1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 349405
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1 Faculty of Pharmacy and Sciences,
Universitas Muhammadiyah Prof.
DR. HAMKA, Islamic Center, Jl.
Delima II/IV, Perumnas Klender,
East Jakarta, 13460, Indonesia
*Corresponding
Ni Putu Ermi Hikmawanti
ermy0907@uhamka.ac.id
Received: 16 April 2022
Accepted: 28 April 2023
Published: 08 May 2023
The effect of extraction methods on total
phenolics and antioxidant activities in
Caulerpa racemosa (Forsskal) J. Agardh
extracts
Efecto de los métodos de extracción sobre los fenoles
totales y las actividades antioxidantes de los extractos de
Caulerpa racemosa (Forsskal) J. Agardh
Sherley , Ni Putu Ermi Hikmawanti* , Mutia Agustin
ABSTRACT
Background: The edible green algae Caulerpa racemosa (Forsskal) J. Agardh (Caulerpaceae),
also known as “sea grape”, is an excellent source of phenolic compounds known for their
activity to reduce free radicals. Objectives: The research aims to evaluate the total phenolic
content and antioxidant activity of C. racemosa (70% ethanol extracts) obtained from different
extraction methods, such as maceration, Soxhlet, and ultrasound. Methods: Total phenolics of
the extracts were determined by the colorimetry method using the Folin-Ciocalteu reagent.
Total phenol content was expressed as mg gallic acid equivalent (GAE) per g extract. The
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used to assess the antioxidant activity
of the extracts. Results: the ethanol extract of C. racemosa obtained from the ultrasonic
methods had the highest phenolic content (39.38 mg GAE/g) compared to other extraction
methods (Soxhlet was 37.31 mg GAE/g and maceration was 22.05 mg GAE/g). The IC 50 value
against DPPH of the C. racemosa ethanol extracts using ultrasonic, Soxhlet, and maceration
was 352.95, 365.73, and 375.84 μg/mL, respectively. Conclusions: the variation of the extraction
methods affected the total phenolics content of C. racemosa ethanol extracts and their
antioxidant activity. We reported here the potential of C. racemosa extracts as an antioxidant
raw material from marine plants for medicinal, nutraceutical, cosmetics, and food products;
however, more research is needed.
Keywords: DPPH, Maceration, Soxhlet, Ultrasound, Marine biota, Phenolic compounds, Sea
grape
ORIGINAL ARTICLE
Published 08 May 2023
Doi: https://doi.org/10.17533/udea.vitae.v30n2a349405
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1 Faculty of Pharmacy and Sciences,
Universitas Muhammadiyah Prof.
DR. HAMKA, Islamic Center, Jl.
Delima II/IV, Perumnas Klender,
East Jakarta, 13460, Indonesia
*Corresponding
Ni Putu Ermi Hikmawanti
ermy0907@uhamka.ac.id
Received: 16 April 2022
Accepted: 28 April 2023
Published: 08 May 2023
The effect of extraction methods on total
phenolics and antioxidant activities in
Caulerpa racemosa (Forsskal) J. Agardh
extracts
Efecto de los métodos de extracción sobre los fenoles
totales y las actividades antioxidantes de los extractos de
Caulerpa racemosa (Forsskal) J. Agardh
Sherley , Ni Putu Ermi Hikmawanti* , Mutia Agustin
ABSTRACT
Background: The edible green algae Caulerpa racemosa (Forsskal) J. Agardh (Caulerpaceae),
also known as “sea grape”, is an excellent source of phenolic compounds known for their
activity to reduce free radicals. Objectives: The research aims to evaluate the total phenolic
content and antioxidant activity of C. racemosa (70% ethanol extracts) obtained from different
extraction methods, such as maceration, Soxhlet, and ultrasound. Methods: Total phenolics of
the extracts were determined by the colorimetry method using the Folin-Ciocalteu reagent.
Total phenol content was expressed as mg gallic acid equivalent (GAE) per g extract. The
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used to assess the antioxidant activity
of the extracts. Results: the ethanol extract of C. racemosa obtained from the ultrasonic
methods had the highest phenolic content (39.38 mg GAE/g) compared to other extraction
methods (Soxhlet was 37.31 mg GAE/g and maceration was 22.05 mg GAE/g). The IC 50 value
against DPPH of the C. racemosa ethanol extracts using ultrasonic, Soxhlet, and maceration
was 352.95, 365.73, and 375.84 μg/mL, respectively. Conclusions: the variation of the extraction
methods affected the total phenolics content of C. racemosa ethanol extracts and their
antioxidant activity. We reported here the potential of C. racemosa extracts as an antioxidant
raw material from marine plants for medicinal, nutraceutical, cosmetics, and food products;
however, more research is needed.
Keywords: DPPH, Maceration, Soxhlet, Ultrasound, Marine biota, Phenolic compounds, Sea
grape
ORIGINAL ARTICLE
Published 08 May 2023
Doi: https://doi.org/10.17533/udea.vitae.v30n2a349405
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 349405Sherley, Ni Putu Ermi Hikmawanti, Mutia Agustin
INTRODUCTION
Substances with one or more unpaired electrons in
the outer orbital are known as free radicals (reactive
oxygen and nitrogen species) (1). The presence of
unpaired electrons makes them reactive, looking
for a partner by attacking and binding electrons to
molecules around it, including cellular components
such as lipids, lipoproteins, proteins, carbohydrates,
ribonucleic acid (RNA), and deoxyribonucleic acid
(DNA) (2). A further consequence of free radical
reactivity is damage to cell structure and function.
This state can lead to oxidative stress, conducting
to illnesses including degenerative diseases,
cardiovascular problems, aging, and cancer (3).
Antioxidants can quench free radicals. The human
body produces antioxidant enzymes, such as
superoxide dismutase (SOD), catalase (CAT), and
glutathione peroxidase (GPx) (1). However, under
oxidative stress conditions, the number of free
radicals is greater than the antioxidants produced
by the body (4). Therefore, antioxidants should be
consumed, whether they are of natural or synthetic
origin (2). Although, when synthetic antioxidants
are used, the potential for toxicity and adverse
consequences must be considered (4). Natural
antioxidants such as phenolics and their derivatives
can be easily found in natural sources, especially
plants (2) or potential species such as those from
the sea (5).
Exploration of the sea and marine biodiversity
provides a new sector in the search for novel natural
products with health advantages for humans (6). The
chlorophyll seaweed Caulerpa racemosa (Forsskal)
J. Agardh is abundant on the coast of Indonesia,
where it is traditionally used as fresh vegetables
(lalapan) and consumed by residents and fishermen
(7). C. racemosa contains phenolic compounds
(8), caulerpenyne, lipids, proteins, and minerals,
making seaweed a potential functional food (7). The
biological activity of C. racemosa has been widely
documented, including larvicidal, antibacterial (9),
and antioxidant (8) properties.
Extraction is the initial stage in studying the
phytochemicals of natural materials (10). The choice
of extraction process must ensure the successful
extraction of the target molecule (11) and maintain
the quality of the resulting extract (12). Different
extraction methods can be used to obtain antioxidant
compounds, both conventional and unconventional.
Conventional extraction methods (including
maceration, percolation, Soxhlet extraction, and
reflux) are often employed to extract phytochemicals
from natural origins, including marine (13). However,
unconventional extractions such as supercritical
carbon dioxide, microwave-assisted, and ultrasonic
extraction have been widely developed and used
to extract natural compounds from seaweed (14).
Information on the best extraction method to
obtain maximum phenolic content in C. racemosa
is still limited. Thus, in this study, the extraction
procedure of C. racemosa was carried out using
several extraction methods, along with testing the
antioxidant activity of the extract.
