KELOMPOK 3: 1.

Elen Vikelavianis (1913206015)

2. Erlisa Maratul ‘Alimah (1913206016)
3. Inang Mahendra (1913206017)
4. Iswari Rahmi A’yuni (1913206018)
5. Ita Rhosida (1913206019)

Rangkuman
Potensi Ekstrak Eucheuma cottonii Sebagai Kandidat Agen Anestesi Ikan
Bahan dan metode
1. Ekstraksi dan penentuan kandungan total fenolik
Rumput laut E. cottonii dipanen pada hari ke-45 sesuai dengan hasil terbaik yang diperoleh dari
penelitian sebelumnya. Rumput laut segar E. cottonii dicuci dengan air mengalir kemudian dikering anginkan
selama 3 hari, dilakukan pengeringan dengan oven pada suhu 50–60 °C selama 10 jam, dilanjutkan dengan
penggilingan menggunakan food grinder dan pengayakan menjadi 100 mesh. Kadar air bubuk tersebut
diperkirakan menurut SNI 01-2891-1992.
Ekstrak air dilakukan dengan mengekstraksi 20 g serbuk E. cottonii dalam 200 mL air dengan kecepatan
120 r/min selama waktu tertentu dalam penangas air yang dikocok (Lab Companion BS-21). Ekstraksi
dilakukan pada suhu 30 °C dan 80 menit dalam dua ulangan. Indikator ekstraksi menghasilkan total fenol.
Dalam penelitian ini ditentukan fenol sebagai senyawa yang mewakili bahan aktif ekstrak E.
cottonii . Kandungan total fenol diidentifikasi menggunakan metode Folin-Ciocealteu. Penentuan kandungan
fenolik total memasukkan asam galat sebagai standar, dan oleh karena itu hasilnya dinyatakan sebagai
persentase setara asam galat (GAE).
2. Uji toksisitas
Uji toksisitas dilakukan dengan menggunakan metode Brine Shrimp Lethality Test (BSLT). Tahapan
tersebut meliputi
a. penetasan larva udang ( Artemia salina ), Telur artemia ditempatkan dalam kotak penetasan, perlahan
diisi dengan air laut hingga setengah volume total dan mencapai ketinggian telur. Kotak ditutup dengan
aluminium foil dan ditempatkan di bawah lampu 5 W (24-28 °C). Setelah 48 jam, larva udang
dikumpulkan menggunakan pipet dan siap untuk uji toksisitas
b. persiapan larutan uji, Ekstrak yang dianalisis disiapkan pada konsentrasi 1000, 500, 100, dan 10 g/mL
air laut.
c. uji toksisitas ekstrak. 10-15 A. salina hidupdimasukkan ke dalam vial uji berisi air laut 100 L, dan
ditambahkan larutan sampel pada konsentrasi 10, 100, 500 dan 1000 g/mL, dilanjutkan dengan
pencampuran hingga homogen. Setiap konsentrasi dilakukan dalam tiga ulangan. Sebagai kontrol dibuat
campuran tanpa penambahan sampel dan didiamkan selama 24 jam, dilanjutkan dengan perhitungan
larva udang yang mati dan hidup. Mortalitas dihitung dengan membandingkan larva yang mati dengan
total larva udang.
Grafik konsentrasi log terhadap kematian diplot. Nilai LC 50 diperoleh dari penarikan garis dari 50%
titik sumbu mortalitas sampai perpotongan dengan garis yang diplot. Titik perpotongan pada sumbu konsentrasi
dicatat, yang menentukan dosis yang diperlukan untuk menyebabkan kematian pada 50% larva, yang
dinyatakan sebagai LC 50 . Suatu zat disebut aktif atau toksik bila LC-nya 50 1000 g/mL.
3. Uji fitokimia
Uji fitokimia kualitatif dilakukan pada flavonoid, tanin, saponin, kuinon, steroid, dan triterpenoid,
mengikuti metode yang digunakan oleh ( Harbourne, 1987 ; Ningsih et al., 2016 ; Suratno, 2016 ). Prosedur
assay adalah sebagai berikut.
 (a) Flavonoid. 2 mL ekstrak E. cottonii diencerkan ke dalam 2 mL metanol, dilanjutkan dengan
penambahan mg bubuk dan 5 tetes HCl pekat. Adanya senyawa flavonoid ditunjukkan dengan
terbentuknya warna merah atau jingga.
(b) Alkaloid. 2 mL kloroform dan 2 mL amonia ditambahkan ke dalam ekstrak E. cottonii , dilanjutkan
dengan penyaringan. Tiga sampai lima tetes terkonsentrasi H2SO4 ditambahkan ke dalam
filtrat. Larutan diaduk hingga terbentuk dua lapisan. Lapisan atas 2,5 mL dipindahkan ke dalam tiga
tabung reaksi. Ketiga larutan tersebut dianalisis dengan 4 tetes pereaksi Mayer, Dragendorff, dan
Wagner. Terbentuknya endapan menunjukkan kandungan alkaloid pada sampel. Reaksi dengan
pereaksi Mayer menghasilkan endapan putih. Reaksi dengan Dragendorff menghasilkan sedimen
berwarna oranye-merah. Reaksi dengan pereaksi Wagner menghasilkan endapan berwarna coklat.
(c) Tanin. Ditambahkan 10 tetes FeCl3 10% ke dalam mL ekstrak E. cottonii tertentu , dilanjutkan
dengan pencampuran selama 1 menit sebelum penambahan 2 tetes HCl 1 N. Ekstrak dikatakan
positif mengandung tanin bila menghasilkan warna hijau kehitaman atau kehitaman. warna biru.
(d) Saponin. mL tertentu E. cottoniiekstrak ditambahkan ke dalam 10 mL air dengan pengocokan 1
menit, dilanjutkan dengan penambahan 2 tetes HCl 1 N. Ekstrak positif mengandung saponin bila
busa yang terbentuk tetap stabil selama 7 menit.
(e) Quinon. NaOH 1 N ditambahkan ke dalam sampel dalam jumlah tertentu, dan diamati perubahan
warna. Reaksi positif ditunjukkan dengan terbentuknya warna kuning.
(f) Steroid dan triterpenoid. 10 tetes CH3COOH glasial dan 2 tetes H2SO4 pekat ditambahkan ke
dalam ekstrak E. cottonii . Larutan dikocok dan dibiarkan selama beberapa menit. Reaksi positif
steroid diidentifikasi ketika warna hijau atau biru dihasilkan, sedangkan reaksi positif triterpenoid
diidentifikasi ketika warna merah atau ungu dihasilkan.
4. Identifikasi senyawa aktif
Identifikasi senyawa aktif dilakukan dengan mengukur senyawa metabolit hasil uji fitokimia secara
kuantitatif dengan spektrofotometer dan LC scanner. Juga, senyawa lain diidentifikasi dengan dan LC-
MS. Identifikasi senyawa dalam ekstrak menggunakan LC-MS dengan kondisi proses sebagai berikut: Kolom
ACQUITY UPLC @ BEH C18 1,7 m; Fase gerak: (a) amonium format, (b) asetonitrit, (c) metanol, dan (d)
air; Aliran: air (+0,01% FA + 0,05% amonia); Volume injeksi: 8 L; Suhu kolom: 28,2 °C; Laju aliran: 0,20
mL/menit; Waktu analisis: 23.20 menit.
5. Analisis data
Hasil dari pengujian dengan LC-MS diproses dalam perangkat lunak MassLynx V4.1. Senyawa yang
ditemukan diinterpretasikan menggunakan Chemspider.com .
 Aquaculture and Fisheries xxx (xxxx) xxx

