Haiga Sophia Gunawan - V3720027

Uji Toksisitas Subkronis Fraksi Etil Asetat Kulit Buah Asam Kandis (Garcinia cowa
Roxb.) terhadap Fungsi Hati dan Ginjal Mencit Putih Betina
Metode
Mencit putih jantan yang berumur 2-3 bulan dengan

berat badan 20-30 gram
Dikelompokkan
Kelompok Kelompok Uji 10 Kelompok Uji 10 Kelompok Uji 10

Kontrol 10 ekor ekor mencit ekor mencit ekor mencit
mencit
Diaklimatisasi selama 7 hari
Hewan uji yang sudah

diaklimatisasi
Perencanaan Dosis dan Pengelompokan Hewan

mencit
Diberikan tween Diberikan larutan Diberikan larutan Diberikan

80 5% uji dosis 500 uji dosis 500 larutan uji
mg/KgBB mg/KgBB dosis 500
mg/KgBB
Hari ke-31 Cek kreatinin

serum
Mencit
Hari ke-61 Cek SPGT
Rasio BB organ
Penyiapan Sediaan Uji
Fraksi etil asetat kulit Ditambahkan Ditambahkan

Tween 80 Aquadest
buah asam kandis
5%
Ditimbang
sesuai dosis
Hasil
Pemeriksaan Fungsi Hati
1 mL reagen I
Ditambahkan
Microbe serum 0,1 ml
Didiamkan 5
menit
Campuran
Ditambahkan
0,25 mL reagen II hingga tercampur

homoghen
Setelah 1 menit, serapan diukur dengan

spektrometer uv-visible pada panjang gelombang
365 nm setiap menit selama 3 menit
Campuran
dihitung selisih rata-rata serapan tiap

menit
Campuran
Kenaikan aktivitas SGPT dapat dihitung

Hasil
Pemeriksaan Fungsi Ginjal
50 µL serum
Dipipet
1 mL reagen I
Ditambahkan dalam tabung dan

diamkan selama 5 menit
0,25 mL reagen II
Dicampurkan
Campuran
Diukur absorban dengan

spertrofotometer uv-visible pada
panjang gelombang 492 nm
Campuran
Dihitung kadar
kreatinin
Hasil
Penetuan Rasio Berat Organ Hati dan Ginjal

mencit
Hari ke-33 dan ke-60 di

lakukan pengambilan darah
mencit organ hati dan ginjal
Mencit
Dibersihkan
Mencit
Ditimbang
Mencit
ditentukan berat organ relatif

terhadap berat masing-masing
hewan
Mencit
Dihitung
Rasio BB organ
Analisis Data
Kadar kreatinin dan perbandingan

rasio berat organ
Dianalisis ANOVA dua arah
Kadar kreatinin dan perbandingan

rasio berat organ
Uji wilayah berganda Duncan’s

Multiple Range Test (DMRT)
Hasil
Latar Belakang
Di bidang kesehatan Indonesia memiliki potensi dalam mengembangkan obat herbal