RESUMEN
Antecedentes: El alga verde comestible Caulerpa racemosa (Forsskal) J. Agardh (Caulerpaceae), también conocida como
“uva de mar”, es una excelente fuente de compuestos fenólicos conocidos por su actividad para reducir los radicales libres.
Objetivos: La investigación pretende evaluar el contenido fenólico total y la actividad antioxidante de C. racemosa (extractos
de etanol al 70%) obtenidos a partir de diferentes métodos de extracción, como maceración, Soxhlet y ultrasonido. Métodos:
Los fenoles totales de los extractos se determinaron por el método colorimétrico utilizando el reactivo de Folin-Ciocalteu. El
contenido total de fenoles se expresó como mg de ácido gálico equivalente (GAE) por g de extracto. Para evaluar la actividad
antioxidante de los extractos se utilizó el radical libre 2,2-difenil-1-picrilhidrazilo (DPPH). Resultados: el extracto etanólico
de C. racemosa obtenido por ultrasonido presentó el mayor contenido fenólico (39,38 mg GAE/g) en comparación con otros
métodos de extracción (Soxhlet fue de 37,31 mg GAE/g y maceración fue de 22,05 mg GAE/g). El valor IC 50 frente a DPPH de
los extractos etanólicos de C. racemosa mediante ultrasonido, Soxhlet y maceración fue de 352,95, 365,73 y 375,84 μg/mL,
respectivamente. Conclusiones: la variación de los métodos de extracción afectó al contenido total de fenoles de los extractos
etanólicos de C. racemosa y a su actividad antioxidante. Aquí reportamos el potencial de los extractos de C. racemosa como
materia prima antioxidante a partir de plantas marinas para productos medicinales, nutracéuticos, cosméticos y alimenticios;
sin embargo, se necesita más investigación.
Palabras clave: DPPH, maceración, Soxhlet, ultrasonido, biota marina, compuestos fenólicos, uva de mar.
INTRODUCTION
Substances with one or more unpaired electrons in
the outer orbital are known as free radicals (reactive
oxygen and nitrogen species) (1). The presence of
unpaired electrons makes them reactive, looking
for a partner by attacking and binding electrons to
molecules around it, including cellular components
such as lipids, lipoproteins, proteins, carbohydrates,
ribonucleic acid (RNA), and deoxyribonucleic acid
(DNA) (2). A further consequence of free radical
reactivity is damage to cell structure and function.
This state can lead to oxidative stress, conducting
to illnesses including degenerative diseases,
cardiovascular problems, aging, and cancer (3).
Antioxidants can quench free radicals. The human
body produces antioxidant enzymes, such as
superoxide dismutase (SOD), catalase (CAT), and
glutathione peroxidase (GPx) (1). However, under
oxidative stress conditions, the number of free
radicals is greater than the antioxidants produced
by the body (4). Therefore, antioxidants should be
consumed, whether they are of natural or synthetic
origin (2). Although, when synthetic antioxidants
are used, the potential for toxicity and adverse
consequences must be considered (4). Natural
antioxidants such as phenolics and their derivatives
can be easily found in natural sources, especially
plants (2) or potential species such as those from
the sea (5).
Exploration of the sea and marine biodiversity
provides a new sector in the search for novel natural
products with health advantages for humans (6). The
chlorophyll seaweed Caulerpa racemosa (Forsskal)
J. Agardh is abundant on the coast of Indonesia,
where it is traditionally used as fresh vegetables
(lalapan) and consumed by residents and fishermen
(7). C. racemosa contains phenolic compounds
(8), caulerpenyne, lipids, proteins, and minerals,
making seaweed a potential functional food (7). The
biological activity of C. racemosa has been widely
documented, including larvicidal, antibacterial (9),
and antioxidant (8) properties.
Extraction is the initial stage in studying the
phytochemicals of natural materials (10). The choice
of extraction process must ensure the successful
extraction of the target molecule (11) and maintain
the quality of the resulting extract (12). Different
extraction methods can be used to obtain antioxidant
compounds, both conventional and unconventional.
Conventional extraction methods (including
maceration, percolation, Soxhlet extraction, and
reflux) are often employed to extract phytochemicals
from natural origins, including marine (13). However,
unconventional extractions such as supercritical
carbon dioxide, microwave-assisted, and ultrasonic
extraction have been widely developed and used
to extract natural compounds from seaweed (14).
Information on the best extraction method to
obtain maximum phenolic content in C. racemosa
is still limited. Thus, in this study, the extraction
procedure of C. racemosa was carried out using
several extraction methods, along with testing the
antioxidant activity of the extract.
RESUMEN
Antecedentes: El alga verde comestible Caulerpa racemosa (Forsskal) J. Agardh (Caulerpaceae), también conocida como
“uva de mar”, es una excelente fuente de compuestos fenólicos conocidos por su actividad para reducir los radicales libres.
Objetivos: La investigación pretende evaluar el contenido fenólico total y la actividad antioxidante de C. racemosa (extractos
de etanol al 70%) obtenidos a partir de diferentes métodos de extracción, como maceración, Soxhlet y ultrasonido. Métodos:
Los fenoles totales de los extractos se determinaron por el método colorimétrico utilizando el reactivo de Folin-Ciocalteu. El
contenido total de fenoles se expresó como mg de ácido gálico equivalente (GAE) por g de extracto. Para evaluar la actividad
antioxidante de los extractos se utilizó el radical libre 2,2-difenil-1-picrilhidrazilo (DPPH). Resultados: el extracto etanólico
de C. racemosa obtenido por ultrasonido presentó el mayor contenido fenólico (39,38 mg GAE/g) en comparación con otros
métodos de extracción (Soxhlet fue de 37,31 mg GAE/g y maceración fue de 22,05 mg GAE/g). El valor IC 50 frente a DPPH de
los extractos etanólicos de C. racemosa mediante ultrasonido, Soxhlet y maceración fue de 352,95, 365,73 y 375,84 μg/mL,
respectivamente. Conclusiones: la variación de los métodos de extracción afectó al contenido total de fenoles de los extractos
etanólicos de C. racemosa y a su actividad antioxidante. Aquí reportamos el potencial de los extractos de C. racemosa como
materia prima antioxidante a partir de plantas marinas para productos medicinales, nutracéuticos, cosméticos y alimenticios;
sin embargo, se necesita más investigación.
Palabras clave: DPPH, maceración, Soxhlet, ultrasonido, biota marina, compuestos fenólicos, uva de mar.
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 349405
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
MATERIALS AND METHODS
Powder preparation of C. racemosa
C. racemosa collected from around the Taman
Mangrove Beach (5°58’30.5” S 106°42’14.2” E), the
northern region of Untung Jawa Island, Kepulauan
Seribu, Indonesia, in February 2020. C. racemosa
was determined at the Research Center for
Oceanography, Indonesian Institute of Sciences,
Jakarta, Indonesia (B-599/IPK.2/IF.07/VII/2020).
Fresh C. racemosa (12 Kg) was cleaned using running
water. The material was dried in direct sunlight with a
black cloth. After drying, the material was powdered
and sieved (15).
Extraction Procedure
1. Maceration
The dried powder (150 g) was put in a glass bottle.
A 70% ethanol solvent was used to macerate
and extract the powder components at a 1:10
powder-solvent ratio (1.5 L). The mixture was
stirred occasionally for the first six h and then left
to stand. After 24 h, the filtrate was collected, and
the residue was re-macerated with a new solvent
for five repetitions (15).
2. Ultrasonic
The dried powder (150 g) was put into a beaker
glass and extracted with 70% ethanol solvent with
the same sample-solvent ratio as in the maceration
method. The extraction process was carried out for
60 min at 30 °C using an ultrasonic bath (Branson
5510, Marshall Scientific LLC., USA) with a frequency
of 40 kHz. The filtrate was collected, and the
residue was re-extracted using a new solvent for
five repetitions (16).
