutrisnanto c, Widodo Farid Ma’rufa,
Hadiyantoc
a Department of Fi sheri es, Faculty of Fisheries and Marine Science, Diponegoro University, Jl. Prof Soedarto, SH, Tembalang, Semarang 50275.
Indonesia
b Department of Chemistry, Faculty of Sceince and Mathematics, Diponegoro University, Jl. Prof Soedarto, SH, Tembalang, Semarang 50275.
Indonesia
c Center of Biomass and Renewable Energy (C-Biore), Depart ment of Chemica l Engineeri ng, Facu lty of En gine ering, Diponegoro University, Jl.
Prof Soedarto, SH, Tembalang, Semarang 50275 . Indonesia
Abstract
• The increase consumption of natural substances has brought about in increasing demand on biological source, including
Spirulina sp. This Spirulina sp. has been recognized to provide some natural substances such as natural colorant, source of
vitamins, protein and some mineral s. Different material stat e of Spiruli na sp. in the for m of fresh and dried form may cause
different quantity and quality of natural substances in microalgae which posses a wide range of change including nutritional and
natural substances. The aims of the study was to investigate the bioactive compounds resulted from different form of Spirulian sp
(fresh and dried) quantitatively. The materials used were fresh and dried Spirulina sp which was extracted by ethanol. Analysis
were subjected for phytochemical screening, flavoniod, phenolic acid and antioxidant activity (DPPH scavenging activity). The
results showed that total flavonoid of fresh and dried are: 25.6615±1.62 and 110.1356±12.5 quercetin/gr extract ; phenolic acid :
2.117±0.99 and 6.92±0.03 GAE/gr extract; IC50 : 33.0755, respectively. Fresh sample of Spirulina platensis shows better results
in bioactive compounds quantitatively and qualitatively compared to that of dried sample, and drying process at 40-50oC does not
give any signi ficant different on nutritional quality of dried sample of Spirulina platensis compared to fresh samples.
© 2014 The Authors. Published by Elsevier B.V.
Peer-review under responsibility of scientific committee of the ICTCRED 2014.
Keywords: phenolic compound; flavonoi d compound; fresh and dried sampl e
* Corresponding author: tagustini@yahoo.com; Telp.: +6224-7474698
© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of scientific committee of the ICTCRED 2014
283
Tri Winarni Agustini et al. / Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
1. Introduction
Many researches have been conducted on bioactive compounds extracted from microalgae[1 – 4]. Some
bioactive compounds can act as natural antioxidant that has been considered as safe and more accepted compared to
that of synthetic antioxidant[5]. Spirulina sp include in the class of cyanobacterium that has been used as a source of
protein, vitamin supplements and health drinks [6 – 8]. Spirulina sp. has a wealth of perfect nutrition, contains 25
kinds of vitamins and minerals for the health and longevity, body regulation, removal of toxins and strengthen the
immune system[(5]
Spirulina platensis is one of cyanobacterium microalgae, multicelluler, and has spiral form that can grow
well in either seawater and fresh water. Spirulina also contains pigment of chlorophyl and carotenoids as well as
phenolic and flavonoid compounds which can act as natural antioxidant [5,8; 9]. Based on previous studies it is
known that the cells of Spirulina platensis biomass will be much more soluble in polar solvents, such as water and
buffer solution compared to the less polar solvents such as acetone or chloroform. The amount of solvent used in the
bioactive extraction greatly affect to the outcome of the extract including the yield obtained, and the purity and
stability of the extract. Spirulina sp. growing in Indonesia is expected to have more antioxidant compounds, because
such microalgae has ability to survive from UV radiation and extreme environmental condition by producing
metabolite seconder. It is able to protect human body from free radical damage, prevent from degenerative diseases
and retard lipid peroxidation on food[5].
Extraction of bioactive compound from micr oalgae can be done by several methods, in cludin g cold
extraction (maceration, sonication) and hot extraction (soxhletation, hot water extraction etc). Extraction method can
affect to the quality and quantity of nutritional and bioactive compounds extracted from microalgae. Soxhletation is
considered more efficient method because increase surface contact between solvent and material, resulting in higher
rendemen and more number of compounds extracted [10]. Therefore it is necessary to select the correct extraction
method used in order to get the optimum condition of extraction. State of the raw material will also affect the
number and quality of extracted compound inside the materials. This study were aimed to compare the nutritional
and bioactive compound (qualitative and quantitatively) extracted from different state of raw material (fresh and
dried Spirulin a).