yang berbasis pada tumbuhan obat karena merupakan salah satu negara yang memiliki
keanekaragaman hayati terbesar. Contohnya tanaman dari genus Garcinia kaya akan
metabolit sekunder terutama triterpen, flavonoid, santon dan phloroglucinol. Senyawa-
senyawa yang telah diisolasi dilaporkan memiliki berbagai aktivitas farmakologis, seperti
aktivitas antikanker, antiinflamasi, antibakteri, antivirus, antijamur, antiHIV, antidepresan,
dan antioksidan. Garcinia cowa Roxb., atau asam kandis, adalah pohon berukuran sedang
dengan buah-buahan yang dapat dimakan, telah digunakan masyarakat sebagai obat disentri,
antipiretik dan anti-inflamasi. Beberapa penelitian melaporkan bahwa senyawa santon,
benzofenon, dan derivat acylphloroglucinol telah berhasil diisolasi dari G. Cowa, dimana
senyawa santon sendiri telah dikenal dengan potensi efek sitotoksiknya. Ekstrak etanol kulit
buah asam kandis (Garcinia cowa Roxb.) diketahui memiliki efek sitotoksik terhadap sel
kanker payudara T47D. Fraksi etil asetat kulit buah asam kandis memiliki efek sitotoksik
terhadap sel kanker serviks HeLa dengan nilai IC50 16,194±3,5019 µg/ mL. Untuk menjamin
keamanan tumbuhan obat, sangatlah penting mengetahui batas keamanan atau ketoksikannya.
Uji toksisitas yang digunakan adalah uji toksisitas subkronis. Data ini digunakan bertujuan
untuk menentukan sifat dan tempat efek toksik dan menentukan kadar tanpa efek samping
yang sering disebut no observed adverse effect level (NOAEL). Penelitian tentang keamanan
fraksi etil asetat kulit buah asam kandis pada tingkat toksisitas sub akut telah dilakukan.
Penelitian sebelumnya mengatakan bahwa fraksi etil asetat kulit buah asam kandis aman
untuk digunakan karena tidak mempengaruhi kadar SGPT hati dari mencit putih secara
langsung setelah pemberian selama 21 hari, namun dipengaruhi secara bermakna terhadap
besaran dosis yang diberikan. Uji toksisitas subkronis adalah uji ketoksikan suatu senyawa
yang diberikan dengan dosis berulang pada hewan uji tertentu, selama 1 sampai 3 bulan. Pada
pengamatan ini yang diperhatikan dalam uji toksisitas sub kronik yaitu fungsi organ seperti
hati dan ginjal setelah pemberian fraksi selama 60 hari. Hati merupakan organ yang berperan
dalam fungsi metabolisme dan ekskresi toksik dalam tubuh. Jika terjadi gangguan hati
ditandai dengan peningkatan aktivitas serum transaminase berupa SGPT (Serum Glutamic
Piruvic Transaminase) pada serum. Ginjal merupakan organ sasaran utama dari efek toksik
karena ginjal menghasilkan urin yang merupakan jalur utama ekskresi toksikan dan
mempunyai volume aliran darah yang tinggi. Salah satu indikator terjadi kerusakan ginjal
adalah terjadi peningkatan atau penurunan kadar kreatinin dalam tubuh maka interpretasi
klinik akan lebih cenderung pada gangguan fungsi ginjal.
Tujuan
Untuk melakukan penelitian lanjutan mengenai uji toksisitas subkronik Fraksi Etil Asetat
Kulit Buah Asam Kandis (Garcinia cowa Roxb.) terhadap Fungsi Hati dan Ginjal Mencit
Putih Betina menggunakan parameter aktivitas SGPT dan rasio berat hati untuk fungsi hati
dan aktivitas kreatinin serum dan rasio berat ginjal untuk aktivitas ginjal.
Hasil
Aktivitas SGPT rata-rata kelompok mencit kontrol dan kelompok yang diberi sediian uji
dengan dosis 500 mg/kgBB, 1000 mg/kgBB berturut-turut adalah 25,888±3.91, 46,918±6,56,
48,733±5,066, sedangkan aktivitas SGPT rata–rata pada hari ke 31, dan 61 berturut–turut
adalah 32,872±3,21, 46,198±5,71. Aktivitas SGPT mencit putih betina dipengaruhi secara
bermakna oleh lama pemberian (p<0,05) dan dosis sediaan uji (p<0,05)
Pengujian fraksi etil asetat kulit buah asam kandis terhadap kadar kreatinin serum
mencit putih betina
Kadar kreatinin serum mencit putih Betina tidak dipengaruhi secara bermakna oleh lama
pemberian (p>0,05) namun dipenagruhi secara bermakna oleh dosis (p>0,05). Kadar
kreatinin rata-rata kelompok mencit kontrol dan kelompok yang diberi sedian uji dengan
dosis 500 mg/kgBB, dan 1000 mg/kgBB berturut-turut adalah 0, 5333 ±0.843, 0,640±0,95,
05,00±0,68. Sedangkan kadar kreatinin rata–rata pada hari ke 31, dan 61 berturut turut adalah
5,777±0,84 dan 5,377±0,481.
Pengujian fraksi etil asetat kulit buah asam kandis terhadap rasio berat organ hati
mencit putih betina
Rasio berat organ hati dipengaruhi secara bermakna oleh lama pemberian (p<0,05) tetapi
tidak dipengaruhi secara bermakna oleh dosis (p>0,05). Rasio organ hati rata-rata kelompok
mencit kontrol dan kelompok yang diberi sedian uji dengan dosis 500 mg/kgBB, 1000
mg/kgBB berturut-turut adalah 0,406±0,003, 0,412±0,002, 0,034±0,007 Sedangkan rasio
organ hati rata–rata pada hari 0,036±0,0015 , 0,043±0,004.
Pengujian fraksi etil asetat kulit buah asam kandis terhadap rasio berat organ ginjal
mencit putih betina
Rasio berat organ ginjal tidak dipengaruhi secara bermakna oleh lama pemberian (p>0,05)
dan tidak di pengaruhi secara bermakna oleh dosis (p>0,05). Sedangkan tidak terdapat
pengaruh yang bermakna terhadap interaksi antara lama pemberian dan dosis (p>0,05). Rasio
organ ginjal rata-rata kelompok mencit kontrol, dan kelompok yang diberi sediaan uji dengan
dosis 500 mg/kgBB, 1000 mg/kgBB berturut-turut adalah 0,036±0,015, 0,012±0,001,
0,015±0,008. Sedangkan rasio organ hati rata-rata pada hari ke 31dan 61 berturut-turut adalah
0,019±0,08 dan 0,026±0,008.
Hasil penelitian pengaruh fraksi etil asetat kulit buah asam kandis terhadap rasio berat
organ ginjal mencit putih betina
Berdasarkan pengujian statistik dengan uji analisis variasi dua arah dari data pengujian fungsi
ginjal diketahui bahwa kadar kreatinin dipengaruhi secara bermakna oleh dosis (p<0,05).
Selain itu interaksi antara faktor lama pemberian dan dosis sediaan uji tidak memberikan
pengaruh yang bermakna (p<0,05). Setelah dilakukan uji lanjut Duncan’s Multiple Range
Test (DMRT) terhadap faktor dosis memang terjadi perbedaan aktivitas kadar kreatinin
serum pada kelompok kontrol dan kelompok dosis.
Kesimpulan
Dosis pemberian fraksi etil asetat kulit buah asam kandis memberikan pengaruh yang
bermakna terhadap aktivitas SGPT dan terhadap kadar kreatinin serum mencit putih betina.
Lama pemberian tidak memberikan pengaruh yang bermakna terhadap peningkatan kadar
kreatinin serum mencit putih betina. Lama pemberian dan dosis pemberian tidak memberikan
pengaruh bermakna terhadap rasio berat hati dan ginjal mencit putih betina (Wahyuni, Putri,
dan Arisanti 2017).
Wahyuni, Fatma Sri, Intan Nedia Putri, dan Dessy Arisanti. 2017. Uji Toksisitas Subkronis
Fraksi Etil Asetat Kulit Buah Asam Kandis (Garcinia cowa Roxb.) terhadap Fungsi
Hati dan Ginjal Mencit Putih Betina. Jurnal Sains Farmasi & Klinis, 3(2):202-212.
TOKSISITAS KHAS
Uji Potensiasi Efek Sedatif-Hipnotik Ekstrak Etanol Kangkung Air (Ipomoea aquatic)
Asal Gambut Kalimantan Selatan
Latar Belakang
Salah satu terapi yang digunakan untuk mengobati gangguan tidur dan ansietas adalah terapi
pengobatan sedatif hipnotik. Pengobatan Sedatif-hipnotik menggunakan golongan obat
depresan Susunan Saraf Pusat (SSP) yang relatif tidak selektif. Efek depresan yang khas
mulai dari sedasi ringan, hipnosis hingga anestesi dan koma. Namun jika digunakan
berkepanjangan akan menyebabkan toksik dan berakibat kematian. Secara empiris tanaman
kangkung dapat bermanfaat sebagai penenang dan bisa menyebabkan kantuk. Ekstrak kasar
kangkung air mengandung alkaloid, steroid, fenol dan hidrokuinon. Senyawa alkaloid
merupakan ligan yang secara selektif dapat berikatan pada GABA binding site, senyawa
flavonoid dan glikosida dapat berikatan pada benzodiazepine binding site sedangkan senyawa
steroid berikatan pada steroid binding site yang merupakan komponen kompleks protein pada
reseptor GABAs yang nantinya mengakibatkan kanal ion klorida terbuka. Hal ini
menyebabkan sel sukar tereksitasi sehingga terjadinya penurunan tonus otot yang ditandai
dengan penurunan aktivitas. Hal inilah yang menyebabkan efek sedatif hipnotik dapat terjadi.
Tujuan
mengetahui efek sedatif hipnotik tanaman Kangkung air yang ada di daerah Gambut
Kalimantan Selatan pada mencit putih jantan galur Balb/C.
Metode
Penelitian menggunakaan metode rotarod dan fireplace test
Penyiapan Ekstrak Pengujian
Pada kontrol positif diberi diazepam (10mg/70KgBB), kontrol negatif (Na-CMC 0,5 %), dan
3 kelompok perlakuan yang diberi ekstrak kangkung air 1 mg/gBB, 2 mg/gBB, dan 4
mg/gBB)
Uji fireplace test Pengujian efek sedative dengan rotarod
Hasil
Hasil analisis uji dengan metode fireplace test menunjukkan adanya perbedaan bermakna
pada kelompok kontrol positif dan kelompok dosis 1 dan negative sedangkan jika
dibandingkan dengan dosis 2 dan 3 menunjukkan perbedaan tidak bermakna. Hasil analisis
uji dengan metode fireplace test pada dosis 3 menunjukkan nilai yang paling tinggi, sehingga
dosis yang paling potensial adalah dosis 3 (Dosis 4 mg/20gBB). Kangkung darat
mengandung berbagai senyawa aktif kalium dan natrium. Kalium dan natrium merupakan
persenyawaan garam bromide yang sifatnya dapat menekan susunan syaraf pusat dan dapat
ditarik kesimpulan bahwa senyawasenyawa inilah yang dapat menyebabkan efek sedasi.
Kesimpulan
1. Terdapat efek sedasi ekstrak etanol kangkung air khas Kalimantan Selatan pada
mencit jantan galur Balb/c dilihat pada jumlah jatuh hewan uji pada rotaryroad dan uji
Fireplace Test.
2. Dosis ekstrak etanol kangkung air khas Kalimantan Selatan dapat meningkatkan efek
sedasi pada mencit jantan pada dosis 4 mg/20gBB.
Astuti, K.I., Dan Fitriyani. 2018. Uji Potensiasi Efek Sedatif-Hipnotik Ekstrak Etanol
Kangkung Air (Ipomoea aquatic) Asal Gambut Kalimantan Selatan. Borneo Journal
Of Pharmascientech, 2(2): 59-64.
Borneo Journal of Pharmascientech, Vol. 02, No. 02, Oktober Tahun 2018
ISSN- Print. 2541 – 3651

ISSN- Online. 2548 – 3897
Research Article
UJI POTENSIASI EFEK SEDATIF-HIPNOTIK EKSTRAK ETANOL KANGKUNG

AIR (Ipomoea aquatic) ASAL GAMBUT KALIMANTAN SELATAN
Potential Test Of Sedative-Hypnotic Effect Extract Ethanol Ipomoea Aquatica