Contents lists available at ScienceDirect

Aquaculture and Fisheries

journal homepage: www.keaipublishing.com/en/journals/aquaculture-and-fisheries

The potential of Eucheuma cottonii extract as a candidate for fish

anesthetic agent
Ninik Purbosari a, *, Endang Warsiki b, Khaswar Syamsu b, Joko Santoso c
a
Study Program of Aquaculture, Politeknik Negeri Lampung, Jalan Soekarno Hatta No 10 Rajabasa Bandar Lampung, 35144, Indonesia
b
Department of Agro-Industrial Engineering, Faculty of Agricultural Technology, IPB University, Jalan Lingkar Akademik Kampus IPB, Darmaga Bogor, 16680,
Indonesia
c
Department of Aquatic Products Technology, Faculty of Fisheries and Marine Science, IPB University, Jalan Lingkar Akademik Kampus IPB, Darmaga Bogor, 16680,
Indonesia

A R T I C L E I N F O A B S T R A C T

Keywords: Red seaweed species Eucheuma cottonii was thought to have potential as an anesthetic agent because it is shown
Active compounds to contain compounds with bioactivity as antimicrobial, antifungal, and also anticancer. There have been no
Eucheuma cottonii studies regarding the active ingredients of anesthetized. The objective of the study was to examine the potential
Fish anesthetic
of E. cottonii extract as a candidate for fish anesthetic based on toxicity, phytochemistry, and active compounds
Phenol
contained in the extract. The stages of research include extraction, toxicity test extracts (BSLT), phytochemical
test, quantitative test compound phytochemical results, and the identification of active compounds with LC-MS.
Test results showed that E. cottonii extract contains a total of phenols of 4.60 ± 0.06 mg/GAE g which is one
presentation of the active ingredient in the extract. The extract had bioactivity with LC50 of 221.71 ± 5.74 μg/
mL. E. cottonii extract contained flavonoids compounds and saponins with a consecutive number of 24.48% and
0.22%. The results of identifying active compounds were found in several compounds that had activity asanti­
depressants or sedatives. E. cottonii extract can be used as a candidate for a fish anesthetic agent.

1. Introduction natural anesthesia agents, leaving no residue, as well as a wider range of

Anesthesia has long been used for the handling and transportation of
live fish in aquaculture activities. Anesthetize conditions during trans­
portation will reduce the level of stress, metabolism, and friction among
fish individuals so that the degree of high livelihood. As long as these
anesthetic agents are used more derived from relatively expensive
chemicals and leave a residue that harmful when consumed such as
tricaine metana-sulfonate/MS-222 ( Bolasina et al., 2017; Chance et al.,
2018), benzocaine (Bizarro et al., 2018) and 2-phenoxyethanol
(Hedayati, 2018). However, synthetic agents are forbidden to be used
regarding security and resistance issues as well as costs that are often a
limiting factor (Brown, 2011). The use of natural agents as anesthetic
agents began to develop among them tuba root (Derris elliptica), nutmeg
Myristica fragrans (Al-Niaeem et al., 2017), and clove oil (Bizarro et al.,
2017). Clove oil proved most effective and commonly used (Akinrotimi
et al., 2016; Saini et al., 2018). Nevertheless, the lack of clove oil is a
narrow range of concentrations and long recovery time (Abdolazizi
et al., 2011; Sutili et al., 2014). Therefore, there is a safer alternative to
doses. One of the potential natural agents that can be used as candidates
for an anesthetic agent is the seaweed that has been proven to have
extensive bioactivity.
Eucheuma cottonii is a species of red seaweed that contains various
active compounds with its bioactivity as an antioxidant (Lim et al., 2015;
Wulandari et al., 2018), antibacterial and antifungal (Selvan et al.,
2014), as well as the anticancer (Namvar et al., 2012). The active
compounds owned by E. cottonii are polyphenols and their derivatives
such as alkaloids, flavonoids, and steroids. Also, there are tannin,
saponin, and terpenoids. Meanwhile, the polyphenols from the brown
seaweed Eclonia cava proved to have a sedative (anesthetic) effect (Cho
et al., 2012); similarly, the compound in the treatment of Laurencia
dendroida has the effect of larvicidal and anesthesia (Neto et al., 2016).
Ekeanyanwu and Njok (2014) reported that the flavonoids extract of
Monodora tenuifolia seed proved toxic for an albino rat. Saponin is a toxic
compound for fish and mollusk (Abele & Lukevics, 2001). The alkaloids
(Jawale, 2014), saponins, tannins, flavonoids, and steroids of ground
plant extracts are proven to be larvacidal and as an anesthetic ingredient

* Corresponding author.
E-mail address: purbosari_nnk@polinela.ac.id (N. Purbosari).