2. Materials and Methods
Materials used is Spirulina platensis in two different form, fresh Spirulina harvested directly from the cultivation
area at Sukoharjo and dr ied Spirulina produced fr om fr esh Spirulina and is dried directly by usin g oven at
temperature 40oC for 10 hours to get final water content below 10%. These two form of Spirulina were then
extracted by using soxhletation (for dried Spirulina) and refluxion combined with sonication (for fresh Spirulina)
and analysed for the nutritional and bioactive compound of proximate analysis, flavonoid, phenolic and antioxidant
activity.
2.1. Extraction of Fresh Spirulina platensis
Fresh sample of Spirulina platensis was extracted by sonication at room temperature, 40 kHz for 1 hour to
destruction the cell wall and followed by Reflux at temperature of 50-60oC for 4 hours. Take 100 mL extract and is
added ethanol with ratio of 2:1 and is used for further analysis.
2.2. Extraction of Dried Spirulina platensis
Dried Spirulina was extracted by soxhletation. Take 100 g of dried sample that has been covered by filter paper and
cotton and put inside the soxhlete apparatus. Ethanol was used as extraction solvent.
2.3. Qualitatif test
The extract of Spirulina was subjected for phytoch emical screening test to observe bioactive compounds contained
in the sample qualitatively[11].
284 Tri Winarni Agustini et al. / Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
2.4. Quantitatif test
Quantitative test was conducted for total phenolic and flavonoid by using Folin-Ciocalteu method[12 – 14].
- Quantitative Analysis for Total Phenolic acid.
Calibration curve of Gallic acid was made as equivalent to total phenolic compound containing in the sample.
Concentration of Gallic acid standards were made by adjusting them according to the extract resulted. Take 0.05 mL
from each concentration, added 0.4 mL aquadest and 2 mL reagen Folin-Ciocalteu and then homogenized. The
homogenate was then left for 8 minutes before added with 1.6 mL Na2CO3 7.5%, and homogenized and left for
another 30 minutes at room temperature until blue colour is performed. The absorbance of this homogenate was
measured by spectrophotometer UV-Vis at 765 nm. The calibration curve was made by correlating gallic acid
concentration (mg/L) with absorbance. Total phenolic was calculated as mg equivalent Gallic acid / gram dried
sample.
- Quantitative Analysis for Total Flavonoid.
Quantitative analysis of Total flavonoid is made by linear regression curve of Quercetin. Quercetin was diluted in
ethanol and make some concentration of 20, 40, 60, 80 ppm. Take 0.5 mL from each concentration, homogenized
them after adding 1.5 mL methanol and 0.1 mL AlCl3 10%. Left the homogenate for 6 minutes, and then add 0.1 mL
Potasium acetate 1M and left for further 6 minutes. The homogenate is diluted up to 5 mL and left for 30 minutes
before measuring the absorbance by using spectrophotometer UV-Vis at 415 nm. Total flavonoid was calculated as
mg equivalen quercetin/gram dried sample.
2.5. Antioxidant activity test
Antioxidant activity test was conducted based on the method of Brand-Williams et al., (1995); waterhouse A (1999);
Orak HH (2006)
2.6. Analysis of proximate
Proximate analysis of Spirulina platensis was conducted on fresh and dried Spirulina by using AOAC (1995 and
1997) methods on water, ash, protein content, and fat content. Carbohydrate was calculated by difference.
3. Results and Discussion
3.1. Qualitative test
The results of phytochemical screening of Spirulina platensis with two different samples form (fresh and dried
Spirulin a) were presented in Table 1.
Table 1. Phytochemical screening Test of Spirulina platensis extract
Extract
Alkaloid
Phenolic
compound
Triterpenoid
and steroid
Flavonoid
Saponin
Dried
-
++
+++
++
++
Fresh
-
++
++
+
-
Note: - bioactive substance in the sample is not detected
+ bioactive substance is detected
Based on the results, there is a positive result for phenolic, triterpenoid and steroid, flavonoid on both samples,
except for saponin only positive on dried sample. Different phytochemical compound will results in their bioactive
activity[15]. Phytochemical compounds can be extracted by using suitable solvent. Polarity of solvent determine the
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Tri Winarni Agustini et al. / Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
extracted phytochemical compound. This study used ethanol, which is considered as polar solvent. It has be proven
that ethanol is safe for food, therefore it is recommended to use ethanol for microalgae extraction which will
consequently safe for further processing.
Alkaloid compound is semi polar and show negative result on ethanol extraction. Alkaloid show negative (not
detected) either on fresh and dried samples. The possibilities are due to alkaloid is considered as alkaline and tends
to difficult to extract its compounds by chemical solvent and there may no alkaloid compound in the samples.