Form Gambut, South Kalimantan.
Karunita Ika Astuti* dan Fitriyanti

STIKES Borneo Lestari, Kelapa Sawit 8 Bumi Berkat, Banjarbaru, Indonesia
*karunitaika@gmail.com
ABSTRAK
Pengobatan Sedatif-hipnotik menggunakan golongan obat depresan Susunan Saraf Pusat (SSP) yang
relatif tidak selektif namun obat dari golongan ini jika digunakan berkepanjangan dapat bersifat toksik
dan menyebabkan kematian. Kangkung air (Ipomoea aquatic) memiliki efek antioksidan yang baik.
Tujuan dari penelitian ini adalah untuk mengetahui kandungan senyawa kimia dan efek tanaman
Kangkung air terhadap Uji sedative-Hipnotik. Sebanyak 30 mencit dalam 5 kelompok uji yaitu
Kelompok control negatif, control positif dengan pemberian diazepam 10 mg/KgBB secara oral,
Kelompok perlakuan dengan ekstrak Kangkung Air dosis 1 mg/KgBB, 2 mg/KgBB, dan $ mg/KgBB
dengan metode rotaryroad dan Fireplace Test. Data yang didapat dianalisis dengan Shapiro-Wilk,
kemudian dilanjutkan uji one way ANOVA dan Post Hoc Tests. Hasil menunjukkan adanaya efek sedasi
ekstrak etanol kangkung air pada dosis 4 mg/20 BB.
Kata kunci: SedatiF-Hipnotik, kangkung air, rotarod.
ABSTRACT
Treatment Sedatives-hypnotics using the class of depressants of Central Nervous System (CNS) are
relatively not selective. However, drugs of this class if used prolonged can be toxic and cause death.
Ipomoea aquatica has a good antioxidant effect. The aim of the test are to the show contains chemical
coumpound and the effect of sedative-hypnotic test. Twenty five male mice divided into 5 group (N=5)
which is called as negative control group, positive control group will be given Diazepam at dose
10mg/KgBW p.o and 3 treatment groups will be given Ipomea Aquatica Ectract (EKA) ad dose 1
mg/KgBW(I); 2 mg/kgBW(II); and 4mg/KgBW(III) p.o, In this study used rotarod and Fireplace method.
Data were analyzed using Saphiro Wilk Test then One ways ANOVA and post hoc test. There is a
sedation effect of Ipomea aquatica Extract at 4 mg/20 BB dose.
Keywords: Sedative-Hypnotive Test, Ipomoea Aquatica, rotarod.
PENDAHULUAN kehilangan waktu tidur diketahui sebagai

Kehidupan yang semakin penyebab ketidakseimbangan dalam
berkembang menuntut seseorang untuk menerima tugas yang melibatkan
bergerak cepat dalam upaya memenuhi memori, pembelajaran, dan alasan logis.
berbagai tuntutan kehidupan, sehingga Salah satu terapi yang digunakan untuk
mengakibatkan kelelahan. Selain itu
59
ISSN- Print. 2541 – 3651

Research Article
mengatasi masalah tersebut adalah terapi alkaloid merupakan ligan yang secara
pengobatan sedatif hipnotik. selektif dapat berikatan pada GABA
Pengobatan Sedatif-hipnotik binding site, senyawa flavonoid dan
menggunakan golongan obat depresan glikosida dapat berikatan pada
Susunan Saraf Pusat (SSP) yang relatif benzodiazepine binding site sedangkan
tidak selektif (Wiria dan Handoko, senyawa steroid berikatan pada steroid
1995). Obat-obatan golongan tersebut binding site yang merupakan komponen
bersifat heterogen secara kimia dengan kompleks protein pada reseptor GABAs
efek farmakologi yang sama, di mana yang nantinya mengakibatkan kanal ion
menghasilkan serangkaian efek depresan klorida terbuka. Hal ini menyebabkan sel
yang khas mulai dari sedasi ringan, sukar tereksitasi sehingga terjadinya
hipnosis hingga anestesi dan koma. penurunan tonus otot yang ditandai
Penggunaan obat ini biasanya dalam dengan penurunan aktivitas (Ikawati,
bersifat ansietas (sedasi) dan kemudahan 2006). Hal inilah yang menyebabkan
untuk tidur (hipnosis) (Katzung, 1994) efek sedatif hipnotik dapat terjadi.
dan pengobatan gangguan tidur, seperti Penelitian yang akan digunakan
insomnia (Siswandono dan Soekardjo, adalah kangkung air yang varietasnya
1995). Namun obat dari golongan ini jika banyak ditemukan dan tumbuh di daerah
digunakan berkepanjangan dapat bersifat rawa dan berair di Kalimantan selatan
toksik dan menyebabkan kematian khususnya daerah Gambut. Penelitian
(Cooper, 2016). menggunakaan metode rotarod dan
Secara empiris tanaman fireplace test sehingga dapat mengetahui
kangkung dimanfaatkan sebagai efek sedatif hipnotik tanaman Kangkung
penenang dan bisa menyebabkan kantuk air yang ada di daerah Gambut
(Anonim, 2001). Ekstrak kasar kangkung Kalimantan Selatan pada mencit putih
air mengandung alkaloid, steroid, fenol jantan galur Balb/C.
dan hidrokuinon (Sabri, 2011). Hidayati
(2013) menyebutkan bahwa senyawa METODOLOGI
alkaloid, flavonoid dan steroid dapat Bahan
menumbulkan efek sedatif. Senyawa
ISSN- Print. 2541 – 3651

Research Article
Bahan yang digunakan pada Alat

penelitian ini adalah kangkung air yang Alat yang digunakan pada
diambil dari Gambut, Kalimantan penelitian ini adalah alat-alat gelas,
Selatan, pelarut etanol 96% (PT. wadah maserasi, pengaduk, rotary
Brataco), akuades, pelarut ekstrak CMC evaporator, kandang mencit, sonde
(Carboksyl Methyl Cellulosa) 0,1%, lambung, gelas beker, neraca analitik,
mencit jantan umur 2-3 bulan, dan pakan gelas ukur, timbangan, rotarod, alat
mencit. fireplace, stopwatch.
Penyiapan Ekstrak Pengujian Mencit Balb/c yang

Pembuatan Ekstrak Etanol Tanaman memenuhi kriteria inklusi diadaptasikan
Kangkung Air dengan membuat haksel dari di laboratorium dengan cara dikandangkan,
batang dan daun kangkung air kemudian diberi pakan BRII 2 kali sehari dan minum
ditimbang dan dimasukkan ke dalam alat 1 kali sehari selama 7 hari. Pada kontrol
maserator. Pada proses ini dilakukan positif diberi diazepam (10mg/70KgBB),
pencampuran sampel dengan pelarut berupa kontrol negatif (Na-CMC 0,5 %), dan 3
etanol 96% dengan perbandingan 1:5 kelompok perlakuan yang diberi ekstrak
sambil diaduk hingga seluruh sampel kangkung air 1 mg/gBB, 2 mg/gBB, dan 4
terbasahi merata dengan pelarut. Kemudian mg/gBB). Perlakuan setelah 30 menit untuk
ditambahkan lagi pelarut hingga tingginya kelompok kontrol positif, dan setelah 45
1 cm di atas permukaan sampel. Ekstraksi menit untuk kelompok kontrol negatif dan
dilakukan selama 3x24 jam dan diaduk 1 3 kelompok perlakuan, mencit diletakkan
kali sehari. Setelah 3x24 jam, filtrat yang pada rotarod (30 rpm) dan dicatat waktu
diperoleh disaring, kemudian diuapkan yang didapat dengan replikasi sebanyak 3
menggunakan rotary evaporator dan kali. Data yang dikumpulkan adalah data
ditimbang hingga diperoleh ekstrak kental primer yang didapat dari waktu (t) yang
dengan bobot tetap. dibutuhkan mencit Balb/c untuk dapat
mempertahankan posisi pada alat rotarod
Persiapan Hewan Uji dan Pengujian yang berputar. hewan percobaan dibagi 5
kelompok.
ISSN- Print. 2541 – 3651