https://doi.org/10.1016/j.aaf.2021.06.003
Received 14 June 2020; Received in revised form 20 May 2021; Accepted 10 June 2021
2468-550X/© 2021 Shanghai Ocean University. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Ninik Purbosari, Aquaculture and Fisheries, https://doi.org/10.1016/j.aaf.2021.06.003
N. Purbosari et al.
(Tsuchiya, 2017). Although several active ingredients contained in the
E. cottonii have been widely reported, there have been no studies related
to the active ingredient of anesthesia, either the effectiveness of the
process of extraction, toxicity and the type of active compounds. Based
on the reports above: (1) saponin and flavonoid compound in E. cottonii
proved toxic for fish; (2) the polyphenol from other types of seaweed are
toxic to fish, while E. cottonii also contains polyphenol compounds, it is
suspected that E. cottonii has activity as a sedative or anesthetic material
with to arrange the dose. Thus the purpose of the study was to examine
the potential of E. cottonii extract as a candidate for a fish anesthetic
agent. Indicator of anesthesia potential seen from toxicity and the
presence of compounds that have anesthetic or antidepressant activity.
2. Materials and methods
2.1. Extraction and determination of total phenolic content
Seaweed E. cottonii used was obtained from Ruguk Village Ketapang
Sub-district South Lampung. It was harvested on the 45th day con­
forming to the best result gained from the previous study. Fresh seaweed
E. cottonii was washed in running water then was air-dried for 3 days.
Subsequently, oven drying at 50–60 ◦ C for 10 h was performed, followed
by grinding using a food grinder and sieving into 100 mesh (Soliman
et al., 2017). The water content of the powder was estimated according
to SNI 01-2891-1992.
The aqueous extract was performed by extracting 20 g E. cottonii
powder in 200 mL water at 120 r/min for a certain time in a shaking
water bath (lab Companion BS-21) (Matanjun et al., 2008). The
extraction was done at 30 ◦ C and 80 min in two replicates. The extrac­
tion indicator was resulting in total phenol.
In this study, phenol was determined as a compound representing the
active ingredient of E. cottonii extract. Total phenol content was iden­
tified using the Folin-Ciocealteu method. Determination of total
phenolic content incorporated gallic acid as a standard, and therefore
the result was expressed as a percentage of gallic acid equivalent (GAE).
Calculation of total phenolic content was done following (Permana
et al., 2016) with a slight modification.
2.2. Toxicity assay
Toxicity assay was carried out using Brine Shrimp Lethality Test
(BSLT) method (Kumala & Sapitri, 2011; Meyer et al., 1982). The stages
included hatchment of shrimp (Artemia salina) larvae, preparation of the
assayed solution, and toxicity assay of the extract. (a) Hatchment of
shrimp (A. salina) larvae. Artemia eggs were placed in a hatchment box.
The box was slowly filled with seawater up to half the total volume and
reaching the height of the eggs. The box was covered with aluminum foil
and was placed under 5 W light (24–28 ◦ C). After 48 h, shrimp larvae
were collected using a pipette and were ready for toxicity assay. (b)
Preparation of assayed solution. The extract analyzed was prepared at
concentrations of 1000, 500, 100, and 10 μg/mL seawater. (c) Toxicity
assay of the extract. 10–15 live A. salina were put into a 100 μL sea
water-containing test vial, and sample solution at concentrations of 10,
100, 500 and 1000 μg/mL was added, followed by mixing until ho­
mogenous. Each concentration was done in three replicates. As a con­
trol, a mixture was prepared without the addition of the sample and was
let to stand for 24 h, followed by calculation of died and live shrimp
larvae. Mortality was calculated by comparing dead larvae with total
shrimp larvae.
A graph of log concentration against mortality was plotted. LC50
value was obtained from drawing a line from a 50% point of mortality
axis until the intersection with the plotted line. The point of the inter­
section at the concentration axis was recorded, which defined a dose
required to cause death for 50% larvae, expressed as LC50. A substance is
called active or toxic when its LC50 ≤ 1000 μg/mL (Meyer et al., 1982).
2
Aquaculture and Fisheries xxx (xxxx) xxx
2.3. Phytochemical assay
The qualitative phytochemical assay was carried out on flavonoids,
tannins, saponins, quinones, steroids, and triterpenoids, following
methods employed by (Harborne, 1987; Ningsih et al., 2016; Suratno,
2016). The procedure of the assay was as follows. (a) Flavonoids. 2 mL
E. cottonii extract was diluted into 2 mL methanol, followed by the
addition of mg powder and 5 drops of concentrated HCl. The presence of
flavonoid compounds was shown by the formation of red or orange
color. (b) Alkaloids. 2 mL chloroform and 2 mL ammonia were added
into E. cottonii extract, followed by filtration. Three to five drops of
concentrated H2SO4 was added into the filtrate. The solution was mixed
until it formed two layers. 2.5 mL top layer was transferred into three
test tubes. The three solutions were analyzed with 4 drops of Mayer,
Dragendorff, and Wagner reagents. The formation of sediment indicated
the alkaloid content of the sample. Reaction with Mayer reagent resulted
in white sediment. Reaction with Dragendorff resulted in orange-red
sediment. Reaction with Wagner reagent resulted in brown sediment.
(c) Tannins. 10 drops of FeCl3 10% were added into a certain mL
E. cottonii extract, followed by mixing for 1 min before the addition of 2
drops HCl 1 N. The extract was said positively containing tannins when
producing a blackish-green or blackish-blue color. (d) Saponins. A
certain mL of E. cottonii extract was added into 10 mL water with 1-min
shaking, followed by the addition of 2 drops HCl 1 N. The extract
positively contained saponins when the formed foams remained stable
for 7 min. (e) Quinones. NaOH 1 N was added into a certain amount of
sample, and color change was observed. A positive reaction was shown
by the formation of a yellow color. (f) Steroids and triterpenoids. 10
drops of glacial CH3COOH and 2 drops of concentrated H2SO4 were
added into an E. cottonii extract. The solutions were shaken and left to
stand for minutes. A positive reaction of steroids was identified when
green or blue color was produced, while a positive reaction of triterpe­
noid was identified when red or purple color was produced.
2.4. Identification of active compounds
The identification of active compounds was carried out by quanti­
tatively measuring metabolite compounds from the result of the
phytochemical assay with spectrophotometer and LC scanner. Also,
other compounds were identified with and LC-MS. Identification of the
compounds in the extract used LC-MS with the following process con­
ditions: Column ACQUITY UPLC @ BEH C18 1.7 μm; Mobile phase: (a)
ammonium formate, (b) acetonitrite, (c) methanol, and (d) water; Flow:
water (+0.01% FA + 0.05% ammonia); Injection volume: 8 μL; Column
temperature: 28.2 ◦ C; Flow rate: 0.20 mL/min; Analysis time: 23.20 min.
2.5. Data analysis
The results from an assay with LC-MS were processed in MassLynx
V4.1 software. The compounds found were interpreted using Chems
pider.com.
3. Results
3.1. Phenolic content as a result of extraction
The total phenolic content of E. cottonii extract was 4.60 ± 0.06 mg/
GAE g, produced at a temperature of 30 ◦ C for 80 min. Total phenolic
represented the active compounds of E. cottonii extract.
3.2. Toxicity
The toxicity assay of E. cottonii showed an L50 value of 221.71 ± 5.74
μg/mL. This suggested that the E. cottonii extract had bioactivity, as
reported by Meyer et al. (1982) that a compound has bioactivity if its
LC50 < 1000 μg/mL. Thus the appropriate dose settings of the extract
N. Purbosari et al. Aquaculture and Fisheries xxx (xxxx) xxx

can serve as an anesthetic agent. 4. Discussion

3.3. Phytochemicals 4.1. Extraction result: total phenol

Results of the phytochemical assay on E. cottonii showed that the
extracts contained secondary metabolite compounds, e.g. flavonoids
and saponins. The result of measuring the number of flavonoids com­
pounds using spectrophotometers and saponins uses a consecutive TLC
scanner of 24.48% and 0.22%.
3.4. Active compounds in E. cottonii extract
Identification of active compounds in E. cottonii extract carried out in
LC-MS exhibited the presence of other compounds as presented in
Table 1. Various compounds have been identified with various bioac­
tive, and many more compounds that have not been exposed may still be
found in this extract.
In extraction, water was selected as a solvent conforming to the pre-
elementary study conducted. Water as a polar solvent has been shown to
produce a relatively high content of active compounds from E. cottonii
extract. Also related to its application in aqueous media, the extract with
a water solvent is the best choice. When compared with the Results of
existing studies, the research provides more value, where water was an
effective solvent in extracting phenols as active compounds. This means
that the extraction process is safer and economical. Total phenol of 4.60
± 0.06 mg/GAE g accrued on 30 ◦ C and 80 min. It means the effective
condition producing phenol. Extraction temperature and time affected
the total phenol content (Sulaiman et al., 2017).
Total phenol is made as an extraction result indicator because in this
research the phenol is defined as a compound representing the active
ingredient in the E. cottonii extract. It refers to several research Results
where polyphenols from brown seaweed E. cava have anesthetic or
sedative activity (Cho et al., 2012). Some other types of seaweed are also
related to polyphenols and derivatives such as alkaloids, flavonoids,

Table 1
Results of E. cottonii active compound identification in LC-MS.
No Molecular Molecular Molecular Retention % Bioactivity References
formula mass (Da) time (min) peak