3. Soxhlet extraction
The dried powder (150 g) was extracted with
250 mL of 70% ethanol as a solvent in a Soxhlet
apparatus. The extraction process was carried out
at 70 °C. Extraction was stopped after the color of
the solvent on the siphon became clear. The filtrate
was collected. The extraction was conducted with
five repetitions (15).
The filtrate obtained from each extraction method
was then concentrated using a vacuum rotary
evaporator (Eyela, Shanghai, China) at 50 °C,
followed by a water bath at the same temperature
until a concentrated dry extract was obtained and
weighed. The yield extraction percentage was
calculated by dividing the weight of the extract
obtained (g) against the dried powder of C.
racemosa (g) multiplied by 100%.
Determination of extract characteristics
We determined the extracts’ characteristics, such
as organoleptic, moisture content, and total ash.
The organoleptic examination includes color, odor,
taste, and texture. Organoleptic observations were
made after the extract was exposed to air for 15
min using the senses (eyes, nose, and tongue)
(17). Determination of water content was carried
out by the gravimetric method using an oven at
105 °C. The total ash content was conducted by
burning 2 g of the extract in a kiln at 600 °C for
five h until it became white ash. These evaluations
conform to the procedures of Indonesian Herbal
Pharmacopoeia (2017) (15) and the World Health
Organization (2011) (12).
Phytochemical Screening
Identification of the chemical content (viz. phenolics,
flavonoids, saponins, triterpenoids/steroids) in the
extracts was carried out following the procedure on
Harborne (1987) (18). Identification of phenolics was
carried out using 5% (w/v) FeCl3 reagent, flavonoids
using magnesium powder and concentrated
hydrochloric acid, saponins using the foam test in
water, and triterpenoids/steroids using Salkowski
(chloroform and concentrated sulfuric acid) and
Liebermann Burchard reagents (chloroform,
concentrated sulfuric acid, and anhydrous acetic
acid). Identification of alkaloids was carried out with
Dragendorff, Mayer, and Hager reagents according
to procedures on Departemen Kesehatan RI (2000)
(19) and tannins using 1% (w/v) gelatin adjusted to
the procedure on Hanani (2015) (20).
Determination of phenolic levels
Determining phenolic levels was carried out using
gallic acid as a standard. This procedure was
initiated by preparing gallic acid stock solution
(Sigma Aldrich, Saint Louis, USA) (500 μg/mL) in
ethanol and an extract stock solution (1000 μg/mL)
in ethanol. Serial concentrations of gallic acid were
prepared at 11, 19, 27, 35, and 43 μg/mL in ethanol
to determine the calibration curve. Meanwhile, the
extract test solution was prepared at 100 μg/mL.
This test solution was diluted from the extract stock
solution. Test procedure brief description: the test
solution of both gallic acid and extract (0.5 ml)
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
MATERIALS AND METHODS
Powder preparation of C. racemosa
C. racemosa collected from around the Taman
Mangrove Beach (5°58’30.5” S 106°42’14.2” E), the
northern region of Untung Jawa Island, Kepulauan
Seribu, Indonesia, in February 2020. C. racemosa
was determined at the Research Center for
Oceanography, Indonesian Institute of Sciences,
Jakarta, Indonesia (B-599/IPK.2/IF.07/VII/2020).
Fresh C. racemosa (12 Kg) was cleaned using running
water. The material was dried in direct sunlight with a
black cloth. After drying, the material was powdered
and sieved (15).
Extraction Procedure
1. Maceration
The dried powder (150 g) was put in a glass bottle.
A 70% ethanol solvent was used to macerate
and extract the powder components at a 1:10
powder-solvent ratio (1.5 L). The mixture was
stirred occasionally for the first six h and then left
to stand. After 24 h, the filtrate was collected, and
the residue was re-macerated with a new solvent
for five repetitions (15).
2. Ultrasonic
The dried powder (150 g) was put into a beaker
glass and extracted with 70% ethanol solvent with
the same sample-solvent ratio as in the maceration
method. The extraction process was carried out for
60 min at 30 °C using an ultrasonic bath (Branson
5510, Marshall Scientific LLC., USA) with a frequency
of 40 kHz. The filtrate was collected, and the
residue was re-extracted using a new solvent for
five repetitions (16).
3. Soxhlet extraction
The dried powder (150 g) was extracted with
250 mL of 70% ethanol as a solvent in a Soxhlet
apparatus. The extraction process was carried out
at 70 °C. Extraction was stopped after the color of
the solvent on the siphon became clear. The filtrate
was collected. The extraction was conducted with
five repetitions (15).
The filtrate obtained from each extraction method
was then concentrated using a vacuum rotary
evaporator (Eyela, Shanghai, China) at 50 °C,
followed by a water bath at the same temperature
until a concentrated dry extract was obtained and
weighed. The yield extraction percentage was
calculated by dividing the weight of the extract
obtained (g) against the dried powder of C.
racemosa (g) multiplied by 100%.
Determination of extract characteristics
We determined the extracts’ characteristics, such
as organoleptic, moisture content, and total ash.
The organoleptic examination includes color, odor,
taste, and texture. Organoleptic observations were
made after the extract was exposed to air for 15
min using the senses (eyes, nose, and tongue)
(17). Determination of water content was carried
out by the gravimetric method using an oven at
105 °C. The total ash content was conducted by
burning 2 g of the extract in a kiln at 600 °C for
five h until it became white ash. These evaluations
conform to the procedures of Indonesian Herbal
Pharmacopoeia (2017) (15) and the World Health
Organization (2011) (12).
Phytochemical Screening
Identification of the chemical content (viz. phenolics,
flavonoids, saponins, triterpenoids/steroids) in the
extracts was carried out following the procedure on
Harborne (1987) (18). Identification of phenolics was
carried out using 5% (w/v) FeCl3 reagent, flavonoids
using magnesium powder and concentrated
hydrochloric acid, saponins using the foam test in
water, and triterpenoids/steroids using Salkowski
(chloroform and concentrated sulfuric acid) and
Liebermann Burchard reagents (chloroform,
concentrated sulfuric acid, and anhydrous acetic
acid). Identification of alkaloids was carried out with
Dragendorff, Mayer, and Hager reagents according
to procedures on Departemen Kesehatan RI (2000)
(19) and tannins using 1% (w/v) gelatin adjusted to
the procedure on Hanani (2015) (20).
Determination of phenolic levels
Determining phenolic levels was carried out using
gallic acid as a standard. This procedure was
initiated by preparing gallic acid stock solution
(Sigma Aldrich, Saint Louis, USA) (500 μg/mL) in
ethanol and an extract stock solution (1000 μg/mL)
in ethanol. Serial concentrations of gallic acid were
prepared at 11, 19, 27, 35, and 43 μg/mL in ethanol
to determine the calibration curve. Meanwhile, the
extract test solution was prepared at 100 μg/mL.
This test solution was diluted from the extract stock
solution. Test procedure brief description: the test
solution of both gallic acid and extract (0.5 ml)
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 349405Sherley, Ni Putu Ermi Hikmawanti, Mutia Agustin
was separately added to 0.5 mL Folin-ciocalteu
reagent (Merck KGaA, Darmstadt, Germany)
(1:10, v/v in water). The mixture was homogenized
using a vortex and allowed to stand for 3 minutes.
Then, 1.5 mL of 7.5% Na2 CO3 was added to the
mixture, homogenized, and incubated for 30 min.
Using a UV-Visible spectrophotometer (UV-1601
Series, Shimadzu), the absorbance of the mixture
was measured at 741 nm. Milligrams of gallic acid
equivalent (GAE) per gram of extract (or mg GAE/g)
represented the overall phenolic content of the
extract (21).
