Antioxidant Activity of N-Hexane and Ethanol Extract of Sponge (Pseudosuberites andrewsi)
3RD SEMINAR OF PHARMACEUTICAL SCIENCE
AND TECHNOLOGY, MAY 31, 2014
Antioxidant Activity of N-Hexane and Ethanol Extract of
Sponge (Pseudosuberites andrewsi)
Oleh:
Azizah Nasution
Suwarti Aris
Rosidah
FACULTY OF PHARMACY
UNIVERSITY OF SUMATERA UTARA
MEDAN, INDONESIA
ANTIOXIDANT ACTIVITY OF n-HEXANE AND ETHANOL EXTRACTS
OF SPONGE (Pseudosuberites andrewsi)
Azizah Nasution*, Suwarti Aris, Rosidah
Faculty of Pharmacy, University of Sumatera Utara,
Medan, Indonesia
*E-mail: nasution.azizah4@gmail.com
Presented at 3rd Seminar of Pharmaceutical Science and Technology, Medan, May
31, 2014
ABSTRACT
Background: Sponges are the excellent sources of compounds with the potential
to function as drugs to treat variety of diseases.
Objective: This study aimed to examine in-vitro antioxidant activity of n-hexane
and ethanol extracts of sponge ( Pseudosuberites andrewsi).
Methods: The collected, cleaned, dried, and powdered sponge was extracted
using n-hexane and ethanol. Ascorbic acid was used as the reference. The
antioxidant activities of the produced extracts with varied concentrations were
examined by adding 1, 1-diphenyl-2-picrylhydrazyl (DPPH) as a radical
scavenger, and allowed for 60 minutes. The absorbances were measured using
ultraviolet visible spectrophotometer at wave length of 516 nm. The same
procedure was performed on vitamin C.
Results: n-hexane and ethanol extracts of sponge had half inhibitory
concentrations (IC50) of 779.05 μg/ml and 1043.29 μg/ml, respectively. Both
extracts had lower antioxidant activity compared to that of vitamin C.
Conclusions: n-hexane of extract of sponge had better antioxidant activity
compared to ethanol extract of the same sponge.
Keywords: Sponge, Antioxidant activity, n-hexane and ethanol extracts
INTRODUCTION
Sponges are the most primitive multicelluar organisms among the species within
the marine animal kingdom. They have existed about 800 years ago (Hentschel et
al, 2002). They are the excellence sources of bioactive metabolites like steroids
with many functions including prevention and treatment of diseases such as
asthma,
rheumatoid
arthritis,
and
variety
of
inflammatory
conditions
(Abdelmohsen et al, 2012). A study indicated that drug sponge bathing was able
to relieve fever caused by exogenous pathogenic factors of infants (Ren et al,
2012). They are also excellent sources of natural preservative of foods and anti
aging component in cosmetics (Suryanarayanan 2012).
Due to the potency of the bioactive secondary metabolites contained in sponges,
researchers in the globe continue undertaking studies on these unique organisms.
A study found that the collection side has an important influence on the
bioactivities of the sponges (Orhan, 2012). Indonesia contains the most diverse
and the largest mangroves area in the world. It reached 20.9% of the global
mangroves area (Spalding et al, 2010; Becking et al, 2013). Therefore research on
sponges from different areas of Indonesia is challenging.
This present study aimed to examine in vitro antioxidant activity of n-hexane and
ethanol extracts of sponge (Pseudosuberites andrewsi) obtained from the west
coastal area of Mandailing Natal, North Sumatera, Indonesia.
METHODS
Ethanol and n-hexane extract preparation
The fresh sponge ( Pseudosuberites andrewsi) used in this study was obtained
from the west coastal area of Mandailing Natal, North Sumatera, Indonesia. Prior
to the study, the sponge was identified at laboratory of ecology, Institute of
Technology Sepuluh November, Surabaya. The collected fresh sponge was first
washed to remove from adhering dirt and then completely dried by using oven at
temperature not more than 60⁰C. The dried sponge was pulverized and stored in a
tightly closed container for further use. As many as 125 grams of the pulverized
sponge were extracted with 2.5 liters of n-hexane for 7 days using a percolator to
separate the lipid soluble components. Its waste was extracted with 2.5 liters of
ethanol for 7 days to separate the crude extract of water-soluble components. Both
the n-hexane and ethanol phases were evaporated using a vacuum pump rotary
evaporator to produce n-hexane extract of sponge (n-HES) and ethanol extract of
sponge (EES), respectively.