Flavonoid Compounds
1 Phthalic anhydride C8H4O3 148.016 16.84 100 Antimicrobial Jafari et al. (2017)
Okada-Junior et al.
(2018)
2 6,7,8,9-tetrahydrobenzo [7]annulen-5- C11H12O 160.212 16.95 50 Anticancer Kaur et al. (2015);
one (1-Benzosuberone) Farghaly et al. (2016)
3 2-Thienyl acetic acid C6H6O2S 142.176 15.42 50 Pesticide Abele and Lukevics
(2001)
Analgesic*, antidepressant*, Singh et al., (2016)
antioxidant
4 9,12,15-Octadecatrien-1-ol C18H32O 264.446 14.28 50 Antidiarrheal Mulyono et al., (2013)
5 3,4-Ethylenedioxythiophene C6H6O2S 142.176 14.28 50 Antitumor Jukić et al. (2017)
6 2-Furoic acid C5H4O3 112.084 0.85 90 Antimicrobial Badr and Barwa (2011)
7 Coumarandione C8H4O3 148.016 0.85 90 Antitubercular Cirillo et al., (2018)
8 2-Benzofuran-4,7-dione C8H4O3 148.016 2.72 80 Antimicrobial Sukdolak et al., (2008)
9 2-Pentyl thiophene C9H14S 154.273 2.72 50 Antimicrobial and antifungal Jaoudeh et al., (2015)
10 N-(2-Cyanophenyl)-2-{[3- C12H10N4OS3 322.429 3.05 50 Analgesic, antifungal Ziyaev and Ismailova
(methylsulfanyl)-1,2,4-thiadiazol-5-yl] (2017)
sulfanyl}acetamide
11 N-(1,3-Benzothiazol-2-yl)-2-[(5-methyl- C12H10N4OS3 322.429 3.05 50 Antifungal Zong et al., (2017)
1,3,4-thiadiazol-2-yl)sulfanyl]acetamide
12 2-(Methylthio)pyrimidine-5- C6H6N2OS 154.190 2.72 98 Antimicrobial Angulwar et al. (2019)
carboxaldehyde Anti-imflammatory Zhang et al., (2017)
13 2,3-Dihydropyrazolo[5,1-b][1,3]thiazole- C6H6N2OS 154.190 17.54 100 anti-inflammatory, Antibacterial, Kryshchyshyn et al.,
6-carbaldehyde anticancer (2018)
Antifungi and antibacterial Karrouchi et al., (2018)
14 1-(1,3-Thiazol-2-yl)-1H-tetrazole C4H3N5S 153.165 2.72 65 Antimicrobial Fadhil and Adnan (2015)
15 4-Chloro-7H-pyrrolo [2,3-d]pyrimidine C6H4ClN3 153.569 2.72 65 Anti parasite Khalaf et al., (2014)
16 4-Chloro-1H-pyrazolo [3,4-b]pyridine C6H4ClN3 153.569 2.72 65 Anticancer, antioxidant Karrouchi et al., (2018)
17 4-Chloro-1H-imidazo [4,5-c]pyridine C6H4ClN3 153.569 2.72 65 Antibacterial Gunreddy et al., (2015)
18 Ammonium (1-hydroxyethyl) C3H10N2OS2 154.254 17.54 40 potential anti-acutemyelogenous Odularu and Ojibade
carbamodithioate leukaemia agents et al., (2019)
Saponin Compounds
1 4(Chloro formyl) morpholine C5H8ClNO2 149.576 13.91 40 AntibacterialAntidepressant* Khammas and Hamood.,
(2017);Can et al. (2017)
2 3-Methyl-2-thiophenecarboxylic acid C6H6O2S 142.176 15.42 40 Antibacterial Al-Wathnani et al.,
(2012)
3 5-Pentyl-1,3,4-thiadiazol-2-amine C7H13N3S 171.263 3.05 40 Antibacterial Kumar et al., (2014)
4 2-(Methylsulfanyl)pyrimidin4- C6H6N2OS 154.190 2.72 98 Analgesik* Sawant and Sarode
carbaldehyde (2011)
5 1-Methyl-6-oxa-3-thiabicyclo [3.1.0] C5H8O3S 148.180 2.72 60 Antifungal Ubaid et al., (2016)
hexane 3,3-dioxide
6 3-Nitro-4,5-dihydro-1,2-oxazol-4-yl 2- C3H4N2O5 148.074 2.72 60 Antimicrobial Demirci et al., (2013)
oxide Antidepressant* Kumar et al. (2013);
Kakkar and Narasimhan
(2019).
7 Methyl 3-oxobutanoate C5H8O3 116.115 17.54 40 Antimicrobial and antioxidant Kumar et al., (2016)