Determination of Antioxidant Activity
Antioxidant ac tivity was determined by the
2,2-diphenyl-1-picrylhydrazyl (DPPH) method (22).
The reference Quercetin solution was prepared
in 100 μg/mL concentration in methanol (Sigma
Aldrich, Saint Louis, USA). This solution was diluted
in methanol to 2, 4, 6, 8, and 10 μg/mL. The stock
solution of extract was prepared in a concentration
of 1,000 μg/mL in methanol and diluted to 100, 200,
300, 400, and 500 μg/mL in methanol. A 200 μL of
quercetin or extract solution were mixed with 1 mL
DPPH (Sigma Aldrich, Saint Louis, USA) 0.639 mM
and 4 mL methanol. The mixture was homogenized
and incubated at room temperature for 30 minutes
in the dark. Absorption was measured at 515.5 nm
using a UV-Vis spectrophotometer (UV-1601 Series,
Shimadzu, Kyoto, Japan). The percentage of DPPH
radical scavenging (%) is calculated by: (absorbance
of the DPPH − absorbance of the sample)/
absorbance of DPPH × 100%. Then, the percentage
of DPPH scavenging (y) is plotted against the sample
concentration (x) to obtain a linear line equation
y = bx±a. The value of 50 is entered as “y” so that
the value of x can be obtained, which is interpreted
as a concentration capable of scavenging 50% DPPH
radical (IC 50 ). The antioxidant activity index (AAI)
was calculated as follows: the ratio of the DPPH
final concentration (μg/mL) to the IC50 value of the
sample (μg/mL). The criteria for the antioxidant
activity of the extracts were measured based on the
AAI values, namely: poor (AAI < 0.5), moderate (AAI
between 0.5 and 1.0), strong (AAI between 1.0 and
2.0), and very strong (AAI > 2.0) (23).
Statistical analysis
Data on the total phenol content of extracts were
tested for homogeneity and normality by the
Kolmogorov-Smirnov test. The differences in total
phenolic content of the three types of extracts were
tested by one-way ANOVA with α=0.05. Meanwhile,
the antioxidant effect of the samples was compared
based on their IC 50 and AAI values descriptively.
RESULTS
We obtained 1.5 Kg dry C. racemosa from 12 Kg
of fresh sample. The yield percentages obtained
from the 150 g of C. racemosa dried powder by
maceration, ultrasonic, and Soxhlet extraction
methods were 13.96 %, 16.03 %, and 17.85%,
respectively. The results of the organoleptic test of
the extracts showed that the three extracts were
odorless, slightly salty, and viscose. The extract
obtained from the Soxhlet extraction was dark
green, while the other extracts were greenish-
brown. Table 1 lists the water content and total ash
in each extract.
Table 1. Water and total ash content of C. racemosa ethanol
extracts
Extraction methods Water content (%) Total ash content (%)
Maceration 7.96 ± 0.84 20.55 ± 0.63
Soxhlet 7.17 ± 0.04 25.71 ± 0.33
Ultrasonic 7.65 ± 0.01 22.74 ± 0.63
Note: Evaluations in triplicate.
The phytochemical screening results showed that all
extracts contain phenolics, flavonoids, tannins and
saponins (Table 2).
Table 2. Phytochemical of C. racemosa ethanol extracts
Extraction methods Phytochemical
Phenolics Flavonoids Tannins Alkaloids Saponins Steroid/tri terpenoids
Maceration + + + - + -
Soxhlet + + + - + -
Ultrasonic + + + - + -
Note: Evaluations in triplicate; (+) = detected; (-) = not detected
was separately added to 0.5 mL Folin-ciocalteu
reagent (Merck KGaA, Darmstadt, Germany)
(1:10, v/v in water). The mixture was homogenized
using a vortex and allowed to stand for 3 minutes.
Then, 1.5 mL of 7.5% Na2 CO3 was added to the
mixture, homogenized, and incubated for 30 min.
Using a UV-Visible spectrophotometer (UV-1601
Series, Shimadzu), the absorbance of the mixture
was measured at 741 nm. Milligrams of gallic acid
equivalent (GAE) per gram of extract (or mg GAE/g)
represented the overall phenolic content of the
extract (21).
Determination of Antioxidant Activity
Antioxidant ac tivity was determined by the
2,2-diphenyl-1-picrylhydrazyl (DPPH) method (22).
The reference Quercetin solution was prepared
in 100 μg/mL concentration in methanol (Sigma
Aldrich, Saint Louis, USA). This solution was diluted
in methanol to 2, 4, 6, 8, and 10 μg/mL. The stock
solution of extract was prepared in a concentration
of 1,000 μg/mL in methanol and diluted to 100, 200,
300, 400, and 500 μg/mL in methanol. A 200 μL of
quercetin or extract solution were mixed with 1 mL
DPPH (Sigma Aldrich, Saint Louis, USA) 0.639 mM
and 4 mL methanol. The mixture was homogenized
and incubated at room temperature for 30 minutes
in the dark. Absorption was measured at 515.5 nm
using a UV-Vis spectrophotometer (UV-1601 Series,
Shimadzu, Kyoto, Japan). The percentage of DPPH
radical scavenging (%) is calculated by: (absorbance
of the DPPH − absorbance of the sample)/
absorbance of DPPH × 100%. Then, the percentage
of DPPH scavenging (y) is plotted against the sample
concentration (x) to obtain a linear line equation
y = bx±a. The value of 50 is entered as “y” so that
the value of x can be obtained, which is interpreted
as a concentration capable of scavenging 50% DPPH
radical (IC 50 ). The antioxidant activity index (AAI)
was calculated as follows: the ratio of the DPPH
final concentration (μg/mL) to the IC50 value of the
sample (μg/mL). The criteria for the antioxidant
activity of the extracts were measured based on the
AAI values, namely: poor (AAI < 0.5), moderate (AAI
between 0.5 and 1.0), strong (AAI between 1.0 and
2.0), and very strong (AAI > 2.0) (23).
Statistical analysis
Data on the total phenol content of extracts were
tested for homogeneity and normality by the
Kolmogorov-Smirnov test. The differences in total
phenolic content of the three types of extracts were
tested by one-way ANOVA with α=0.05. Meanwhile,
the antioxidant effect of the samples was compared
based on their IC 50 and AAI values descriptively.
RESULTS
We obtained 1.5 Kg dry C. racemosa from 12 Kg
of fresh sample. The yield percentages obtained
from the 150 g of C. racemosa dried powder by
maceration, ultrasonic, and Soxhlet extraction
methods were 13.96 %, 16.03 %, and 17.85%,
respectively. The results of the organoleptic test of
the extracts showed that the three extracts were
odorless, slightly salty, and viscose. The extract
obtained from the Soxhlet extraction was dark
green, while the other extracts were greenish-
brown. Table 1 lists the water content and total ash
in each extract.
Table 1. Water and total ash content of C. racemosa ethanol
extracts
Extraction methods Water content (%) Total ash content (%)
Maceration 7.96 ± 0.84 20.55 ± 0.63
Soxhlet 7.17 ± 0.04 25.71 ± 0.33
Ultrasonic 7.65 ± 0.01 22.74 ± 0.63
Note: Evaluations in triplicate.
The phytochemical screening results showed that all
extracts contain phenolics, flavonoids, tannins and
saponins (Table 2).