On the other hand, phenolic compound showed positive result. Phenolic compound is considered as big moleculs
and is composed from various str uctures with main characteristic of aromatic chain that has hydroxyl[14]. Phenolic
is considered as acid with hydrogen can be easily remove. The most phenolic compound obtained in plants has low
antioxidant activity but there is no side effect resulted as do for synthetic antioxidant[5, 16].
Triterpenoid and steroid showed positive results and high content of such compound were found in the samples.
Triterpenoid and steroid are considered as non polar, therefor they can be extracted well by using non polar.
However, the results showed positive, eventhougt ethanol used as extraction solvent is considered as polar solvent.
This is because moment dipole of polar and semi polar compound will do induction to the non polar moleculs which
has no dipole, so that non polar compound can dissolve in semi polar and polar solvent. This statement is also
supported [17] who stated that one factor causing non polar compound dissolved in semi polar and polar solvent due
to there is moment dipol between polar and semi polar that will do induction non polar moleculs which has no
dipole so that there is electrostatic power resulted from polar and semi polar compounds.
Flavonoids is considered as polar compound and there is positive result for both samples. Ethanol as polar sovent
can dissolve the flavonoids present in the samples.
Saponin showed positive result from dried samples only. Saponin is a polar substance, so that it can be easily
dissolved in ethanol as polar solvent. But the presence of saponin in fresh sample is very small so that it can not be
detected qualitatively. Saponin mainly in form of glicoside and is considered as polar substance[18]. Formation of
foam in saponin test is a proven evident that saponin is present in the sample which can be converted into glucose
and other compounds. In addition, saponin is glycoside complex with high molecular weight and present in plants,
bacteria and low level animal[19].
3.2. Quantitative test
Total Phenolic and total flavonoid of Spirulina platensis extract form fresh and dried form were presente in
Table 2 and Table 3.
Table 2 . Quantitative test of total phenolic of Spirulina platensis extract (fresh and dried form)
Sample
Quercetin/g extract
Dried sample
Fresh sam ple
1st
2nd
Wet basis
Dried
basis
1st
2nd
Wet basis
Dried
basis
EEt
101,29
118,98
110,14 ± 12,50
119,43
26,81
24,52
25,66± 1,62
469,96
EEA
213,70
232,16
222,93 ± 13,05
241,74
27,39
21,90
24,65± 3,88
451,47
EA
182,02
225,87
203,94 ± 31
221,15
14,64
13,85
14,25± 0,58
260,99
Based on Table 2 and Table 3, it is obviously shown that fresh sample contained higher phenolic and flavonoid
compounds compared to that of dried sample quantitatively. The big differ ent of phenolic and flavonoid compoun ds
resulted from fresh and dried sample were mainly due to the diferent in water content between this two samples.
Fresh sample has water content almost 12 times (94.54%) than dried sample (7.78%). Flavonoid is polar compound,
so that it can be easily dissolve in water[20]. In addition, flavonoid generally is attached to sugar group which
consequently flavonoid can be easily dissolve in water or any other polar solvent[21].
286 Tri Winarni Agustini et al. / Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
Table 3. Quantitative test of total flavonoid of Spirulina platensis extract (fresh and dried form)
Type of
Solvent
GAE/g extract
Dried sample
Wet sample
1st
2nd
Wet basis
Dried basis
1st
2nd
Wet basis
Dried
basis
Eet
6,89
6,94
6,92± 0,03
7,50
2,82
1,41
2,11±0,99
38,64
EEA
15,43
5,49
10,46± 7,02
11,34
2,83
1,26
2,04±1,10
37,36
EA
19,69
8,10
13,89± 8,19
15,06
4,66
2,28
3,47±1,68
63,55
3.3. Antioxidant Activity
Antioxidant activity of the Spirulina platensis samples were analysed by using method of [12] and the results is
presented in Fig.1 and Fig.2. Antioxidant activity of the samples were determined by using DPPH (Diphenil pycril
hydrazil) method. Etil acetat is considered as semi polar solvent and suitable to extract the bioactive compound in
Spirulina [9]. However, the antioxidant activity resulted is very low. It means that the type of solvent not only
important for getting high yield but also it is very important to consider the activity of bioactive extracted.
Utilisation of other solvent is necessary to carried out, for example by using ethanol to extract bioactive compound
from Spirulina platensis may give better result for their activity.
Based on Fig.1 and 2, it is obvious that the higher concentration of extr act, the higher precentage of inhibition. This
is in line with the result that the higher concentration of extract, the higher percentage of inhibition[22]. This is
because on high cencentration of extr act, the higher antioxidant present in the sample so that consequently the level
of free radical inhibition by antioxidant contained in the sample is also higher. Antioxidant activity can be measured
by the number of reducing intencity purple color of DPPH which is linier with reduction of DPPH concentration.