3
N. Purbosari et al. Aquaculture and Fisheries xxx (xxxx) xxx

steroids, tannins, saponin, and terpenoids with various bioactivities extract is also a prospective anesthetic agent for fish.
(Mohapatra et al., 2016).
Total phenolic content is different in different species, and even can 4.3. Compounds of phytochemical analysis
be different in the same species as they are affected by the growing
environment of the seaweed. Various literature related to the number of The phytochemical test results showed that the extract of E. cottonii
phenols in various plant extracts that have anesthetic activity is pre­ contains saponins and flavonoid compounds. The use of water as a sol­
sented in Table 2. vent extract in this study suggests that both of these compounds can be
According to Results from previous studies presented in Table 2, the taken effectively by water. However, different findings were reported by
E. cottonii extract showed a lower total phenolic content than that from previous studies. Secondary metabolite flavonoid has been effectively
the same species reported by (Yanuarti et al., 2017). Besides, the extract extracted with ethyl acetic solvent (Ebrahimzadeh et al., 2018) and
studied exhibited a higher total phenolic content when compared to methanol (Chai et al., 2015; Ganesan et al., 2008). Flavonoids are the
extract of different seaweeds, either green seaweed Caulerpa (Shalaby & largest polyphenol group which have a broad spectrum and biological
Shanab, 2013) or seagrass plants (Permana et al., 2016). However, the activity associated with free-radical scavenging and antioxidant (Panche
results of this study were considered better in terms of economic, safety, et al., 2016). Flavonoids have amphiphilic structures as well as local
and environmental aspects, as the present study used water as a solvent anesthetics (Tsuchiya, 2017).
while previous studies used organic solvents which were relatively Saponins are glycoside compounds showing a soap-like property due
expensive and left residues. Based on Table 2, it was noticed that to its capability to form foams when being shaken in the water, for which
terrestrial plant materials showed higher phenolic contents as compared it is called as a natural surfactant. In addition to the water, other solvents
to aquatic products, even though some materials were extracted using that can be used for saponin extraction are hexane, ethyl acetic, and
organic solvents (Zhu, 2009). The best effectivity was suggested by methanol (Septiana & Asnani, 2012). Saponins can kill protozoans and
derris root with water as an extraction solvent. Therefore, following the mollusks and act as antifungal and antiviral agents (Addisu & Assefa,
background of this study in exploring the potential of aquatic products, 2016). Saponin components are responsible for local anesthetic effects
the use of water solvent could be recommended for the extraction of for fish and mollusca. It can cause the plasmolysis of the red blood cells
E. cottonii as a prospective anesthetic. and death to the fish, while flavonoids can render a calming effect
(Tsuchiya, 2017).
The existence of the compound flavonoids and saponins in the
4.2. Toxicity of E.cottonii extract
extract of E. cottonii in this study showed that the extract can be used as a
candidate as a fish anesthetic agent. It supported several previous
The toxic properties of E. cottonii extract allow its use as an anesthetic
research results where flavonoids serve as antioxidants, anti-cancer,
ingredient by regulating its dose (Stehly & Gingerich, 2001). The
anti-inflammatory, and also is a modulation neuroreceptor that will
toxicity of E. cottonii extract of 221.71 ± 5.74 μg/mL was higher than
affect the brain and nerve control that regulates conditions awareness
control, clove oil, showing LC50 of 268.99 ± 1.60 μg/mL. Based on these
(related to anesthesia). Another example, the flavonoids in propolis are
data, the extract of E. cottonii is expected to give a higher toxic effect
shown to be instrumental in increasing some of the biochemical markers
than the clove. Likewise, E. cottonii extract is also expected to give a
associated with oxidative stress in the fish brain. Saponins are toxic to
higher anesthetic effect than the widely used clove oil. The toxicity
fish and amphibians. Saponins can also kill protozoa and mollusks and
shown was higher than that shown by another natural ingredient, bee
act as antiviral and anti-fungal (Addisu & Assefa, 2016).
pollen, reporting a range of 292.53 ± 60.70 μg/mL (Chowdhury et al.,
2017) and was nearly similar to the toxicity of derris root (Derris tri­
4.4. Compounds active in E. cottonii extract
foliate), 211.31 μg/mL (Jiang et al., 2012). As derris root has been
proven to serve as a fish anesthetic, it can be suggested that E. cottonii
Subsequently, an assay to investigate other compounds contained in
the E. cottonii extract was performed in LC-MS. Table 1 showed the
Table 2
various active compound found in the E. cottonii extract. The numerous
Total phenolic content of different plant extracts.
active compounds and extensive bioactivity in the E. cottonii extract
Source of active Solvent Total phenolic References have supported several existing research results. The earliest alleged
compound (mg /GAE g)
extracts that contain antifungal compounds and pesticides also anti-
Caulerpa lentillifera Ethanol 1.30 Nguyen et al. depressant compounds that have an anesthesia activity. The active
(2011) compound “andrographolide” in sambiloto (Andrographis paniculata)
Spirulina plantesis methanol 1.23 Shalaby and
water 0.55 Shanab (2013)
leaves are often used as a fish anesthetic in addition to giving a larvicidal
water:methanol 0.89 activity (Thangavel et al., 2015). Derris root showing larvicidal function
= 50:50 in addition to serving as an insecticide (Jiang et al., 2012; Yi et al., 2012)
Lamun Cymodocea methanol 25.73 ± 1.41 Permana et al. is also frequently used as a fish anesthetic (Olufayo, 2009).
sp (2016)
The main components of the compounds shown are calculated based
E. cottonii ethyl acetic 134.33 Yanuarti et al.
methanol 141.00 (2017) on the percentage of peaks in the chromatogram. The compounds dis­
Sargassum water, (0.11 ± 0.01)– Sedjati et al. (2017) cussed are the flavonoid and saponin class compounds that are thought
methanol, (1.36 ± 0.01) to be responsible for the anesthetic process, namely their bioactivity as
ethanol an antidepressant or analgesic. The main flavonoid class compounds
Sargassum methanol 74.63 Lailiyah et al.
hexane 35.85 (2014)
were 2-Thienyl acetic acid with a high peak of 50% at 15.42 retention
Clove oil ethanol 1300 ± 0.52 Rorong (2008) time, while the main saponin class compounds were 2- (Methylsulfanyl)
Nutmeg fruit ethanol 183.56 ± 8.18 Fawwas et al. pyrimidin4-carbaldehyde, 3-Nitro-4,5-dihydro-1,2-oxazole-4-yl 2-
(2018) oxide, and (Chloro formyl) morpholine with peak lengths of 98%, 60%,
Derris (Derris water 70.90 ± 1.05 Pukumpuang et al.
and 40% at retention times 2.72, 2.72 and 13.91, respectively (Table 1).
scandes) (2012)
ethanol 41.97 ± 12.49 This study showed that the E. cottonii extract contains several antide­
Root of tuba (Derris water 78.84 ± 0.01 Kumkrai et al. pressant or anesthetic compounds so it makes one of the reasons that
riticulata) (2015) E. cottonii can be used as a candidate for an anesthetic agent.
Root of tuba (Derris water 48.85 ± 1.78 Simlai et al. (2017) A variety of compounds identified has raised a new question whether
trifoliata)
the compounds would show an anesthetic activity following saponins