Research Article
Uji fireplace test menggunakan Pengujian efek sedative dengan

hewan uji yang diletakkan kedalam tabung rotarod dilakukan dengan menghitung
kaca, hewan normal akan berusaha lompat jumlah jatuh mencit pada alat rotaryroad.
keluar dari tabung dalam waktu 30 detik Selain itu juga dicatat waktu jatuhnya
sedangkan hewan abnormal yang telah selama 1 menit. Hasil yang didapat dari
memiliki efek sedatif akan keluar tabung semua kelompok dapat dilihat pada gambar
kaca lebih dari 30 detik (Almaner dkk., 1. Hasil yang didapat ke dalam analisa spss
2012). Pengamatan dilakukan dengan versi 18. Hasil analisis menunjukkan
melihat waktu lompat hewan keluar dari adanya perbedaan bermakna pada
tabung setiap rentang waktu pengujian. perlakuan antar kelompok Perlakuan
kelompok kontrol negatif dengan kelompok
dosis dan kontrol juga menunjukkan
adanya perbedaan yang bermakna.
HASIL DAN PEMBAHASAN
Gambar 1. Rerata jatuh mencit pada rotaryroad
Hasil analisis uji dengan metode fireplace test menunjukkan adanya perbedaan
bermakna pada kelompok kontrol positif dan kelompok dosis 1 dan negative sedangkan jika
dibandingkan dengan dosis 2 dan 3 menunjukkan perbedaan tidak bermakna, di mana nilai
tertinggi ada pada kelompok dosis 3, dilihat pada rentang korelasi angka loncat mencit pada
fireplace test yang cukup tinggi antar kelompok. Sehingga dapat dikatakan dosis paling
potensial pada ekstrak etanol kangkung air dilihat keseluruhan pada uji yang dilakukan adalah
pada dosis kelompok 3 (Dosis 4 mg/20gBB).
dapat dilihat pada gambar 2.
ISSN- Print. 2541 – 3651

Research Article
Gambar 2. Rerata jatuh mencit pada rotaryroad
Kankung air memiliki kekerabatan klorida sehingga menyebabkan

dengan Kangkung darat (Ipomea hiperpolarisasi sel. Kondisi sel yang sulit
reptansPoir.). Anggara (2009) menemukan terdepolarisasi ini akan menyebabkan
bahwa kangkung darat memiliki efek sedasi menurunnya eksibilitas sehingga
dengan waktu jatuh pada rotaryroad yang memberikan efek hipnotik dan rileks. Sutio
lebih cepat dibandingkan kontrol negatif. (2012) menyebutkan bahwa kuersetin
Kangkung darat mengandung berbagai (flavonoid) pada kangkung dapat berefek
senyawa aktif kalium dan natrium. Kalium pada sistem saraf pusat dengan memacu
dan natrium merupakan persenyawaan pusat inhibisi pada formation reticularis
garam bromide yang sifatnya dapat dan memodulasi reseptor GABA dan ligan-
menekan susunan syaraf pusat dan dapat ion gated channel sehingga dapat
ditarik kesimpulan bahwa senyawa- menghambat impuls penghantar dan
senyawa inilah yang dapat menyebabkan menyebabkan perlambatan reaksi.
efek sedasi. Setiawan (2011) menyebutkan
kandungan kalium dan natrium yang tinggi KESIMPULAN
dalam kangkung akan berikatan dengan Kesimpulan yang didapat dari
bromida, sehingga membentuk penelitian ini adalah :
persenyawaan garam bromide. Garam 1. Terdapat efek sedasi ekstrak etanol
bromide inilah yang memicu rangsangan kangkung air khas Kalimantan Selatan
pusat inhibisi di formatio reticularis otak pada mencit jantan galur Balb/c dilihat
dan selanjutnya akan berikatan dengan pada jumlah jatuh hewan uji pada
reseptor GABA dan terbukanya saluran rotaryroad dan uji Fireplace Test
ISSN- Print. 2541 – 3651

Research Article
2. Dosis ekstrak etanol kangkung air khas (Mitragyna speciosa Korth.) Pada
Mencit Jantan Galur Balb/c. Skripsi.
Kalimantan Selatan dapat Universitas Tanjungpura. Pontianak.
meningkatkan efek sedasi pada mencit Ikawati, Z., 2006, Pengantar Farmakologi
Molekuler, Gadjah Mada Unibersity
jantan pada dosis 4 mg/20gBB. Press, Yogyakarta
Katzung BG, 2004, Farmakologi Dasar
dan klinik. Buku 2. Edisi 8. Jakarta:
DAFTAR PUSTAKA Salemba Medika; 25-53
Alnamer, R., Alaoui, K., Bouidida, E. H., Sabri Sudirman, 2011, Aktivitas
Benjouad, A. dan Cherrah, Y., Antioksidan dan Komponen
2012, Sedative and Hypnotic Komponen Bioaktif Kangkung Air
Activities of the Methanolic and (Ipomoea aquatica Forsk.), Skripsi,
Aqueous Extracts of Lavandula Bogor: Institut Pertanian Bogor,
officinalis from Morocco, Research Setiawan I., 2011, Efek Hipnotik Ekstrak
Article, Advanecsh Etanol Kangkung (Ipomoea
Pharmacological Sciences, Morocco Aquatica Forsk.) pada Mencit Swiss
Anggara R., 2009, Pengaruh Ekstrak Webster Jantan Yang Diinduksi
Kangkung Darat (Ipomea Reptans Fenobarbital, Jurnal Medika Plant,
Poir.)Terhadap Efek Sedasi Pada 2(1), Bandung.
Mencit Balb/C, Karya Tulis Ilmiah, Siswandono dan Soekardjo. 1995. Kimia
Universitas Dipenogoro, Semarang. Medisinal. Surabaya: Penerbit
Cooper J. Toxicity, Sedative-Hypnotics Airlangga University Press.
[Online]. 2016 [cited on August Sutio, R. 2012. Pengaruh Kukusan Daun
4,2009]. Available from URL: Kangkung Air (Ipomoea aquatica)
http://emedicine.medscape.com/artic terhadap Kewaspadaan dan
le/818430-Overview Ketelitian pada Pria Dewasa.
Hidayati, A. 2013. Uji Efek Sedatif Ekstrak
N-Heksan Dari Daun Kratom
Wiria, M.S.S dan Handoko, T., 1995,

Hipnotik-Sedatif dan Alkohol, dalam
Ganiswarna, S.G., (Eds),
Farmakologi dan Terapi, Edisi IV,
124-137, Bagian Farmakologi
Universitas Indonesia, Jakarta
Ju r n al S a i n s Farm asi & Kl in is , 3 (2), 202-212
Jurnal Sains Farmasi & Klinis

(p- ISSN: 2407-7062 | e-ISSN: 2442-5435)
diterbitkan oleh Ikatan Apoteker Indonesia - Sumatera Barat

homepage: http://jsfkonline.org
Uji Toksisitas Subkronis Fraksi Etil Asetat Kulit Buah Asam

Kandis (Garcinia cowa Roxb.) terhadap Fungsi Hati dan Ginjal
Mencit Putih Betina
{Sub-chronic toxicity evaluation of ethyl acetate fraction of fruit rind of “asam kandis”
(Garcinia cowa Roxb.) against liver and kidney function of female white mice}
Fatma Sri Wahyuni1, Intan Nedia Putri1, & Dessy Arisanti2