Antioxidant activity test of n-HES and EES
N-HES and EES at varied concentrations (50, 100, 200, and 400 μg/ml) were
prepared in 25 ml-volumetric flasks. Their antioxidant activities were examined
with 40 μg/ml of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) by adding 5 ml of DPPH
200 μg/ml as a radical scavenger, and allowed for 60 minutes (Molyneux, 2004).
Their absorbances were measured using ultraviolet visible spectrophotometer at
wave length of 516 nm. The same procedure was performed on vitamin C.
Inhibition of DPPH (%) was calculated as follows
Inhibition (%) = (Ablank – Asample)/Ablank x 100%
in which:
Ablank = absorbance of the blank
Asample = absorbance of the sample
Linear regression analysis was performed to obtain the association between
absorbances and inhibited concentrations of n-HES and EES. The half inhibitory
concentration (IC50) of each of the extracts was calculated by applying the linear
equation obtained in the regression analysis. The same procedure was performed
for vitamin C to obtain its IC50 . The lower the IC50 obtained, the higher the
scavenging activity.
RESULTS AND DISCUSSION
Based on the identification of the sponge performed at laboratory of ecology,
Institute of Technology Sepuluh November, Surabaya indicated that the
systematic of the sponge is as follows:
Kongdom
: Animalia
Phylum
: Porifera
Class
: Demospongiae
Order
: Hadromerida
Familia
: Suberitidae
Genus
: Pseudosuberites
Species
: Pseudosuberites andrewsi
Photograph of the sponge is shown in Figure 1. As shown in Figure 1, it has a pale
yellow color with fragile consistency. Its height reaches about 40 millimeters and
width of about 25 millimeters. Sponges have numerous tiny pores on their surface.
Figure 1. Photograph of Pseudosuberites andrewsi
Eight hundred and sixty grams of the fresh sponge resulted in 269 grams (31%) of
dried sponge. One hundred and twenty five grams of the dried sponge resulted in
2.875 grams of n-hexane extract. The n-hexane extract had a yellow brownish
color with thick consistency. The ethanol extract produced from extraction of the
waste of n-hexane extraction was 3.560 grams with dark brown color and thick
consistency.
The linear equations obtained for the n-HES and EES were y = 0.060485x +
2.87925 and y = 0.048630x + 0.52650, respectively. The N-HES and EES had
half inhibitory concentrations of 779.05 μg/ml and 1043.29 μg/ml, respectively.
The difference in their scavenging activities may be affected by the compounds
extracted into both n-hexane and ethanol. Further studies are required to elucidate
chemical structures extracted into both extracts. Vitamin C had IC50 of 3.8 μg/mL.
Both N-HES and EES had lower antioxidant activity compared to that of vitamin
C. Previous studies proved that the bioactivities of the sponges are associated with
the collection site (Orhan et al., 2012). Studies to identify factors associated with
the induction the secondary metabolite production are still limited (Koopmans et
al, 2009).
However, few studies indicated that bioactive metabolite
concentrations differ from one environment to another. Sponges are the hosts for a
large community of microorganisms, which reach up to 50 to 60% of the biomass
of the sponge host. Subsequently, it has been reported that the produced
therapeutically bioactive compounds are associated with the microbes and fungi
that colonized the sponges, along with the geographic location (Remya et al,
2010). The present study was only an initial exploration of the possible potency of
sponge as a source of antioxidant. Though, this study provides useful information
to consider the future selection of sponge collection site as raw material in drug
development. Isolation of active compounds extracted into N-HES and EES as
well as well as their pharmacologic effects need to be performed.
CONCLUSIONS
Based on the present study, it could be concluded that:
1) The sponge studied is Pseudosuberites andrewsi with pale yellow color.
Its height reaches about 40 millimeters, width of about 25 millimeters and
fragile consistency.
2) N-hexane extract of the sponge had better antioxidant activity compared to
those of ethanol extract.
REFERENCES
1. Abdelmohsen, U. R., Szesny, M., Othman, E. M., Schirmeister, T., Grond, S.,
Stopper, H., & Hentschel, U. (2012). Antioxidant and anti-protease activities
of diazepinomicin from the sponge-associated Micromonospora strain
RV115.Marine drugs, 10 (10), 2208-2221.