4
N. Purbosari et al.
and flavonoids or would show other activities. The presence of com­
pounds with antidepressant activity strengthens the chance of this
extract as a candidate for anesthetic. Besides that, a great number of
active molecules found in E. cottonii extract with wide bioactivity makes
E. cottonii a good source for pharmacological and health substances. This
becomes a new opportunity for future studies.
However, in general, the results of this research can be said to be
better than the economical, safety, and environmental-friendly side
because the solvent used is water, where the results of other studies
using organic solvents that have a higher price and leave a residue.
Based on the background of this research, to explore the results of the
water, then E. cottonii with solvent water can be recommended as a total
source of phenol which is potentially used as anesthesia.
5. Conclusions
The effective conditions for producing phenol from E. cottonii extract
were extraction at 30 ◦ C and 80 min with the highest amount of phenol
of 4.60 ± 0.06 mg/GAE g. The extract’s toxicity was 221.71 ± 5.74 μg/
mL, indicating that the extract had bioactivity. The phytochemical re­
sults showed that the extract contained 24.48% and 0.22% of flavonoids
and saponins, respectively. The identification results of the compounds
in the extract indicated the presence of several compounds in the
saponin and flavonoid groups that were antidepressants. Based on these
conditions, E. cottonii extract can be used as a safe natural ingredient as
an anesthetic agent.
CRediT author statement
Ninik Purbosari: Conceptualization, Methodology, Investigation,
Validation, Data curation, Software, Writing – original draft; Endang
Warsiki:Conceptualization, Investigation, Validation, Data curation,
Writing – review & editing; Khaswar Syamsu: Conceptualization,
Investigation, Validation, Data curation; Joko Santoso: Conceptualiza­
tion, Investigation, Validation, Data curation.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgment
We appreciate our collegue, Tabligh Permana for his assistance
during data analysis.
References
Abdolazizi, S., Ghaderi, E., Naghdi, N., & Kamangar, B. B. (2011). Effects of clove oil as
an anesthetic on some hematological parameters of Carassius auratus. Journal of
Aquaculture Research & Development, 2(1), 1–3.
Abele, E., & Lukevics, E. (2001). Furan and thiopene oximes: Synthesis, reactions, and
biological activity (review). Chemistry of Heterocyclic Compounds, 37(2), 141–169.
Addisu, S., & Assefa, A. (2016). Role of plant containing saponin on livestock production;
a review. Advances in Biological Research, 10(5), 309–314.
Akinrotimi, O., Ansa, E., & Edun, O. (2016). Effectiveness of clove seed extracts as
anaesthetics in transportation of Tilapia guineensis juveniles. Journal of Aquaculture
Enginering and Fisheries Research, 2(2), 67–75.
Al-Niaeem, K. S., Mohammed, F. A., & Al-Hamadany, Q. H. (2017). The anaesthetic effect
of nutmeg powder, Myristica fragrans on young common carp, Cyprinus carpio.
Biological and Applied Environmental Research, 1(2), 279–286.
Al-Wathnani, H., Ismet, A., Tahmaz, R. R., Al-Dayel, T. H., & Bakir, M. A. (2012).
Bioactivity of natural compounds isolated from cyanobacteria and green algae
against human pathogenic bacteria and yeast. Journal of Medicinal Plants Research, 6
(18), 3425–3433.
Angulwar, J. A., Khansole, G. S., & Bhosale, V. N. (2019). Novel synthesis and
antimicrobial activity of 7-Substituted derivatives of 7-(methylthio)-5- oxo-2-phenyl-
5H-[1,3,4] thiadiazolo[3,2-b]-pyrimidine-6-carbonitrile. Asian Journal of Organic &
Medicinal Chemistry, 4(1), 40–45.
Badr, S. M., & Barwa, R. M. (2011). Synthesis of some new [1,2,4] triazolo[3,4b][1,3,4]
thiadiazines and [1,2,4]triazolo[3,4-b][1,3,4] thiadiazoles starting from 5-nitro-2-
furoic acid and evaluation of their antimicrobial activity. Bioorganic & Medicinal
Chemistry, 9(15), 4506–4512.
5
Aquaculture and Fisheries xxx (xxxx) xxx
Bizarro, Y. W. S., Navarro, F. K. S. P., & Navarro, R. D. (2018). Clove oil, benzocaine and
sodium chloride concentrations during the transport simulation of nile tilapia.
Revista Verde de Agroecologia e Desenvolvimento Sustentável, 13(1), 106–111.
Bolasina, S. N., Azevedo, A.d., & Petry, A. C. (2017). Comparative efficacy of benzocaine,
tricaine methanesulfonate and eugenol as anesthetic agents in the guppy Poecilia
vivipara. Aquaculture Reports, 6, 56–60.
Brown, L. A. (2011). Anaesthesia for fish. Vietfish, 8(2), 68–70.
Can, N. O., Cevik, U. A., Saglik, B. N., Ozkay, Y., Atli, O., Baysal, M., Ozkay, U. D., &
Can, O. D. (2017). Pharmacological and toxicological screening of novel
benzimidazole-morpholine derivatives as dual-acting inhibitors. Molecules, 22
(1374), 1–20.
Chai, T. T., Kwek, M. T., Ismail, N. I. M., Ooi, J. L. S., Amri, A. Y., Manan, F. A.,
Law, Y. C., & Wong, F. C. (2015). Antioxidant activities of methanol extract and
solvent fractions of marine macroalga, Avrainvillea erecta (Berkeley) A. Gepp and E.S.
Gepp (Dichotomosiphonaceae). Tropical Journal of Pharmaceutical Research, 14(3),
503–509.
Chance, R., Cameron, G. A., Fordyce, M., Noguera, P., Wang, T., Collins, C.,
Secombes, C. J., & Collet, B. (2018). Effects of repeated anaesthesia on gill and
general health of Atlantic salmon, Salmo salar. Journal of Fish Biology, 93(6),
1069–1081.
Chowdhury, M. A. R., Manirujjaman, & Haq, M. M. (2017). Phytochemical and
pharmacological activity of Myristica fragrans houtt (myristicaceae). International
Journal of Toxicological and Pharmacological Research, 9(1), 56–63.
Cho, S., Yang, H., Jeon, Y. J., Lee, C. J., Jin, Y. H., Baek, N. I., Kim, D., Kang, M.,
Yoon, M., Yong, H., Shimizu, M., & Han, D. (2012). Phlorotannin of edible brown
seaweed Ecklonia cava kjellman induced sleep via positive allosteric modulation of
gamma-aminobutyric acid type A-benzodiazepine receptor: A novel neurological
activity of seaweed polyphenols. J.of Food Chem., 132, 1133–1142.
Cirillo, D., Borroni, E., Festoso, I., Monti, D., Romeo, S., Mazier, D., & Verotta, L. (2018).
Synthesis and antimycobacterial activity of (+)- usnic acid conjugates. Arch. Pharm.
Chemistry of Life Science, 351(12), 1–9.
Demirci, S., Basoglu, S., Bozdereci, A., & Demirbas, N. (2013). Preparation and
antimicrobial activity evaluationof some new bi- and triheterocyclic azoles.
Medicinal Chemistry Research, 22, 4930–4945.
Ebrahimzadeh, M. A., Khalili, M., & Dehpour, A. A. (2018). Antioxidant activity of ethyl
acetate and methanolic extracts of two marine algae, Nannochloropsis oculata and
Gracilaria gracilis-an in vitro assay. Brazilian Journal of Pharmaceutical Sciences, 54(1),
1–6.
Ekeanyanwu, R. C., & Njok, O. U. (2014). Acute and subacute oral toxicity study on the
flavonoid rich fraction of Monodora tenuifolia seed in albino rats. Asian Pacific Journal
of Tropical Biomedicine, 4(3), 194–202.
Fadhil, Z., & Adnan, S. (2015). Synthesis and identification of some new tetrazole
derivates from 4,5-dichloro imidazole and study of their biological activity. Journal
of Chemistry Chemical Engineering, 9, 347–352.
Farghaly, T. A., Gomhaa, S. M., Sayed, A. R., & Khedr, M. A. (2016). Hydrazonoyl halides