1
Fakultas Farmasi Universitas Andalas
2
Fakultas Kedokteran Universitas Andalas
Keywords: ABSTRACT: The sub-chronic toxicity testing of ethyl acetate fraction of fruit rind of “asam kandis”
sub-chronic toxicity; (Garcinia cowa Roxb.) to the liver and kidney function has been carried out to female white mice. A total
ethyl acetat fraction; of 18 female white mice aged 2-3 months weighing 20-30 grams are used as test animals. Animals were
Garcinia cowa; cowa divided into three groups: one control group and two treatment groups. Groups were given daily ethyl
mangosteen; fruit rind. acetate fraction at the doses of 500 and 1000 mg/kg orally for 60 days. Parameters observed were the
activity of SGPT and liver weight ratio to observe the liver function; serum creatinine level and kidney
weight ratio to determine kidney function. Data of SGPT, serum creatinine and the weight ratio of
liver and kidney were analyzed by two-way ANOVA. Results show that the activity of SGPT and serum
creatinine level were directly affected by the dose (p<0.05), while the organ weight ratio of liver and kidney
were not affected by the dose and duration of administration (p>0.05). The study concluded that the dosage
of ethyl acetate fraction of the fruit rind of G. cowa had a significant effect on the activity of SGPT
and serum creatinine level of female white mice. The duration of administration did not give a significant
effect on the increase of serum creatine level as well as the organ weight ratio of liver and kidney of mice.
Kata Kunci: ABSTRAK: Pengujian toksisitas subkronis fraksi etil asetat kulit buah asam kandis (Garcinia
toksistas sub kronik; cowa Roxb.) terhadap fungsi hati dan ginjal mencit putih betina telah dilakukan. Sebanyak 18 ekor
fraksi etil asetat; mencit putih betina berusia 2-3 bulan dengan berat badan 20-30 gram digunakan sebagai hewan
Garcinia cowa; asam uji. Hewan dibagi menjadi 3 kelompok yaitu 1 kelompok kontrol dan 2 kelompoj perlakuan yang
kandis; kulit buah. diberi fraksi etil asetat kulit buah asam kandis dengan dosis 500 dan 1000mg/kgBB sekali sehari
secara oral selama 60 hari. Parameter yang diamati yaitu aktivitas SGPT dan rasio berat hati
untuk fungsi hati dan aktivitas kreatinin serum dan rasio berat ginjal untuk aktivitas ginjal. Data
aktivitas SGPT, kreatinin serum dan rasio berat organ hati dan ginjal dianalisis dengan ANOVA
dua arah. Hasil penelitian menunjukan bahwa aktivitas SGPT, Kreatinin serum dipengaruhi
secara langsung oleh dosis (p<0,05) dan untuk rasio berat hati dan ginjal tidak dipengaruhi secara
langsung oleh dosis dan lama pemberian (p>0,05). Dapat disimpulkan bahwa dosis pemberian
fraksi etil asetat kulit buah asam kandis memberikan pengaruh yang bermakna terhadap aktivitas
SGPT dan kadar kreatinin serum mencit putih betina. Lama pemberian tidak memberikan
pengaruh yang bermakna terhadap peningkatan kadar kreatinin serum mencit putih betina dan
rasio berat hati dan ginjal mencit putih betina.
*Corresponding Author: Fatma Sri Wahyuni (Fakultas Farmasi Article History:

Universitas Andalas, Kampus Limau Manis, Kec. Pauh, Kota Received: 23 Mar 2017 Accepted: 03 May 2017
Padang, Sumbar 21563). email: fatmasriwahyuni@gmail.com Published: 21 May 2017 Available online: 30 May 2017
202
Uji Toksisitas Subkronis Fraksi Etil Asetat Kulit Buah… | Wahyuni, dkk.
PENDAHULUAN keamanan terhadap G. cowa. Meskipun obat

herbal sudah dimanfaatkan sejak lama namun
Indonesia merupakan salah satu negara yang penggunaannya belum sepenuhnya aman, sehingga
memiliki keanekaragaman hayati terbesar. Hal sangatlah penting mengetahui batas keamanan
ini tentu memiliki potensi dalam pengembangan atau ketoksikannya. Untuk mengevaluasi suatu
obat herbal yang berbasis pada tumbuhan obat zat kimia perlu dikenali bahayanya dengan
dalam usaha kemandirian di bidang kesehatan. mengumpulkan dan menyusun data toksisitas.
Dewasa ini telah di lakukan penelitian dimana Keamanan obat menjadi salah satu faktor terpenting
terdapat tumbuhan menghasilkan senyawa yang perlu diperhatikan dalam pengembangan
metabolit sekunder dengan struktur molekul dan dan penggunaan obat herbal. Keamanan obat juga
aktivitas biologi yang beraneka ragam. Beberapa menjadi salah satu syarat dalam pelaksanaan uji
senyawa yang telah terbukti memiliki aktivitas praklinik obat herbal. Uji yang biasanya dilakukan
sebagai antikanker, antara lain golongan alkaloid, adalah uji toksisitas yang meliputi uji toksisitas
terpenoid, flavonoid, xanthon, dan kumarin [1]. akut, sub akut, sub kronik dan kronik [8]. Data
Tanaman dari genus Garcinia (guttiferae) ini digunakan bertujuan untuk menentukan sifat
telah diteliti secara luas secara fitokimia dan dan tempat efek toksik dan menentukan kadar
biologis [2]. Genus Garcinia kaya akan metabolit tanpa efek samping yang sering disebut no observed
sekunder terutama triterpen, flavonoid, santon adverse effect level (NOAEL). Salah satu kelebihan
dan phloroglucinol. Senyawa-senyawa yang telah penelitian ini adalah kita dapat menggunakan
diisolasi dilaporkan memiliki berbagai aktivitas satu atau beberapa dosis yang relatif tinggi yang
farmakologis, seperti aktivitas antikanker, anti- dapat menginduksi tanda-tanda toksisitas. Tanda
inflamasi, antibakteri, antivirus, antijamur, anti- tanda ini akan membantu menunjukan secara tepat
HIV, antidepresan, dan antioksidan [3]. organ sasaran dan efek khusus yang disebabkan
Garcinia cowa Roxb., atau yang lebih dikenal oleh dosis [9].
dengan nama asam kandis, adalah pohon berukuran Penelitian tentang keamanan fraksi etil asetat
sedang dengan buah-buahan yang dapat dimakan, kulit buah asam kandis pada tingkat toksisitas
telah digunakan masyarakat sebagai obat disentri, sub akut telah dilakukan. Penelitian sebelumnya
antipiretik dan anti-inflamasi. Beberapa penelitian mengatakan bahwa fraksi etil asetat kulit buah
melaporkan bahwa senyawa santon, benzofenon, asam kandis aman untuk digunakan karena tidak
dan derivat acylphloroglucinol telah berhasil mempengaruhi kadar SGPT hati dari mencit putih
diisolasi dari G. cowa [2,4], dimana senyawa secara langsung setelah pemberian selama 21 hari,
santon sendiri telah dikenal dengan potensi efek namun dipengaruhi secara bermakna terhadap
sitotoksiknya [5]. Ekstrak etanol kulit buah asam besaran dosis yang diberikan. Kadar kreatinin
kandis (Garcinia cowa Roxb.) diketahui memiliki mencit putih dipengaruhi secara bermakna oleh
efek sitotoksik terhadap sel kanker payudara lama pemberian dan dosis fraksi etil asetat kulit
T47D [6]. Fraksi etil asetat kulit buah asam buah asam kandis [10]. Untuk menguji keamanan
kandis memiliki efek sitotoksik terhadap sel kanker fraksi lebih lanjut di perlukan pengujian lanjutan
serviks HeLa dengan nilai IC50 16,194±3,5019 µg/ akan keamanan fraksi etil asetat kulit buah asam
mL [7]. kandis ini.
Sebagai bahan obat herbal baru dan akan Pada penelitian ini dilakukan penelitian
digunakan oleh masyrakat, perlu dilakukan kajian lanjutan yang merupakan uji toksisitas sub kronik.
Jurnal Sains Farmasi & Klinis | Vol. 03 No. 02 | Mei 2017 203
Uji toksisitas subkronis adalah uji ketoksikan suatu Makanan dan minuman diberikan secukupnya.
senyawa yang diberikan dengan dosis berulang Mencit yang digunakan adalah mencit yang sehat
pada hewan uji tertentu, selama 1 sampai 3 bulan dan tidak mengalami perubahan berat badan lebih
[9]. Pada pengamatan ini yang diperhatikan dari 10% dan secara visual menunjukkan perilaku
dalam uji toksisitas sub kronik yaitu fungsi organ yang normal [16].
seperti hati dan ginjal setelah pemberian fraksi
selama 60 hari [11]. Hati merupakan organ yang Perencanaan Dosis dan Pengelompokan Hewan
berperan dalam fungsi metabolisme dan ekskresi Dosis sediaan uji yang diberikan kepada hewan
toksik dalam tubuh [12]. Jika terjadi gangguan uji ditentukan berdasarkan penelitian sebelumnya.
hati ditandai dengan peningkatan aktivitas serum Dosis sediaan uji yang diberikan kepada hewan
transaminase berupa SGPT (Serum Glutamic uji adalah 500 dan 1000 mg/kgBB. Sediaan uji
Piruvic Transaminase) pada serum [13]. Ginjal diberikan secara oral dengan frekuensi pemberian
merupakan organ sasaran utama dari efek toksik 1 kali sehari selama 60 hari [11]. Untuk kontrol
karena ginjal menghasilkan urin yang merupakan hanya diberikan larutan tween 80 5%.
jalur utama ekskresi toksikan dan mempunyai Hewan uji dikelompokkan menjadi 4 kelompok.
volume aliran darah yang tinggi [14]. Salah satu Masing-masing kelompok terdiri dari 10 ekor
indikator terjadi kerusakan ginjal adalah terjadi mencit, dimana perlakuan dilakukan selama 60
peningkatan atau penurunan kadar kreatinin hari. Cek kreatinin serum, SGPT dilakukan pada
dalam tubuh maka interpretasi klinik akan lebih hari, ke-31 dan ke-61 serta diambil organ hati dan
cenderung pada gangguan fungsi ginjal [15]. ginjal untuk menentukan rasio berat organ.
Pada penelitian ini akan dilakukan penelitian
lanjutan uji toksisitas sub kronik fraksi etil asetat Penyiapan Sediaan Uji
kulit buah asam kandis (Garcinia cowa Roxb.) Sediaan uji dibuat dengan melarutkan
terhadap mencit putih betina. Parameter yang fraksi etil asetat kulit buah asam kandis dengan
diamati adalah penentuan aktivitas SGPT, kadar menggunakan Tween 80 5% dan aquadest.
kreatinin serum serta perbandingan rasio berat Berat ekstrak yang akan dilarutkan ditimbang
organ hati dan ginjal. berdasarkan dosis yang direncanakan Volume
sediaan uji yang akan diberikan secara oral ke
METODE PENELITIAN dalam tubuh mencit adalah 1% dari berat badan.
Pengambilan darah mencit dilakukan pada hari ke-
Penyiapan Hewan Uji 31, dan ke 61 (tiap kelompok terdiri dari 3 ekor
Hewan percobaan yang digunakan adalah mencit) dengan cara memotong pembuluh darah
mencit putih jantan yang berumur 2-3 bulan dibagian leher [11]. Darah ditampung dengan
dengan berat badan 20-30 gram sebanyak 28 microtube 1,5 mL, didiamkan selama 15 menit,
ekor untuk tiap kelompoknya, dan belum pernah kemudian disentrifus dengan kecepatan 3000 rpm
mengalami perlakuan terhadap obat. Hewan selama 20 menit untuk mendapatkan serum. Serum
percobaan dibagi dalam 4 kelompok yang terdiri dipisahkan dengan cara dipipet dan digunakan
dari 3 kelompok uji dan 1 kelompok kontrol. untuk pengujian SGPT, kadar kreatinin serum dan
Sebelum digunakan, semua mencit penentuan rasio berat organ hati dan ginjal.
diaklimatisasi selama 7 hari untuk membiasakan
hewan berada pada lingkungan percobaan.
204 Jurnal Sains Farmasi & Klinis | Vol. 03 No. 02 | Mei 2017
Pemeriksaan Fungsi Hati (As2 - As1)