[23]. Such reduction was due to reaction between DPPH and hydrogen atom released and characterised by changing
from purple to yellow color of DPPH.
Fig.1. Antioxidant activity of Spirulina sp (dried form)
y = 0.1083x + 32.281
R² = 0.9849
0
10
20
30
40
50
0 20 40 60 80 100 120
% Inhibition
Concentration (μg/mL)
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Tri Winarni Agustini et al. / Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
Fig.2. Antioxidant activity of Spirulina sp (fresh form)
The result of antioxidant activity of the samples by using DPPH scavenging activity method is represented by IC50,
meaning that concentration of extract required to inhibit 50% free radical DPPH. Antioxidant activity is performed
by IC50 which define as concentration of sample required to inhibit 50% free radical of DPPH[5, 24]. IC50 of
Spirulina platensis extracted with ethanol extraction for fresh and dried sample and quercetin as control is presented
in Table 4.
Table 4. IC50 of extracted Spirulina platensis in fresh and dried samples compared to quercetine.
Sample
IC50 (ppm)
Quercetine
21.616
Dried sample
33.075
Fresh sample
163.61
Based on Table 4, it is shown that IC50 of fresh and dried samples are different compared to quercetine. Dried
sample of Spirulina platensis perform high IC50 of 33.075 ppm, which is considered as very strong antioxidant. For
the fresh sample, IC50 is 163.61 ppm and it is considered as weak antioxidant. Both two samples have lower IC50
compared to Quercetine (21.616 ppm). Study has been conducted on DPPH scavenging activity of Spirulina extract
compared to Vitamin C and E which perform that IC50 was around 40 ppm that is comparable to our result[5].
According to research result, a compound can be characterized as very strong antioxidant (IC50 < 50 ppm), strong
antioxidant (IC50: 50 – 100 ppm), middle (IC50: 100 – 150 ppm), weak (IC50: 150 – 200 ppm), and very weak (IC50:
> 200 ppm). Moreover, Andyani et al.(2008) reported that a compound is considered to have antioxidant activity if it
has IC50 less than 200 ppm[22].
From this results, there is a correlation between antioxidant activity and phytochemical screening test in which dried
sample of Spirulina gave more higher extracted bioactive compound compared to fresh sample and consequently
perform high antioxidant activity.
3.4. Proximate analysis
Fresh sample and dried sample of Spirulina have different proximate data as shown on Table 5. Based on the results
it was obviously found that fresh sample perform comparatively the same nutritional value compared to that of dried
sample of Spirulina. Protein and ash content of dried sample is higher than fresh sample. In this case th e effect of
heating process applied during processin g of dried Spirulina does not give significant effect to nutritional decrease
of dried sample. The dried sample was subjected to heating process at 40-50oC for 10 hours, which is considered to
have very less effect to the nutritional quality of protein. Protein would subject to denaturation at temperature of
70oC which consequently results in nutritional damage of the sample. In this study, heating process took place at
fairly low temperature and thus processing can maintain the nutritional quality of dried sample.
y = 0.3167x + 39.525
R² = 0.9883
0
10
20
30
40
50
60
70
80
0 20 40 60 80 100 120
% Inhibition
Concentration (μg/mL)
288 Tri Winarni Agustini et al. / Procedia Environmental Sciences 23 ( 2015 ) 282 – 289
Table 5. Proximate analysis of Spirulina platensis (fresh and dried samples)
Concentration of compound
(%)
Dried sample
Fresh sample
Wet basi s
Dried basis
Wet basi s
Dried basis
Water
7.78
-
94.54
-
Protein
67.18
72.85
3.28
60.07
Lipid
2.64
2.86
0.27
4.94
Carbohydrate
11.74
12.73
1.47
26.92
Ash
10.66
11.56
0.44
8.06
However, this dr ying process gave effect to the number of bioactive compoun ds present in dried sample as shown
on Table 2 and Table 3. Dried sample perform to have less number of bioactive compounds compared to that of
fresh sample. This phenomenon can be described that some of bioactive compound are mainly heat-sensitive
compound, so that they will perform decreasing number due to heating process even at low temperature.
4. Conclusion
• Fresh sample of Spirulina platensis shows better results in bioactive compounds quantitatively and
qualitatively compared to that of dried sample, and
• Drying process at 40-50oC does not give any significant different on nutritional quality of dried sample of
Spirulina platensis compared to fresh samples.
5. Ackowledgement
We are ackowledged and thank you very much to Directorate Genderal of Higher Education for this honorable grant
and participation of Neoalgae in contributing Spirulina and cooperation for further work.
This study is supported by Directorate Genderal of Higher Education through National Competitve Grant of MP3EI,
fiscal year 2013-2014.
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