as precursors for synthesis of bioactive thiazole and thiadiazole derivatives:
Synthesis, molecular docking and pharmacological study. Current Organic Synthesis,
13, 1–11.
Fawwaz, M., Nurdiansyah, S., & Baits, M. (2018). Potensi daun pala (Myristica fragrans
houtt) sebagai sumber fenolik. [The potential of nutmeg leaves (Myristica fragrans
houtt) as a phenolic source]. Jurnal Fitofarmaka Indonesia, 4(1), 212–214.
Ganesan, P., Kumar, C. S., & Bhaskar, N. (2008). Antioxidant properties of methanol
extract and its solvent fractions obtained from selected Indian red seaweeds.
Bioresource Technology, 99, 2717–2723.
Gunreddy, A. R., Maroju, R. C., Pochampalli, J., & Eppakayala, L. (2015). Antimicrobial
activity of oxadiazole, thiazolidin-4-one and azetidin-2-one derivatives of 1H-
Imidazo[4,5-b]pyridine. Research Journal of Pharmaceutical. Biological and Chemical
Sciences, 6(1), 1402–1406.
Harborne, J. (1987). Metode fitokimia. Penuntun cara modern menganalisis tumbuhan. Edisi
kedua [Chemical methods Modern way guide to analyzing plants. 2nd ed]. ITB Pr.
Bandung.
Hedayati, A. A. (2018). Effects of 2-phenoxyethanol (2-PE) anesthesia on some
haematological and biochemical indices of silver carp (Hypophthalmichthys molitrix).
Iranian Journal of Fisheries Sciences, 17(1), 1–10.
Jafari, E., Najafabadi, N. T. J., Najafabadi, A. J., Poorirani, S., Hassanzadeh, F., &
Rizi, S. S. (2017). Synthesis and evaluation of antimicrobial activity of cyclic imides
derived from phthalic and succinic anhydrides. Research in Pharmaceutical Sciences,
12(6), 526–534.
Jaoudeh, C. A., Baydoun, S., & Arnold, N. (2015). Essential oil and bioactivity of the
Ziziphora canescens benth. Growing wild in Lebanon. Pharmacognosy Communications,
5(4), 265–269.
Jawale, C. S. (2014). Larvicidal activity of some saponin containing plants against the
dengue vector Aedes aegypti. Trends in Biotechnology Research, 3(1), 1–11.
Jiang, C., Liu, S., He, W., Luo, X., Zhang, S., Xiao, Z., Qiu, X., & Yin, H. (2012). A new
prenylated flavanone from Derris trifoliata Lour. Molecules. 17, 657–663.
Jukić, M., Rastija, V., Opačak-Bernardi, T., Stolić, I., Krstulović, L., Bajić, M., & Glavaš-
Obrovac, L. (2017). Antitumoractivity of 3,4-ethylenedioxythiophene derivatives
and quantitative structure-activity relationship analysis. Journal of Molecular
Structure, 1133, 66–73.
Kakkar, S., & Narasimhan, B. (2019). A comprehensive review on biological activities of
oxazole derivatives. BMC Chemistry, 13(16), 1–24.
Karrouchi, K., Radi, S., Ramli, Y., Taoufik, J., Mabkhot, Y. N., Al-aizari, F. A., & Ansar, M.
(2018). Synthesis and pharmacological activities of pyrazole derivatives: A review.
Molecules, 23(134), 1–85.
N. Purbosari et al.
Kaur, M., Kohli, S., Sandhu, S., Bansal, Y., & Bansal, G. (2015). Coumarin: A promising
scaffold for anticancer agents. Anti-Cancer Agents in Medicinal Chemistry, 15(8),
1032–1048.
Khalaf, A. I., Huggan, J. K., Suckling, C. J., Gibson, C. L., Stewart, K., Giordani, F.,
Barrett, M. P., Wong, P. E., Barrack, K. L., & Hunter, W. N. (2014). Structure-based
design and synthesis of antiparasitic pyrrolopyrimidines targeting pteridine
reductase. Journal of Medicinal Chemistry, 57, 6479–6494.
Khammas, S. J., & Hamood, A. J. (2017). Synthesis and evaluation of the biological
activity of new 4-(2-Chloroacetyl) morpholine derivatives. Journal of Chemical and
Pharmaceutical Research, 9(9), 146–158.
Kryshchyshyn, A., Roman, O., Lozynskyi, A., & Lesyk, R. (2018). Thiopyrano [2,3-d]
thiazoles as new efficient scaffolds in medicinal chemistry. Scientia Pharmaceutica, 86
(26), 1–24.
Kumala, S., & Sapitri, D. W. (2011). Phytochemical screening and toxicological
evaluation using brine shrimp lethality test (BSLT) of some fractions of prasman
leaves (Eupatorium triplinerve v) extract. Indonesian Journal of Cancer
Chemoprevention, 2(1), 194–197.
Kumar, J., Chawla, G., Akhtar, M., Sahu, K., Rathore, V., & Sahhu, S. (2013). Design,
synthesis and pharmacological evaluation ofsome novel derivatives of 1-{[3-(furan-
2-yl)-5-phenyl-4,5-dihydro-1,2-oxazol-4-yl]methyl}-4-methyl piperazine. Arabian
Journal of Chemistry, 10(1), 141–149.
Kumar, S., Sharmaa, S. K., Jaina, S., Kumara, A., & Jain, N. (2014). Synthesis and
antibacterial activities of some N-(p-substituted benzylidene)-5-pentyl-1,3,4-
thiadiazole-2-amines. Der Pharma Chemica, 6(1), 85–89.
Kumkrai, P., Weeranantanapan, O., & Chudapongse, N. (2015). Antioxidant,
α-glucosidase inhibitory activity andsub-chronic toxicity of Derris reticulata extract:
Its antidiabetic potential. BMC Complementary and Alternative Medicine, 15(35), 1–10.
Lailiyah, A., Adi, T. K., Hakim, A., & Yusnawan, E. (2014). Kapasitas antioksidan dan
kandungan total senyawa fenolik ekstrak kasar alga coklat Sargassum cristaefolium
Dari pantai Sumenep Madura. [Antioxidant capacity and total content of phenolic
compounds in the crude extract of brown algae Sargassum cristaefolium from the
coast of Sumenep Madura]. ALCHEMY, 3(1), 18–30.
Lim, C. L., Koh, R. Y., Haw, T. Y., & Boudville, L. A. (2015). Antioxidant activity of the
sea bird nest (Eucheuma cottonii) and its radical scavenging effect on human
keratinocytes. Journal of Medical Bioengineering, 4(6), 46–465.
Matanjun, P., Mohamed, S., Mustapha, N. M., Muhammad, K., & Ming, C. H. (2008).
Antioxidant activities and phenolics content of eight species of seaweeds from north
Borneo. Journal of Applied Phycology, 20(4), 367–373.
Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jasobsen, L. B., Nichols, D. E., &
McLaughlin, J. L. (1982). Brine shrimp: A convenient general bioassay for active
plant contituents. Journal of Medicinal Plants Research, 45, 31–34.
Mohapatra, L., Bhattamisra, S. K., Panigrahy, R. C., & Parida, S. K. (2016). Evaluation of
the antioxidant, hypoglycaemicand antidiabetic activities of some seaweed collected
from the east coast of India. Biomedical & Pharmacology Journal, 9(1), 365–375.
Mulyono, N., Lay, B. W., Ocktreya, L., & Rahayu, S. (2013). Antidiarrheal activity of Apus
bamboo (Gigantochloa apus) leaf extract and its bioactive compounds. American
Journal of Microbiology, 4(1), 1–8.
Namvar, F., Mohamed, S., Fard, S. G., Behravan, J., Mustapha, N. M., Alitheen, N. B. M.,
& Othman, F. (2012). Polyphenol-rich seaweed (Eucheuma cottonii) extract
suppresses breast tumour via hormone modulation and apoptosis induction. Food
Chemistry, 130(2), 376–382.
Neto, O. S., Gomes, S. A., Soares, A. R., da Machado, S. F. L., Samuels, R. I., Rodrigo, N.,
da Fonseca, R. N., Menezes, J. S., da Moraes, J. L. C., Campos, E., Mury, F. B., &
Silva, J. R. (2016). Larvicidal potential of the halogenated sesquiterpene (+)-
bbtusol, isolated from the Alga Laurencia dendroidea J. Agardh (Ceramiales:
Rhodomelaceae), against the dengue vector mosquito Aedes aegypti (Linnaeus)
(Diptera:Culicidae). Marine Drugs, 14(2), 1–14.
Nguyen, V. T., Uenag, J. P., & Tsai, G. J. (2011). Proximate composition, total phenolic
content, and antioxidant activity of seagrape (Caulerpa lentillifera). Journal of Food
Science, 76(7), C950–C958.
Ningsih, D. R., Zusfahair, & Kartika, D. (2016). Identifikasi senyawa metabolit sekunder