2. Becking, L. E., Cleary, D. F., & de Voogd, N. J. (2013). Sponge species
composition, abundance, and cover in marine lakes and coastal mangroves in
Berau, Indonesia.
3. Ganjar, I.G., and Rohman, A. (2007). Kimia Farmasi Analisis , Yogyakarta:
Pustaka Pelajar. Page 220-222.
4. Hentschel, U., Hopke, J., Horn, M., Friedrich, A. B., Wagner, M., Hacker, J.,
& Moore, B. S. (2002). Molecular evidence for a uniform microbial
community in sponges from different oceans. Applied and Environmental
Microbiology, 68 (9), 4431-4440.
5. Koopmans, M., Martens, D., & Wijffels, R. H. (2009). Towards commercial
production of sponge medicines. Marine drugs, 7 (4), 787-802.
6. Molyneux, P. (2004). The use of the stable free radical diphenylpicrylhydrazyl
(DPPH)
for
estimating
antioxidant
activity. Songklanakarin
J
Sci
Technol, 26 (2), 211-219.
7. Orhan, I. E., Ozcelik, B., Konuklugil, B., Putz, A., Kaban, U. G., & Proksch,
P. (2012). Bioactivity Screening of the Selected Turkish Marine Sponges and
Three Compounds from Agelas oroides. Records of Natural Products , 6 (4).
8. Thomas, T. R. A., Kavlekar, D. P., & LokaBharathi, P. A. (2010). Marine
drugs from sponge-microbe association—A review. Marine Drugs , 8 (4),
1417-1468.
9. Ren, X. Q., Xiang, Y. Q., & MA, J. H. (2012). Clinical Study of Drug Sponge
Bathing for Reliveing Fever of Wind-Heat Caused by Exogenous Pathogenic
Factors for Infants. Chinese Archives of Traditional Chinese Medicine , 11 ,
036.
10. Rosidah, Yam, M.F., Sadikun, A., dan Asmawi, M.Z. (2008). Antioxidant
Potential of Gynura procumbens. Pharmaceutical Biology. 46(9): 616-625.
11. Spalding, M., Kainuma, M., & Collins, L. (2010). World atlas of mangroves .
Earth scan.
12. Suryanarayanan, T. S. (2012). The diversity and importance of fungi
associated with marine sponges.
AND TECHNOLOGY, MAY 31, 2014
Antioxidant Activity of N-Hexane and Ethanol Extract of
Sponge (Pseudosuberites andrewsi)
Oleh:
Azizah Nasution
Suwarti Aris
Rosidah
FACULTY OF PHARMACY
UNIVERSITY OF SUMATERA UTARA
MEDAN, INDONESIA
ANTIOXIDANT ACTIVITY OF n-HEXANE AND ETHANOL EXTRACTS
OF SPONGE (Pseudosuberites andrewsi)
Azizah Nasution*, Suwarti Aris, Rosidah
Faculty of Pharmacy, University of Sumatera Utara,
Medan, Indonesia
*E-mail: nasution.azizah4@gmail.com
Presented at 3rd Seminar of Pharmaceutical Science and Technology, Medan, May
31, 2014
ABSTRACT
Background: Sponges are the excellent sources of compounds with the potential
to function as drugs to treat variety of diseases.
Objective: This study aimed to examine in-vitro antioxidant activity of n-hexane
and ethanol extracts of sponge ( Pseudosuberites andrewsi).
Methods: The collected, cleaned, dried, and powdered sponge was extracted
using n-hexane and ethanol. Ascorbic acid was used as the reference. The
antioxidant activities of the produced extracts with varied concentrations were
examined by adding 1, 1-diphenyl-2-picrylhydrazyl (DPPH) as a radical
scavenger, and allowed for 60 minutes. The absorbances were measured using
ultraviolet visible spectrophotometer at wave length of 516 nm. The same
procedure was performed on vitamin C.
Results: n-hexane and ethanol extracts of sponge had half inhibitory
concentrations (IC50) of 779.05 μg/ml and 1043.29 μg/ml, respectively. Both
extracts had lower antioxidant activity compared to that of vitamin C.
Conclusions: n-hexane of extract of sponge had better antioxidant activity
compared to ethanol extract of the same sponge.