Scr = x 2 mg/dL
Pengujian pengaruh fraksi etil asetat kulit (Ast2 - Ast1)
buah asam kandis terhadap fungsi hati dengan Keterangan:
penentuan aktivitas SGPT. Sebanyak 1 mL reagen Scr: Kadar kreatinin dalam serum (mg/dL)
I ditambahkan ke dalam microtube serum 100 µL As1: Absorban sampel pada menit pertama
(0,1 mL), kemudian didiamkan 5 menit. Selanjutnya As2: Absorban sampel yang diukur 2 menit setelah As1
ditambahkan 0,25 mL reagen II hingga tercampur Ast1: Absorban larutan standar kreatinin pada menit
homogen. Setelah 1 menit, serapan diukur dengan pertama
spektrometer uv-visible pada panjang gelombang Ast2: Absorban larutan standar kreatinin pada 2 menit
365 nm setiap menit selama 3 menit, kemudian setelah Ast1
dihitung selisih rata-rata serapan tiap menit. 2 mg/dL: Konsentrasi larutan kreatinin standard.
Kenaikan aktivitas SGPT dapat dihitung dengan
rumus: Penetuan Rasio Berat Organ Hati dan Ginjal
Pada hari ke-30 dan ke-60, (tiap kelompok
Aktivitas SGPT (U/L) = ΔA/menit x F terdiri dari 3 ekor mencit) setelah di lakukan
pengambilan darah mencit organ hati dan ginjal di
Keterangan: ambil lalu di bersihkan dan di timbang. Selanjutnya
ΔA/menit: perubahan aktivitas rata-rata per menit di tentukan berat organ relatif terhadap berat
F:faktor 3235 (untuk pengukuran pada panjang masing-masing hewan. Kemudian di dapatkan
gelombang 365 nm) rasio berat organ dengan rumus:
(Abs Tes 2-Abs Tes 1)-(Abs test 3-Abs Tes 2) Berat organ (g)
ΔA/menit = Rasio Berat Badan =
2 Berat badan mencit (g)
Pemeriksaan Fungsi Ginjal Analisis Data