Table 2. Phytochemical of C. racemosa ethanol extracts
Extraction methods Phytochemical
Phenolics Flavonoids Tannins Alkaloids Saponins Steroid/tri terpenoids
Maceration + + + - + -
Soxhlet + + + - + -
Ultrasonic + + + - + -
Note: Evaluations in triplicate; (+) = detected; (-) = not detected
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 349405
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
The total phenolic content of extracts was determined
using the calibration curve of gallic acid (y = 0.0111x
+ 0.1466; R 2 = 0.9994.) (Table 3). Based on the one-
way ANOVA test, there was a significant difference
in the total phenolic content of the three extracts
obtained by maceration, Soxhlet, and ultrasonic
methods (α = 0.05). The highest total phenolic
content was found in the extract produced from
the ultrasonic-assisted extraction method of 39.38 ±
0.36 mgGAE/g (ultrasonic > Soxhlet > maceration).
Table 3. Total phenolics content of C. racemosa ethanol extracts
Extraction methods Total phenolics content (mgGAE/g)
Maceration 22.05 ± 0.05 *
Soxhlet 37.31 ± 0.41*
Ultrasonic 39.38 ± 0.36*
Note: Evaluations in triplicate. * means a significant difference in the average
total phenolic content based on the three groups of extraction methods (α=0.05).
The DPPH technique was used to assess the
antioxidant activity of the C. racemosa ethanolic
extracts. The IC 50 value of C. racemosa ethanol
extract produced from the three extraction methods
(>300 μg/mL) showed weak antioxidant activity
against DPPH radicals when compared to quercetin
(6.15 ± 0.07 μg/mL). In this study, AAI values of
the C. racemosa ethanolic extracts obtained by
maceration, Soxhlet, and ultrasonic were 0.67, 0.69,
and 0.71, respectively. Meanwhile, the AAI value of
quercetin was 41.04. An AAI value of around 0.5-1.0
indicates a moderate antioxidant of the 70% ethanol
extract of C. racemosa (23).
6.15
375.84 352.95 365.73
0
100
200
300
400
Samples
Quercetin
Maceration
Ultrasonic
Soxhlet
Figure 1. IC50 value of C. racemosa ethanol extracts and quercetin
DISCUSSION
Marine is a source of natural materials with
important chemical content with potential use in
medicine, nutraceuticals, cosmetical, and food.
Marine metabolites can be extracted and isolated
by various techniques (13). This study evaluated
the characteristics, total phenolic content, and
antioxidant activity of the ethanol extract of C.
racemosa using three different extraction methods:
maceration, Soxhlet, and ultrasonic.
One of the characteristics of the extract that is
determined is the water content. Based on the
results obtained, the water content of the three
C. racemosa extracts was at <10%. It meant
that the extracts still met the quality standard of
extract according to Farmakope Herbal Indonesia
(Indonesia Herba Pharmacopeia) (17). High water
content in the extract can cause contamination of
microorganisms, which can affect the quality of the
extract (12). Caulerpa spp. naturally grows on the
coast. Fresh Caulerpa spp. predominantly contains
water, up to 94.84% (24). According to Tapotubun
(2018), drying fresh C. lentillifera under indirect
sunlight, covering them with a black cloth can
drastically reduce this water content. However, the
dried marine algae are still high in water content at
9.22 to 18.22% (24).
Seaweed, including C. racemosa, has a very high
mineral content (7). The total ash content of the
three extracts was >20%. The ash content in
other Caulerpaceae species (C. veravelensis, C.
Scalpelliformis, and C. racemosa) was 24.20–33.70%
(25); meanwhile, in C. lentillifera was around 40.66-
41.83% (24). Total ash indicates the presence of
material remaining after ignition. This material can
come from the internal or external (environment)
(26). A high total ash content indicates the risk of
heavy metal contamination such as Hg, Pb, Cd, etc.
However, it is better to carry out further qualitative
and quantitative identification to ascertain the type
and amount of heavy metal in the extract.
C. racemosa contains important phytochemicals,
such as phenolic, flavonoids, tannins, saponins,
coumarins, carbohydrates (27), fatty acids, and
methyl esters (16). The choice of solvent is crucial
for selectively extracting the target compound (11).
In addition, safety and toxicity considerations must
also be considered (28). Ethanol 70% is a mixture
of water-ethanol solvent that is known to be able
to extract phenolic compounds. This solvent can
increase phenolic extraction compared to the
solvents used individually (29). Other chemicals, such
as tannins, polyacetylenes, flavonols, terpenoids,
sterols, and alkaloids, may be extracted using
ethanol (30).
The percentage yield of the extract obtained from
Soxhlet is the highest compared to other extraction
methods (Soxhlet > ultrasonic > maceration).
However, the highest phenolic content of the C.
racemosa extract was found in the extract produced
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
The total phenolic content of extracts was determined
using the calibration curve of gallic acid (y = 0.0111x
+ 0.1466; R 2 = 0.9994.) (Table 3). Based on the one-
way ANOVA test, there was a significant difference
in the total phenolic content of the three extracts
obtained by maceration, Soxhlet, and ultrasonic
methods (α = 0.05). The highest total phenolic
content was found in the extract produced from
the ultrasonic-assisted extraction method of 39.38 ±
0.36 mgGAE/g (ultrasonic > Soxhlet > maceration).
Table 3. Total phenolics content of C. racemosa ethanol extracts
Extraction methods Total phenolics content (mgGAE/g)
Maceration 22.05 ± 0.05 *
Soxhlet 37.31 ± 0.41*
Ultrasonic 39.38 ± 0.36*
Note: Evaluations in triplicate. * means a significant difference in the average
total phenolic content based on the three groups of extraction methods (α=0.05).
The DPPH technique was used to assess the
antioxidant activity of the C. racemosa ethanolic
extracts. The IC 50 value of C. racemosa ethanol
extract produced from the three extraction methods
(>300 μg/mL) showed weak antioxidant activity
against DPPH radicals when compared to quercetin
(6.15 ± 0.07 μg/mL). In this study, AAI values of
the C. racemosa ethanolic extracts obtained by
maceration, Soxhlet, and ultrasonic were 0.67, 0.69,
and 0.71, respectively. Meanwhile, the AAI value of
quercetin was 41.04. An AAI value of around 0.5-1.0
indicates a moderate antioxidant of the 70% ethanol
extract of C. racemosa (23).
6.15
375.84 352.95 365.73
0
100
200
300
400
Samples
Quercetin
Maceration
Ultrasonic
Soxhlet
Figure 1. IC50 value of C. racemosa ethanol extracts and quercetin
DISCUSSION
Marine is a source of natural materials with
important chemical content with potential use in
medicine, nutraceuticals, cosmetical, and food.
Marine metabolites can be extracted and isolated
by various techniques (13). This study evaluated
the characteristics, total phenolic content, and
antioxidant activity of the ethanol extract of C.
racemosa using three different extraction methods:
maceration, Soxhlet, and ultrasonic.
One of the characteristics of the extract that is
determined is the water content. Based on the
results obtained, the water content of the three
C. racemosa extracts was at <10%. It meant
that the extracts still met the quality standard of
extract according to Farmakope Herbal Indonesia
(Indonesia Herba Pharmacopeia) (17). High water
content in the extract can cause contamination of
microorganisms, which can affect the quality of the
extract (12). Caulerpa spp. naturally grows on the
coast. Fresh Caulerpa spp. predominantly contains
water, up to 94.84% (24). According to Tapotubun
(2018), drying fresh C. lentillifera under indirect
sunlight, covering them with a black cloth can
drastically reduce this water content. However, the
dried marine algae are still high in water content at
9.22 to 18.22% (24).
Seaweed, including C. racemosa, has a very high
mineral content (7). The total ash content of the
three extracts was >20%. The ash content in
other Caulerpaceae species (C. veravelensis, C.
Scalpelliformis, and C. racemosa) was 24.20–33.70%
(25); meanwhile, in C. lentillifera was around 40.66-
41.83% (24). Total ash indicates the presence of
material remaining after ignition. This material can
come from the internal or external (environment)
(26). A high total ash content indicates the risk of
heavy metal contamination such as Hg, Pb, Cd, etc.