Keywords: Sponge, Antioxidant activity, n-hexane and ethanol extracts
INTRODUCTION
Sponges are the most primitive multicelluar organisms among the species within
the marine animal kingdom. They have existed about 800 years ago (Hentschel et
al, 2002). They are the excellence sources of bioactive metabolites like steroids
with many functions including prevention and treatment of diseases such as
asthma,
rheumatoid
arthritis,
and
variety
of
inflammatory
conditions
(Abdelmohsen et al, 2012). A study indicated that drug sponge bathing was able
to relieve fever caused by exogenous pathogenic factors of infants (Ren et al,
2012). They are also excellent sources of natural preservative of foods and anti
aging component in cosmetics (Suryanarayanan 2012).
Due to the potency of the bioactive secondary metabolites contained in sponges,
researchers in the globe continue undertaking studies on these unique organisms.
A study found that the collection side has an important influence on the
bioactivities of the sponges (Orhan, 2012). Indonesia contains the most diverse
and the largest mangroves area in the world. It reached 20.9% of the global
mangroves area (Spalding et al, 2010; Becking et al, 2013). Therefore research on
sponges from different areas of Indonesia is challenging.
This present study aimed to examine in vitro antioxidant activity of n-hexane and
ethanol extracts of sponge (Pseudosuberites andrewsi) obtained from the west
coastal area of Mandailing Natal, North Sumatera, Indonesia.
METHODS
Ethanol and n-hexane extract preparation
The fresh sponge ( Pseudosuberites andrewsi) used in this study was obtained
from the west coastal area of Mandailing Natal, North Sumatera, Indonesia. Prior
to the study, the sponge was identified at laboratory of ecology, Institute of
Technology Sepuluh November, Surabaya. The collected fresh sponge was first
washed to remove from adhering dirt and then completely dried by using oven at
temperature not more than 60⁰C. The dried sponge was pulverized and stored in a
tightly closed container for further use. As many as 125 grams of the pulverized
sponge were extracted with 2.5 liters of n-hexane for 7 days using a percolator to
separate the lipid soluble components. Its waste was extracted with 2.5 liters of
ethanol for 7 days to separate the crude extract of water-soluble components. Both
the n-hexane and ethanol phases were evaporated using a vacuum pump rotary
evaporator to produce n-hexane extract of sponge (n-HES) and ethanol extract of
sponge (EES), respectively.
Antioxidant activity test of n-HES and EES
N-HES and EES at varied concentrations (50, 100, 200, and 400 μg/ml) were
prepared in 25 ml-volumetric flasks. Their antioxidant activities were examined
with 40 μg/ml of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) by adding 5 ml of DPPH
200 μg/ml as a radical scavenger, and allowed for 60 minutes (Molyneux, 2004).
Their absorbances were measured using ultraviolet visible spectrophotometer at
wave length of 516 nm. The same procedure was performed on vitamin C.
Inhibition of DPPH (%) was calculated as follows
Inhibition (%) = (Ablank – Asample)/Ablank x 100%
in which:
Ablank = absorbance of the blank
Asample = absorbance of the sample
Linear regression analysis was performed to obtain the association between
absorbances and inhibited concentrations of n-HES and EES. The half inhibitory
concentration (IC50) of each of the extracts was calculated by applying the linear
equation obtained in the regression analysis. The same procedure was performed
for vitamin C to obtain its IC50 . The lower the IC50 obtained, the higher the
scavenging activity.
RESULTS AND DISCUSSION
Based on the identification of the sponge performed at laboratory of ecology,
Institute of Technology Sepuluh November, Surabaya indicated that the
systematic of the sponge is as follows:
Kongdom
: Animalia
Phylum
: Porifera
Class
: Demospongiae
Order
: Hadromerida
Familia
: Suberitidae
Genus
: Pseudosuberites
Species
: Pseudosuberites andrewsi
Photograph of the sponge is shown in Figure 1. As shown in Figure 1, it has a pale
yellow color with fragile consistency. Its height reaches about 40 millimeters and
width of about 25 millimeters. Sponges have numerous tiny pores on their surface.
Figure 1. Photograph of Pseudosuberites andrewsi
Eight hundred and sixty grams of the fresh sponge resulted in 269 grams (31%) of
dried sponge. One hundred and twenty five grams of the dried sponge resulted in
2.875 grams of n-hexane extract. The n-hexane extract had a yellow brownish
color with thick consistency. The ethanol extract produced from extraction of the
waste of n-hexane extraction was 3.560 grams with dark brown color and thick
consistency.