serta uji aktivitas daun sirsak sebagai antibakteri.[Identification of secondary
metabolite compounds and the activity test of soursop leave as antibacterial].
Molekul, 11(1), 101–111.
Okada-Junior, C. Y., Monteiro, G., Batista, V. S., de Souza, J. O., Souza, G. E.,
Bueno, R. V., Oliva, G., Nascimento-Júnior, N. M., Guido, R. V. C., & Yassuo, V. S. B.
(2018). Phthalimide derivatives with bioactivity against Plasmodium falciparum:
Synthesis, evaluation, and computational studies involving bc1 cytochrome
inhibition. ACS Omega, 3, 9424–9430.
Olufayo, M. O. (2009). Haematological characteristics of Clarias gariepinus (Burchell
1822) juveniles exposed to Derris elliptica root powder. African Journal of Food,
Agriculture, Nutrition and Development, 9(3), 920–933.
Panche, A. N., Diwan, A. D., & Chandra, S. R. (2016). Flavonoids: An overview. Journal of
Nutritional Science.5(7), 1–15.
Permana, A. H. C., Husni, A., & Budhiyanti, S. A. (2016). Aktivitas antioksidan dan
toksisitas ekstrak lamun Cymodocea sp. [Antioxidant activity and toxicity of seagrass
extract Cymodocea sp]. Jurnal Teknologi Pertanian, 17(1), 37–46.
Pukumpuang, W., Thongwai, N., & Tragoolpua, Y. (2012). Total phenolic contents,
antibacterial and antioxidant activities of some Thai medicinal plant extracts.
Journal of Medicinal Plants Research, 6(35), 4953–4960.
Aquaculture and Fisheries xxx (xxxx) xxx
Rorong, J. A. (2008). Uji aktivitas antioksidan Dari daun cengkeh (Eugenia carryophyllus)
dengan metode DPPH. [Assay of the antioxidant activity of the Clove leaf (Eugenia
carryophyllus) with the DPPH method]. Jurnal Chemical Processing, 1(2), 111–116.
Saini, V. P., Kamble, A. D., Ojha, M. L., & Raosaheb, S. S. (2018). Anesthetic efficacy of
clove oil in the transportation of carp (Cyprinus carpio) seed. Journal of Entomology
and Zoology Studies, 6(5), 2397–2402.
Sawant, R., & Sarode, V. (2011). Synthesis, spectral characterization and analgesic
activity of 2-methylthio-1,4-dihydropyrimidines. Iranian Journal of Pharmacheutical
Research, 10(4), 733–739.
Sedjati, S., Suryono, Santosa, A., Supriyantini, E., & Ridlo, A. (2017). Aktivitas
antioksidan dan kandungan senyawa fenolik makroalga coklat Sargassum sp. Jurnal
Kelautan Tropis, 20(2), 117–123.
Selvan, B. K., Piriya, P. S., Chandrasekhar, M., & Vennison, S. J. (2014). Macro algae
(Eucheuma cottonii and Sargassum sp.) are reservoirs of biodisel and bioactive
compounds. Journal of Chemical and Pharmaceutical Sciences, 2, 62–70.
Septiana, A. T., & Asnani, A. (2012). Kajian sifat fisikokimia ekstrak rumput laut coklat
Sargassum duplicatum menggunakan berbagai pelarut dan metode ekstraksi. [Study of
physicochemical properties of brown seaweed extract Sargassum duplicatum using various
solvents and extraction methods]. AGROINTEK, 6(1), 22–28.
Shalaby, E. A., & Shanab, S. M. M. (2013). Comparison of DPPH and ABTS assays for
determining antioxidant potential of water and methanol extracts of. Spirulina
platensis. Indian Journal of Geo-Marine Sciences, 42(5), 556–564.
Simlai, A., Gangwar, A., Ghonge, S. A., & Roy, A. (2017). Antimicrobial and antioxidative
activities in the stem extracts of. Derris trifoliata, a mangrove shrub. ournal of
Pharmaceutical Research International, 17(3), 1–10.
Singh, N., Ranjana, R., Kumari, M., & Kumar, B. (2016). A review on biological activities
of hydrazone derivatives. International Journal of Pharmaceutical Chemistry Research,
8(3), 162–166.
Soliman, J. L., Lim, N. R. E. G., Mercado, J. P. D., Lagurin, M. R. C., Felix, C. B., &
Ubando, A. T. (2017). Effects of temperature and pressure on the vacuum drying
characteristics of red seaweed Kappaphycus alvarezii. In IEEE 9th international
conference on humanoid, nanotechnology, information technology, communication and
control, environment and management (HNICEM).
Stehly, G. R., & Gingerich, W. H. (2001). Evaluation of AQUI-STM (Efficacy and minimum
toxic concentration) as a fish anaesthetic/sedative for public aquaculture in the
United States. Aquaculture Research, 30(5), 365–372.
Sulaiman, I. S. C., Mahiran, B., Masoumi, H. R. F., Chee, W. J., Ashari, S. E., & Ismail.
(2017). Effects of temperature, time, and solvent ratio on the extraction of phenolic
compounds and the anti-radical activity of Clinacanthus nutans Lindau leaves by
response surface methodology. Chemistry Central Journal, 11(54), 1–11.
Suratno. (2016). Skrining fitokimia ekstrak ethanol mikroalga Spirulina platensis yang
berpotensi sebagai antibakteri. [Phytochemical screening of microbial extracts of
microalgae Spirulina platensis as potentially antibacterial]. Jurnal Surya Medika, 1(2),
26–33.
Sutili, F. J., Gressler, L. T., & Baldisserotto, B. (2014). Clove oil, eugenol effective
anesthetics for silver catfish, other Brazilian species. Global Aquaculture Advocate,
71–72.
Thangavel, M., Umavathi, S., Thangam, Y., Thamaraiselvi, A., & Ramamurthy, M.
(2015). GC-MS analysis and larvicidal activity of Andrographis paniculata (Burm.F)
Wall. Ex Nees. Against the dengue vector Aedes aegypti (L) (Diptera: Culicidae).
International Journal Current Microbiolology Applied Science, 4, 392–403.
Tsuchiya, H. (2017). Anesthetic agents of plant origin: A review of phytochemicals with
anesthetic activity. Molecules, 22(1369), 1–34.
Ubaid, J. M., Hussein, H. M., & Hameed, I. H. (2016). Determination of bioactive
chemical composition of Callosobruchus maculutus and investigation of its anti-fungal
activity. International Journal of Pharmacognosy and Phytochemical Research, 8(8),
1293–1299.
Wulandari, D., Kilawati, Y., & Fadjar, M. (2018). Activity of compounds on seaweed
Eucheuma cottonii extract as antioxidant candidate to prevent effects of free radical in
water pollution. Research Journal of Life Science Res. J. Life Sci., 5(3), 173–182.
Yanuarti, R., Nurjanah, Anwar, E., & Hidayat, T. (2017). Profil fenolik dan aktivitas
antioksidan Dari ekstrak rumput laut Turbinaria conoides dan Eucheuma cottonii.
[Phenolic profiles and antioxidant activities of Turbinaria conoides dan Eucheuma
cottonii seaweed extracts]. Jurnal Pengolahan Hasil Perikanan Indonesia. 2017, 20
(2), 230–237.
Yi, F., Zou, C., Hu, Q., & Hu, M. (2012). The joint action of destruxins and botanical
insecticides (Rotenone, Azadirachtin, and Paeonolum) against the cotton aphid, Aphis
gossypii glover. Molecules, 17, 7533–7542.
Zhang, Y., Luo, L., Han, C., Handeng, L., Chen, D., Shen, G., Wu, K., Pan, W., & Ye, F.
(2017). Design, synthesis, and biological activity oftetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidinederivatives as anti-inflammatory agents. Molecules, 22(11), 1–22.
Zhu, W. (2009). Cytotoxic and antioxidative phenolic compounds from the traditional
Chinese medical plant. Myristica fragrans. Planta Medica, 75, 1241–1245.
Ziyaev, A. A., & Ismailova, D. S. (2017). Biological activity of 5-(2,3,4-pyridyl)-1,3,4-
oxadiazol-2- thiones and their derivatives. World Journal of Pharmaceutical Research,
6(4), 52–77.
Zong, G., Yan, X., Bi, J., Jiang, R., Qin, Y., Yuan, H., Lu, H., Dong, Y., Jin, S., & Zhang, J.
(2017). Synthesis, fungicidal evaluation and 3D-QSAR studies of novel 1,3,4-thia­
diazole xylofuranose derivatives. PloS One, 12(7), 1–16.