However, it is better to carry out further qualitative
and quantitative identification to ascertain the type
and amount of heavy metal in the extract.
C. racemosa contains important phytochemicals,
such as phenolic, flavonoids, tannins, saponins,
coumarins, carbohydrates (27), fatty acids, and
methyl esters (16). The choice of solvent is crucial
for selectively extracting the target compound (11).
In addition, safety and toxicity considerations must
also be considered (28). Ethanol 70% is a mixture
of water-ethanol solvent that is known to be able
to extract phenolic compounds. This solvent can
increase phenolic extraction compared to the
solvents used individually (29). Other chemicals, such
as tannins, polyacetylenes, flavonols, terpenoids,
sterols, and alkaloids, may be extracted using
ethanol (30).
The percentage yield of the extract obtained from
Soxhlet is the highest compared to other extraction
methods (Soxhlet > ultrasonic > maceration).
However, the highest phenolic content of the C.
racemosa extract was found in the extract produced
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 349405Sherley, Ni Putu Ermi Hikmawanti, Mutia Agustin
by the ultrasonic-assisted extraction method
compared to the Soxhlet and maceration methods
(Table 3). Based on the statistical analysis results with
the one-way ANOVA test, there was a significant
difference in the total phenolic content of the three
extracts (α=0.05). Traditional extraction techniques
(such as Soxhlet or reflux) typically use a sizable
number of solvents, a prolonged extraction period,
and high temperatures (29). Meanwhile, ultrasonic is
considered an alternative extraction method that is
effective and efficient in extracting marine-derived
compounds (13). Ultrasonic waves can cause cell
wall rupture, increasing solvent penetration in cells.
The use of ultrasonic in phytochemical extraction is
based on the physicochemical concept of acoustic
cavitation, a phenomenon where ultrasonic waves
cause bubbles that grow and burst in a liquid
media (16). The ultrasonic-assisted extraction of
components from natural materials became the
more effective and efficient method in our research.
However, the optimal extraction conditions of
ultrasonic extraction vary for each species and must
be evaluated separately.
The DPPH (2,2-diphenyl-1-picr ylhydrazyl) is
highly reactive and captures electrons from other
compounds to become stable (1). The DPPH method
describes the mechanism of action of antioxidants
with Single Electron Transfer (SET) (2), and it is used
to determine the antioxidants’ scavenging ability.
This method is valid, simple, accurate, sensitive,
and economical. Both pure chemicals and complex
samples may have their antioxidant capacity
measured (33). In addition, it is quite reproducible
compared to 2,2’-azino-bis (3-ethylbenzothiazoline-
6-sulfonic acid (ABTS), even though the reactivity
of DPPH with radicals (alkyl) (34). The inability to
attain a steady state with varying antioxidant/DPPH
ratios due to the nonlinearity of the time response
curve limits its use (1). Based on this study, the
antioxidant in the 70% ethanol extract sample was
moderate compared to quercetin. Determination of
antioxidant activity with other mechanisms of action
can still be determined on extracts that provide the
highest phenolic content.
CONCLUSIONS
Our findings showed that the extraction process
influenced the phenolic content obtained from
C. racemosa. A non-conventional method, such
as ultrasonic extraction, has many advantages in
terms of the efficiency and effectiveness of phenolic
recovery. However, C. racemosa extract showed
weak antioxidant activity compared to quercetin.
The use of C. racemosa as a source of medicinal,
nutraceutical, and marine food ingredients still
needs further study.
CONFLICT OF INTEREST
None to declare
ACKNOWLEDGEMENT
The authors thank Dr. apt. Supandi, M.Si. and Unit
Pembina dan Pengembang Publikasi Ilmiah (UPPI)/
Scientific Publication Supervisor and Developer Unit,
Universitas Muhammadiyah Prof. DR. HAMKA, who
have assisted and supervised the preparation of the
draft of this article.
AUTHORS’ CONTRIBUTIONS
Ni Putu Ermi Hikmawanti and Sherley participated
in the conceptualization of the study, supervisor
the experimental phase, discussion of the results,
and final writing of the manuscript. Mutia Agustin
carried out the experimental part and participated
in the discussion.
REFERENCES
1. Singh S, Singh RP. In vitro methods of assay of antioxidants:
An overview. Food Rev Int. 2008;24(4):392–415. https://doi.
org/10.1080/87559120802304269.
2. Moharram HA, Youssef MM. Methods for Determining the
Antioxidant Activity: A Review. Alexandria J Food Sci Technol.
2014;11(1):31–42.
3. Phaniendra A, Jestadi DB, Periyasamy L. Free Radicals: Properties,
Sources, Targets, and Their Implication in Various Diseases. Indian
J Clin Biochem. 2015;30(1):11–26.
4. Gulcin İ. Antioxidants and antioxidant methods: an updated
over view. Arch Toxicol. 2020;94(3):651–715. ht tps://doi.
org/10.1007/s00204-020-02689-3
5. Balakrishnan D, Kandasamy D, Nithyanand P. A review on
antioxidant activity of marine organisms. Int J ChemTech Res.
2014;6(7):3431–6.
6. Sansone C, Brunet C. Marine algal antioxidants. Antioxidants.
2020;9(3):11–4. https://doi.org/10.3390/antiox9030206.
7. Fithriani D. Opportunities and Challenges for Developing
Caulerpa racemosa as Functional Foods. 1st Int Symp Aquat Prod
Process 2013. 2015;1. http://dx.doi.org/10.18502/kls.v1i0.90.
8. Yap WF, Tay V, Tan SH, Yow YY, Chew J. Decoding antioxidant and
antibacterial potentials of Malaysian green seaweeds: Caulerpa
racemosa and Caulerpa lentillifera. Antibiotics. 2019;8(3). https://
doi.org/10.3390/antibiotics8030152.
9. Nagaraj SR, Osborne JW. Bioactive compounds from Caulerpa
racemosa as a potent larvicidal and antibacterial agent. Front
by the ultrasonic-assisted extraction method
compared to the Soxhlet and maceration methods
(Table 3). Based on the statistical analysis results with
the one-way ANOVA test, there was a significant
difference in the total phenolic content of the three
extracts (α=0.05). Traditional extraction techniques
(such as Soxhlet or reflux) typically use a sizable
number of solvents, a prolonged extraction period,
and high temperatures (29). Meanwhile, ultrasonic is
considered an alternative extraction method that is
effective and efficient in extracting marine-derived
compounds (13). Ultrasonic waves can cause cell
wall rupture, increasing solvent penetration in cells.
The use of ultrasonic in phytochemical extraction is
based on the physicochemical concept of acoustic
cavitation, a phenomenon where ultrasonic waves
cause bubbles that grow and burst in a liquid
media (16). The ultrasonic-assisted extraction of
components from natural materials became the
more effective and efficient method in our research.
However, the optimal extraction conditions of
ultrasonic extraction vary for each species and must
be evaluated separately.
The DPPH (2,2-diphenyl-1-picr ylhydrazyl) is
highly reactive and captures electrons from other
compounds to become stable (1). The DPPH method
describes the mechanism of action of antioxidants
with Single Electron Transfer (SET) (2), and it is used
to determine the antioxidants’ scavenging ability.
This method is valid, simple, accurate, sensitive,
and economical. Both pure chemicals and complex
samples may have their antioxidant capacity
measured (33). In addition, it is quite reproducible
compared to 2,2’-azino-bis (3-ethylbenzothiazoline-
6-sulfonic acid (ABTS), even though the reactivity
of DPPH with radicals (alkyl) (34). The inability to
attain a steady state with varying antioxidant/DPPH
ratios due to the nonlinearity of the time response
curve limits its use (1). Based on this study, the
antioxidant in the 70% ethanol extract sample was
moderate compared to quercetin. Determination of
antioxidant activity with other mechanisms of action
can still be determined on extracts that provide the
highest phenolic content.