Pengujian pengaruh fraksi etil asetat Data dari hasil penelitian pada aktivitas SGPT,
kulit buah asam kandis terhadap fungsi ginjal kadar kreatinin dan perbandingan rasio berat organ
dengan penentuan kadar kreatinin serum. Kadar dianalisa secara statistik dengan metoda analisis
kreatinin serum diukur dengan cara memipet variasi (ANOVA) dua arah. Analisa kemudian
50 µL serum ke dalam tabung reaksi. Kemudian dilanjutkan dengan uji wilayah berganda Duncan’s
1 mL reagen I ditambahkan ke dalam tabung Multiple Range Test (DMRT) untuk mengetahui
dan diamkan selama 5 menit. Sebanyak 0,25 mL adanya perbedaan yang bermakna pada masing-
reagen II dicampurkan dengan baik menggunakan masing kelompok perlakuan.
vortex. Pengukuran absorban sampel dilakukan
dengan spertrofotometer uv-visible pada panjang HASIL DAN DISKUSI
gelombang 492 nm. Absorban sampel diukur pada
menit pertama (As1) dan pengukuran selanjutnya Pengujian fraksi etil asetat kulit buah asam kandis
dilakukan pada menit ketiga (As2). Kadar kreatinin terhadap aktivitas SGPT mencit putih betina
serum ditentukan dengan rumus: Aktivitas SGPT mencit putih betina
dipengaruhi secara bermakna oleh lama pemberian
(p<0,05) dan dosis sediaan uji (p<0,05). Aktivitas
g1
Gambar 1. Pengaruh fraksi etil asetat kulit buah asam kandis terhadap
aktivitas SGPT mencit putih jantan
SGPT rata-rata kelompok mencit kontrol dan tidak dipengaruhi secara bermakna oleh dosis
kelompok yang diberi sediian uji dengan dosis (p>0,05). Rasio organ hati rata-rata kelompok
500 mg/kgBB, 1000 mg/kgBB berturut-turut mencit kontrol dan kelompok yang diberi sedian
adalah 25,888±3.91, 46,918±6,56, 48,733±5,066, uji dengan dosis 500 mg/kgBB, 1000 mg/kgBB
sedangkan aktivitas SGPT rata–rata pada hari ke berturut-turut adalah 0,406±0,003, 0,412±0,002,
31, dan 61 berturut–turut adalah 32,872±3,21, 0,034±0,007 Sedangkan rasio organ hati rata–rata
46,198±5,71. pada hari 0,036±0,0015 , 0,043±0,004.
Pengujian fraksi etil asetat kulit buah asam kandis Pengujian fraksi etil asetat kulit buah asam kandis
terhadap kadar kreatinin serum mencit putih terhadap rasio berat organ ginjal mencit putih betina
betina Rasio berat organ ginjal tidak dipengaruhi
Kadar kreatinin serum mencit putih Betina secara bermakna oleh lama pemberian (p>0,05)
tidak dipengaruhi secara bermakna oleh lama dan tidak di pengaruhi secara bermakna oleh dosis
pemberian (p>0,05) namun dipenagruhi secara (p>0,05). Sedangkan tidak terdapat pengaruh
bermakna oleh dosis (p>0,05). Kadar kreatinin yang bermakna terhadap interaksi antara lama
rata-rata kelompok mencit kontrol dan kelompok pemberian dan dosis (p>0,05). Rasio organ
yang diberi sedian uji dengan dosis 500 mg/kgBB, ginjal rata-rata kelompok mencit kontrol, dan
dan 1000 mg/kgBB berturut-turut adalah 0, 5333 kelompok yang diberi sediaan uji dengan dosis
±0.843, 0,640±0,95, 05,00±0,68. Sedangkan kadar 500 mg/kgBB, 1000 mg/kgBB berturut-turut
kreatinin rata–rata pada hari ke 31, dan 61 berturut- adalah 0,036±0,015, 0,012±0,001, 0,015±0,008.
turut adalah 5,777±0,84 dan 5,377±0,481. Sedangkan rasio organ hati rata-rata pada hari ke
31dan 61 berturut-turut adalah 0,019±0,08 dan
Pengujian fraksi etil asetat kulit buah asam kandis 0,026±0,008.
terhadap rasio berat organ hati mencit putih betina Penggunaan obat tradisional sebenarnya dapat
Rasio berat organ hati dipengaruhi secara menimbulkan efek yang tidak diharapkan. Keadaan
bermakna oleh lama pemberian (p<0,05) tetapi ini ditimbulkan oleh produk yang mungkin toksik
atau telah terkontaminasi. Menurut WHO efek hati dan ginjal mencit putih betina. Uji toksisitas
toksik dari suatu senyawa tergantung pada dosis sebelumnya didapatkan bahwa pada beberapa
dan durasi pemakaiaan obat [17]. Pengembangan parameter seperti kadar SGPT, kadar kreatinin
suatu obat tradisional dari tumbuhan harus terlebih serum, rasio berat hati dan ginjal dipengaruhi
dahulu diuji keamanannya sebelum diajukan secara bermakna oleh pemberian fraksi etil asetat
sebagai fitofarmaka, karena obat tradisional ini kulit buah asam kandis ini [10]. Pada uji subkronis
merupakan senyawa asing bagi tubuh, sehingga ini akan mengamati dan mengevaluasi keseluruhan
sangatlah penting mengetahui ketoksikannya efek yang merugikan setelah pemberian fraksi etil
[19]. Untuk menilai keamanan tersebut dilakukan asetat kulit buah asam kandis dalam kurun waktu
serangkaian uji toksisitas. 2 bulan pada organ hati dan ginjal mencit putih
Salah satu syarat tumbuhan dapat dijadikan betina [21]. Uji toksisitas subkronik dirancang
sebagai obat herbal adalah diuji tingkat untuk mengetahui spektrum efek toksik serta
keamanannya terlebih dahulu, diantaranya melalui hubungan dosis dan toksisitas pada pemberian
uji toksisitas akut dan uji toksisitas subkronik. secara berulang dalam jangka waktu 2 bulan (90
Toksisitas akut (jangka pendek) adalah pemberian hari) [22].
bahan kimia ada hewan coba dengan jumlah yang Sediaan uji yang digunakan adalah fraksi etil
semakin meningkat dalam kurun waktu 14 hari asetat dari kulit buah asam kandis (Garcinia cowa
hingga hewan percobaan tersebut mati. Sedangkan Roxb). Berdasarkan penelitian sebelumnya telah
toksisitas subkronik adalah pemberian bahan kimia dilakukan pemeriksaan kandungan metabolit
dengan jangka waktu panjang hingga timbulnya sekunder terhadap kulit buah asam kandis
efek yang merugikan kesehatan [20]. menunjukan adanya senyawa golongan flavonoid
Uji toksisitas sub kronik fraksi etil asetat kulit dan fenolik [7]. Fraksi etil asetat kulit buah
buah asam kandis ini dilakukan sebagai penelitian asam kandis (Garcinia cowa Roxb.) memiliki efek
lanjutan dari penelitian uji toksisitas sub akut fraksi sitotoksik terhadap sel kanker serviks HeLa dengan
etil asetat kulit buah asam kandis terhadap organ nilai IC50 16,194±3,5019 µg/mL [7]. Dengan
g2
Gambar 2. Pengaruh fraksi etil asetat kulit buah asam kandis terhadap
kadar kreatinin serum mencit putih betina
g3
Gambar 3. Hasil penelitian pengaruh fraksi etil asetat kulit buah