The linear equations obtained for the n-HES and EES were y = 0.060485x +
2.87925 and y = 0.048630x + 0.52650, respectively. The N-HES and EES had
half inhibitory concentrations of 779.05 μg/ml and 1043.29 μg/ml, respectively.
The difference in their scavenging activities may be affected by the compounds
extracted into both n-hexane and ethanol. Further studies are required to elucidate
chemical structures extracted into both extracts. Vitamin C had IC50 of 3.8 μg/mL.
Both N-HES and EES had lower antioxidant activity compared to that of vitamin
C. Previous studies proved that the bioactivities of the sponges are associated with
the collection site (Orhan et al., 2012). Studies to identify factors associated with
the induction the secondary metabolite production are still limited (Koopmans et
al, 2009).
However, few studies indicated that bioactive metabolite
concentrations differ from one environment to another. Sponges are the hosts for a
large community of microorganisms, which reach up to 50 to 60% of the biomass
of the sponge host. Subsequently, it has been reported that the produced
therapeutically bioactive compounds are associated with the microbes and fungi
that colonized the sponges, along with the geographic location (Remya et al,
2010). The present study was only an initial exploration of the possible potency of
sponge as a source of antioxidant. Though, this study provides useful information
to consider the future selection of sponge collection site as raw material in drug
development. Isolation of active compounds extracted into N-HES and EES as
well as well as their pharmacologic effects need to be performed.
CONCLUSIONS
Based on the present study, it could be concluded that:
1) The sponge studied is Pseudosuberites andrewsi with pale yellow color.
Its height reaches about 40 millimeters, width of about 25 millimeters and
fragile consistency.
2) N-hexane extract of the sponge had better antioxidant activity compared to
those of ethanol extract.
REFERENCES
1. Abdelmohsen, U. R., Szesny, M., Othman, E. M., Schirmeister, T., Grond, S.,
Stopper, H., & Hentschel, U. (2012). Antioxidant and anti-protease activities
of diazepinomicin from the sponge-associated Micromonospora strain
RV115.Marine drugs, 10 (10), 2208-2221.
2. Becking, L. E., Cleary, D. F., & de Voogd, N. J. (2013). Sponge species
composition, abundance, and cover in marine lakes and coastal mangroves in
Berau, Indonesia.
3. Ganjar, I.G., and Rohman, A. (2007). Kimia Farmasi Analisis , Yogyakarta:
Pustaka Pelajar. Page 220-222.
4. Hentschel, U., Hopke, J., Horn, M., Friedrich, A. B., Wagner, M., Hacker, J.,
& Moore, B. S. (2002). Molecular evidence for a uniform microbial
community in sponges from different oceans. Applied and Environmental
Microbiology, 68 (9), 4431-4440.
5. Koopmans, M., Martens, D., & Wijffels, R. H. (2009). Towards commercial
production of sponge medicines. Marine drugs, 7 (4), 787-802.
6. Molyneux, P. (2004). The use of the stable free radical diphenylpicrylhydrazyl
(DPPH)
for
estimating
antioxidant
activity. Songklanakarin
J
Sci
Technol, 26 (2), 211-219.
7. Orhan, I. E., Ozcelik, B., Konuklugil, B., Putz, A., Kaban, U. G., & Proksch,
P. (2012). Bioactivity Screening of the Selected Turkish Marine Sponges and
Three Compounds from Agelas oroides. Records of Natural Products , 6 (4).
8. Thomas, T. R. A., Kavlekar, D. P., & LokaBharathi, P. A. (2010). Marine
drugs from sponge-microbe association—A review. Marine Drugs , 8 (4),
1417-1468.
9. Ren, X. Q., Xiang, Y. Q., & MA, J. H. (2012). Clinical Study of Drug Sponge
Bathing for Reliveing Fever of Wind-Heat Caused by Exogenous Pathogenic
Factors for Infants. Chinese Archives of Traditional Chinese Medicine , 11 ,
036.
10. Rosidah, Yam, M.F., Sadikun, A., dan Asmawi, M.Z. (2008). Antioxidant
Potential of Gynura procumbens. Pharmaceutical Biology. 46(9): 616-625.
11. Spalding, M., Kainuma, M., & Collins, L. (2010). World atlas of mangroves .
Earth scan.
12. Suryanarayanan, T. S. (2012). The diversity and importance of fungi
associated with marine sponges.