CONCLUSIONS
Our findings showed that the extraction process
influenced the phenolic content obtained from
C. racemosa. A non-conventional method, such
as ultrasonic extraction, has many advantages in
terms of the efficiency and effectiveness of phenolic
recovery. However, C. racemosa extract showed
weak antioxidant activity compared to quercetin.
The use of C. racemosa as a source of medicinal,
nutraceutical, and marine food ingredients still
needs further study.
CONFLICT OF INTEREST
None to declare
ACKNOWLEDGEMENT
The authors thank Dr. apt. Supandi, M.Si. and Unit
Pembina dan Pengembang Publikasi Ilmiah (UPPI)/
Scientific Publication Supervisor and Developer Unit,
Universitas Muhammadiyah Prof. DR. HAMKA, who
have assisted and supervised the preparation of the
draft of this article.
AUTHORS’ CONTRIBUTIONS
Ni Putu Ermi Hikmawanti and Sherley participated
in the conceptualization of the study, supervisor
the experimental phase, discussion of the results,
and final writing of the manuscript. Mutia Agustin
carried out the experimental part and participated
in the discussion.
REFERENCES
1. Singh S, Singh RP. In vitro methods of assay of antioxidants:
An overview. Food Rev Int. 2008;24(4):392–415. https://doi.
org/10.1080/87559120802304269.
2. Moharram HA, Youssef MM. Methods for Determining the
Antioxidant Activity: A Review. Alexandria J Food Sci Technol.
2014;11(1):31–42.
3. Phaniendra A, Jestadi DB, Periyasamy L. Free Radicals: Properties,
Sources, Targets, and Their Implication in Various Diseases. Indian
J Clin Biochem. 2015;30(1):11–26.
4. Gulcin İ. Antioxidants and antioxidant methods: an updated
over view. Arch Toxicol. 2020;94(3):651–715. ht tps://doi.
org/10.1007/s00204-020-02689-3
5. Balakrishnan D, Kandasamy D, Nithyanand P. A review on
antioxidant activity of marine organisms. Int J ChemTech Res.
2014;6(7):3431–6.
6. Sansone C, Brunet C. Marine algal antioxidants. Antioxidants.
2020;9(3):11–4. https://doi.org/10.3390/antiox9030206.
7. Fithriani D. Opportunities and Challenges for Developing
Caulerpa racemosa as Functional Foods. 1st Int Symp Aquat Prod
Process 2013. 2015;1. http://dx.doi.org/10.18502/kls.v1i0.90.
8. Yap WF, Tay V, Tan SH, Yow YY, Chew J. Decoding antioxidant and
antibacterial potentials of Malaysian green seaweeds: Caulerpa
racemosa and Caulerpa lentillifera. Antibiotics. 2019;8(3). https://
doi.org/10.3390/antibiotics8030152.
9. Nagaraj SR, Osborne JW. Bioactive compounds from Caulerpa
racemosa as a potent larvicidal and antibacterial agent. Front
7Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 349405
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
Biol (Beijing). 2014;9:300–5. https://doi.org/10.1007/s11515-014-
1312-4.
10. Mosić M, Dramićanin A, Ristivojević P, Milojković-Opsenica D.
Extraction as a critical step in phytochemical analysis. J AOAC
Int. 2020;103(2):365–72. https://doi.org/10.5740/jaoacint.19-0251.
11. Abubakar AR, Haque M. Preparation of Medicinal Plants: Basic
Extraction and Fractionation Procedures for Experimental
Purposes. J Pharm Bioallied Sci. 2020;12(1):1–10. https://doi.
org/10.4103/jpbs.JPBS_175_19.
12. World Health Organization. Quality control methods for herbal
materials. World Health Organization.; 2011.
13. Grosso C, Valentão P, Ferreres F, Andrade PB. Alternative and
efficient extraction methods for marine-derived compounds.
Mar Drugs. 2015;13(5):3182–230. https://doi.org/10.3390/
md13053182.
14. Cotas J, Leandro A, Monteiro P, Pacheco D, Figueirinha A,
Goncąlves AMM, et al. Seaweed phenolics: From extraction to
applications. Mar Drugs. 2020;18(8). https://doi.org/10.3390/
md18080384.
15. Kementerian Kesehatan RI. Farmakope Herbal Indonesia (FHI).
2nd ed. Jakarta: Kementerian Kesehatan RI; 2008.
16. Carreira-Casais A, Otero P, Garcia-Perez P, Garcia-Oliveira
P, Pereira AG, Carpena M, et al. Benefits and drawbacks of
ultrasound-assisted extraction for the recovery of bioactive
compounds from marine algae. Int J Environ Res Public Health.
2021;18(17). https://doi.org/10.3390/ijerph18179153.
17. Ministry of Health Republic of Indonesia. Farmakope Herbal
Indonesia (Indonesian Herbal Pharmacopoeia). II. Jakarta:
Ministry of Health Republic of Indonesia; 2008. 221 p.
18. Harborne J. Metode Fitokimia: Penentuan Cara Modern
Menganalisis Tumbuhan. Padmawinata, Kosasih; Soediro I, editor.
Bandung: ITB Press; 1987.
19. Departemen Kesehatan RI. Parameter Standar Umum Ekstrak
Tumbuhan. Jakarta: Departemen Kesehatan RI; 2000. 17 p.
20. Hanani E. Analisis Fitokimia. Jakarta: Buku Kedokteran EGC; 2015.
34–45 p.
21. Hikmawanti NPE, Hanani E, Sapitri Y, Ningrum W. Total
Phenolic Content and Antioxidant Activity of Different Extracts
of L. Leaves. Pharmacogn J. 2020;12(6):1311–6. https://doi.
org/10.5530/pj.2020.12.180.
22. Molyneux P. The Use of the Stable Free Radical Diphenylpicryl-
hydrazyl (DPPH) for Estimating Antioxidant Activity. Songklanakarin
J Sci Technol. 2004;26(2):211–9.
23. Scherer R, Godoy HT. Antioxidant activity index (AAI) by the
2,2-diphenyl-1-picrylhydrazyl method. Food Chem. 2009;112(3):654–
8. https://doi.org/10.1016/j.foodchem.2008.06.026.
24. Tapotubun AM. Komposisi Kimia Rumput Laut ( Caulerpa
lentillifera) dari Perairan Kei Maluku dengan Metode Pengeringan
Berbeda. J Pengolah Has Perikan Indones. 2018;21(1):13–23.
https://doi.org/10.17844/jphpi.v21i1.21257.
25. Kumar M, Gupta V, Kumari P, Reddy CRK, Jha B. Assessment of
nutrient composition and antioxidant potential of Caulerpaceae
seaweeds. J Food Compos Anal. 2011;24(2):270–8. https://doi.
org/10.1016/j.jfca.2010.07.007.
26. World Health Organization. Quality control methods for medicinal
plant materials World Health Organization Geneva. Geneva:
World Health Organization; 1998. Available from: https://apps.
who.int/iris/handle/10665/41986
27. Raj A, Mala K, Prakasam A. Phytochemical analysis of marine
macroalgae Caulerpa racemosa (J. Agardh) (Chlorophyta-
Caulerpales) from Tirunelveli District, Tamilnadu, India. J Glob
Biosci. 2015;4(8):3055–67.
28. Houghton PJ, Raman A . L aborator y Handbook for the
Fractionation of Natural Extracts. 1st ed. Laboratory Handbook for
the Fractionation of Natural Extracts. Springer-Science+Business
Media, B.V.; 1998. 14–16, 24–31, 39–52 p.
29. Getachew AT, Jacobsen C, Holdt SL. Emerging technologies
for the extraction of marine phenolics: Opportunities and
challenges. Mar Drugs. 2020;18(8):1–22. https://doi.org/10.3390/
MD18080389.
30. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H. Phytochemical screening
and Extraction: A Review. Int Pharm Sci. 2011;1(1):98–106.
31. Ngamwonglumlert L, Devahastin S, Chiewchan N. Natural
colorants: Pigment stability and extraction yield enhancement via
utilization of appropriate pretreatment and extraction methods.
Crit Rev Food Sci Nutr. 2015;57(15):3243–59. http://dx.doi.org/1
0.1080/10408398.2015.1109498.
32. Molnár É, Dóra Rippel-Pethő !, Bocsi R. Solid-Liquid Extraction
of Chlorophyll From Microalgae From Photoautotroph Open-Air
Cultivation. Hungarian J Ind Chem Veszprém. 2013;41(2):119–22.
https://doi.org/10.1515/511.
33. Boligon A A. Technical Evaluation of Antioxidant Activity.
Med Chem (Los Angeles). 2014;4(7):517–22. ht tps://doi.
org/10.4172/2161-0444.1000188.
34. Sadeer NB, Montesano D, Albrizio S, Zengin G, Mahomoodally MF.
The versatility of antioxidant assays in food science and safety—
chemistry, applications, strengths, and limitations. Antioxidants.
2020;9(8):1–39. https://doi.org/10.3390/antiox9080709.
The effect of extraction methods on total phenolics and antioxidant activities in Caulerpa racemosa (Forsskal) J. Agardh extracts
Biol (Beijing). 2014;9:300–5. https://doi.org/10.1007/s11515-014-
1312-4.
10. Mosić M, Dramićanin A, Ristivojević P, Milojković-Opsenica D.
Extraction as a critical step in phytochemical analysis. J AOAC
Int. 2020;103(2):365–72. https://doi.org/10.5740/jaoacint.19-0251.
11. Abubakar AR, Haque M. Preparation of Medicinal Plants: Basic
Extraction and Fractionation Procedures for Experimental
Purposes. J Pharm Bioallied Sci. 2020;12(1):1–10. https://doi.
org/10.4103/jpbs.JPBS_175_19.
12. World Health Organization. Quality control methods for herbal
materials. World Health Organization.; 2011.
13. Grosso C, Valentão P, Ferreres F, Andrade PB. Alternative and
efficient extraction methods for marine-derived compounds.
Mar Drugs. 2015;13(5):3182–230. https://doi.org/10.3390/
md13053182.
14. Cotas J, Leandro A, Monteiro P, Pacheco D, Figueirinha A,
Goncąlves AMM, et al. Seaweed phenolics: From extraction to
applications. Mar Drugs. 2020;18(8). https://doi.org/10.3390/
md18080384.
15. Kementerian Kesehatan RI. Farmakope Herbal Indonesia (FHI).
2nd ed. Jakarta: Kementerian Kesehatan RI; 2008.
16. Carreira-Casais A, Otero P, Garcia-Perez P, Garcia-Oliveira
P, Pereira AG, Carpena M, et al. Benefits and drawbacks of
ultrasound-assisted extraction for the recovery of bioactive
compounds from marine algae. Int J Environ Res Public Health.
2021;18(17). https://doi.org/10.3390/ijerph18179153.
17. Ministry of Health Republic of Indonesia. Farmakope Herbal
Indonesia (Indonesian Herbal Pharmacopoeia). II. Jakarta:
Ministry of Health Republic of Indonesia; 2008. 221 p.
18. Harborne J. Metode Fitokimia: Penentuan Cara Modern
Menganalisis Tumbuhan. Padmawinata, Kosasih; Soediro I, editor.
Bandung: ITB Press; 1987.
19. Departemen Kesehatan RI. Parameter Standar Umum Ekstrak
Tumbuhan. Jakarta: Departemen Kesehatan RI; 2000. 17 p.
20. Hanani E. Analisis Fitokimia. Jakarta: Buku Kedokteran EGC; 2015.
34–45 p.
21. Hikmawanti NPE, Hanani E, Sapitri Y, Ningrum W. Total
Phenolic Content and Antioxidant Activity of Different Extracts
of L. Leaves. Pharmacogn J. 2020;12(6):1311–6. https://doi.
org/10.5530/pj.2020.12.180.
22. Molyneux P. The Use of the Stable Free Radical Diphenylpicryl-
hydrazyl (DPPH) for Estimating Antioxidant Activity. Songklanakarin
J Sci Technol. 2004;26(2):211–9.
23. Scherer R, Godoy HT. Antioxidant activity index (AAI) by the
2,2-diphenyl-1-picrylhydrazyl method. Food Chem. 2009;112(3):654–
8. https://doi.org/10.1016/j.foodchem.2008.06.026.
24. Tapotubun AM. Komposisi Kimia Rumput Laut ( Caulerpa
lentillifera) dari Perairan Kei Maluku dengan Metode Pengeringan
Berbeda. J Pengolah Has Perikan Indones. 2018;21(1):13–23.
https://doi.org/10.17844/jphpi.v21i1.21257.
25. Kumar M, Gupta V, Kumari P, Reddy CRK, Jha B. Assessment of
nutrient composition and antioxidant potential of Caulerpaceae
seaweeds. J Food Compos Anal. 2011;24(2):270–8. https://doi.
org/10.1016/j.jfca.2010.07.007.
26. World Health Organization. Quality control methods for medicinal
plant materials World Health Organization Geneva. Geneva:
World Health Organization; 1998. Available from: https://apps.
who.int/iris/handle/10665/41986
27. Raj A, Mala K, Prakasam A. Phytochemical analysis of marine
macroalgae Caulerpa racemosa (J. Agardh) (Chlorophyta-
Caulerpales) from Tirunelveli District, Tamilnadu, India. J Glob
Biosci. 2015;4(8):3055–67.
28. Houghton PJ, Raman A . L aborator y Handbook for the
Fractionation of Natural Extracts. 1st ed. Laboratory Handbook for
the Fractionation of Natural Extracts. Springer-Science+Business
Media, B.V.; 1998. 14–16, 24–31, 39–52 p.
29. Getachew AT, Jacobsen C, Holdt SL. Emerging technologies
for the extraction of marine phenolics: Opportunities and
challenges. Mar Drugs. 2020;18(8):1–22. https://doi.org/10.3390/
MD18080389.
30. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H. Phytochemical screening
and Extraction: A Review. Int Pharm Sci. 2011;1(1):98–106.
31. Ngamwonglumlert L, Devahastin S, Chiewchan N. Natural
colorants: Pigment stability and extraction yield enhancement via
utilization of appropriate pretreatment and extraction methods.
Crit Rev Food Sci Nutr. 2015;57(15):3243–59. http://dx.doi.org/1
0.1080/10408398.2015.1109498.
32. Molnár É, Dóra Rippel-Pethő !, Bocsi R. Solid-Liquid Extraction
of Chlorophyll From Microalgae From Photoautotroph Open-Air
Cultivation. Hungarian J Ind Chem Veszprém. 2013;41(2):119–22.
https://doi.org/10.1515/511.
33. Boligon A A. Technical Evaluation of Antioxidant Activity.
Med Chem (Los Angeles). 2014;4(7):517–22. ht tps://doi.
org/10.4172/2161-0444.1000188.
34. Sadeer NB, Montesano D, Albrizio S, Zengin G, Mahomoodally MF.
The versatility of antioxidant assays in food science and safety—
chemistry, applications, strengths, and limitations. Antioxidants.
2020;9(8):1–39. https://doi.org/10.3390/antiox9080709.