asam kandis terhadap rasio berat organ hati mencit putih betina
melihat besarnya IC50 yang dimiliki fraksi etil perbedaan yang nyata antara hewan uji yang diberi
asetat kulit buah asam kandis ini memungkinkan perlakuan.
bahwa fraksi ini memiliki potensi sebagai anti Organ vital yang diamati pada penelitian ini
kanker. adalah organ hati dan ginjal. Hati merupakan organ
Pada penelitian ini hewan yang digunakan vital yang terlibat dalam proses metabolisme tubuh.
adalah mencit putih betina. Faktor hormonal yang Hati juga mempunyai peranan penting dalam
terdapat pada mencit betina tidak mengganggu proses detoksifikasi. Hati dapat mengaktifkan atau
parameter yang digunakan pada penelitian sehingga menonaktifkan zat zat yang masuk ke dalam tubuh.
mencit betina dapat digunakan sebagai hewan uji. Ginjal juga merupakan organ yang vital bagi
Mencit juga mempunyai waktu pengujian pendek, tubuh, oleh sebab itu sering dijadikan parameter
hewan mudah didapat, mudah dalam pemeliharaan pengamatan untuk uji toksisitas suatu obat.
dan perlakuan, dapat beradaptasi dengan baik Ginjal mempunyai aliran darah yang besar karena
terhadap keadaan laboratorium, memerlukan zat berfungsi menjaga homoeostatik tubuh dengan
uji dalam jumlah kecil dan biaya lebih terjangkau cara mengatur keseimbangan elektrolit tubuh,
dibandingkan dengan hewan lainnya. Pemberian mengatur keseimbangan asam basa, dan mengatur
obat diberikan secara oral karena tidak menyakitkan osmolaritas tubuh. Ginjal mengekresikan zat
bagi hewan serta sesuai dengan penggunaan yang terlarut dan membuang hasil metabolisme
biasa dilakukan oleh manusia [14]. sehingga zat zat yang kiranya tidak berguna bagi
Penentuan dosis dalam penelitian ini dilakukan tubuh akan dibawa ke ginjal dalam jumlah yang
dengan mengikuti penelitian uji toksisitas subakut besar.
yang telah dilakukan sebelumnya [10]. Hewan Paramater yang digunakan untuk melihat
dikelompokkan menjadi empat kelompok uji dengan fungsi hati adalah kadar Serum Glutamic Pyruvic
masing masing dosis kontrol, 500 mg/kgBB, 1000 Transaminase (SGPT). SGPT merupakan enzim
mg/kgBB dan 2000 mg/kgBB. Adanya kelompok aminotransferase yang dibuat dalam sel hati
kontrol bertujuan untuk membandingkan hasil (hepatosit) sehingga keberadannya hanya terdapat
kelompok uji sehingga dapat melihat adanya pada organ hati. Bila terjadi kerusakan enzim
ini akan keluar dari sel hati dan masuk kedalam sebelumnya dikatakan bahwa hewan uji mampu
sistem peredaran darah. Adanya enzim ini di darah bertahan pada dosis 8000 mg/kgBB [10]. Namun
mengindikasikan adanya kerusakan sel-sel hati kenyataan yang ditemui pada penelitian ini, hewan
[23]. Paramater yang digunakan untuk melihat uji tidak dapat bertahan hidup setelah pemberian
fungsi ginjal dapat berupa peningkatan kadar berulang dosis 2000 mg/kgBB. Hal ini dapat terjadi
kreatinin darah. Kreatinin ada 2 yaitu kreatinin karena pada penelitian pendahuluan, dosis tinggi
serum dan kreatinin klirens. Pada penelitian ini hanya diberikan sekali. Dan pada penelitian ini,
kadar kreatinin serum merupakan metoda spesifik dosis yang diberikan memang tidak setinggi dosis
untuk melihat aktifitas organ ginjal. Kreatinin saat uji pendahuluan, namun karena pemberian di
merupakan hasil metabolism otot, dan dieksresikan berikan berulang membuat terjadinya akumulasi
secara konstan melalui ginjal. Sehingga kerusakan pada tubuh hewan uji dan menyebabkan toksik
ginjal, akan berdampak pada kadar kreatinin kepada hewan uji.
serum. Pengamatan lain yang terlihat pada penilitian
Penggunaan dosis pada penelitian ini berubah uji toksistas subkronik fraksi etil asetat kulit buah
dari rancangan dosis yang akan digunakan asam kandis ini adalah terjadinya perubahan warna
sebelumnya. Sebelumnya dosis yang dirancang mata pada 1 bagian mata pada hewan uji yang
adalah kontrol, dosis 500 mg/kgBB, 1000 mg/ diberi perlakuan dosis 1000 mg/kgBB. Perubahan
kgBB dan 2000 mg/kgBB. Pada perjalanan terjadi di akhir masa hidup hewan uji. Awalnya
penelitian diketahui bahwa hewan uji tidak mampu mata hewan uji akan berubah warna menjadi
bertahan hidup bila diberikan dosis 2000 mg/ lebih pucat dan akhirnya tertutup. Terjadinya
kgBB. Dari 20 hewan uji yang disediakan untuk penurunan fungsi pengelihatan ini kemungkinan
kelompok dosis uji 2000 mg/kgBB. Hanya 3 ekor disebabkan oleh dosis pemberiaan sediaan uji yang
hewan uji yang dapat bertahan hidup. Hal ini tinggi sehingga berpengaruh kepada mata hewan
dimungkinkan terjadi karena dosis terlalu tinggi uji. Penurunan bobot berat badan juga ditemukan
sehingga toksik untuk hewan uji. Pada penelitian pada kelompok ini. Penurunan bobot berat badan
g4
Gambar 4. Hasil penelitian pengaruh fraksi etil asetat kulit buah

asam kandis terhadap rasio berat organ hati mencit putih betina
kemungkinan terjadi karena sediaan uji yang nyata antara letak subset dari kontrol dan dosis 500
terlalu asam sehingga melukai saluran cerna mg/kgBB juga 1000 mg/kgBB. Sedangkan untuk
hewan uji dan menyebabkan hewan uji kehilangan dosis 500 mg/kgBB dan 1000 mg/kgBB tidak
nafsu makan. terdapat perbedaan bermakna aktivitas rata rata
Bila diamati, hewan uji yang sehat dan dapat kadar SGPT karena masih terletak pada subset
bertahan hidup kebanyakan ditemukan pada yang sama. Artinya tidak ada pengaruh atau efek
kelompok yang diberikan sediaan uji sebesar 500 yang bermakna yang terjadi akibat pemberian dosis
mg/kgBB. Pada kelompok ini berat badan hewan yang berbeda. Terjadinya perbedaaan aktivitas
uji tidak menunjukan penurunan yang signifikan. rata rata kadar SGPT untuk kelompok kontrol
Dan banyak nya hewan uji yang bertahan hidup dan kelompok yang diberi sediaan dikarenakan
lebih banyak daripada hewan uji kelompok dosis pada dosis kontrol tidak diberikan sediaan uji.
1000 mg/kgBB. Hal ini kemungkinan terjadi Sediaan uji meningkatkan proses metabolisme
karena perbedaan kadar dosis yang diberikan. Hal- hati hewan uji sehingga terjadinya kerusakan hati
hal diluar harapan yang terjadi pada penelitian ini dan sel sel hati mengalami lisis. Enzim GPT yang
kemungkinan terjadi karena pemilihan dosis yang dimana dalam keadaan normal berada di dalam sel
terlalu besar untuk sediaan uji yang menggunakan saat terjadinya lisis sel hati akan keluar dan masuk
fraksi. fraksi berbeda dengan ekstrak, fraksi ke dalam sirkulasi darah, sehingga kadar SGPT
merupakan bentuk yang sederhana dari ekstrak menjadi tinggi dalam darah. Dengan hasil uji lanjut
sehingga sebaiknya pada penggunaan fraksi dosis ini menandakan bahwa dengan bertambahnya
yang digunakan juga kecil. dosis yang diberikan memberikan peningkatan
Aktivitas SGPT pada organ hati dilihat SGPT kepada hewan uji.
dengan mereaksikan darah mencit dengan reagen Menurut penelitian uji toksisitas subakut
I dan reagen II. Serapan dari kedua reaksi ini akan sebelumnya dikatakan bahwa tidak adanya
dilihat dan dibaca serapannya di bawah mikroskop. pengaruh yang berarti terhadap dosis maupun
Hasil penelitian ini menunjukkan bahwa pemberian waktu pemberian terhadap kadar SGPT. Setelah
fraksi etil asetat kulit buah asam kandis dengan dilakukan uji toksisitas subkronik baru diketahui
variasi dosis kontrol, 500 mg/kgBB, 1000 mg/ bahwa pemberian sediaan uji secara berulang
kgBB, dan 2000 mg/kgBB pengamatan dilakukan dapat memberikan peningkatan kadar SGPT
pada hari ke 31 dan 61. Parameter yang dilihat kepada hewan uji.
untuk mengetahui fungsi hati hewan uji adalah Berdasarkan pengujian statistik dengan uji
kadar SGPT. Kadar SGPT dapat menggambarkan analisis variasi dua arah dari data pengujian fungsi
kerusakan hati yang disebabkan oleh sediaan uji. ginjal diketahui bahwa kadar kreatinin dipengaruhi
Menurut hasil pengolahaan statistik hasil secara bermakna oleh dosis (p<0,05). Selain itu
yang didapat dari perhitungan kadar SGPT bahwa interaksi antara faktor lama pemberian dan dosis
aktivitas SGPT dipengaruhi secara bermakna oleh sediaan uji tidak memberikan pengaruh yang
dosis juga dipengaruhi secara bermakna oleh lama bermakna (p<0,05). Setelah dilakukan uji lanjut
waktu pemberian. Namun interaksi antar dosis dan Duncan’s Multiple Range Test (DMRT) terhadap
lama pemberian tidak mempengaruhi kadar SGPT faktor dosis memang terjadi perbedaan aktivitas
secara bermakna. Setelah dilakukan uji lanjut kadar kreatinin serum pada kelompok kontrol dan
dengan menggunakan Duncan’s Multiple Range kelompok dosis.
Test (DMRT). Terlihat bahwa terdapat perbedaan Pada penelitian ini nilai kreatinin serum yang
diberi ekstrak menunjukan aktifitas rata rata Rasio organ ginjal juga menjadi salah satu
kreatinin serum yang lebih tinggi dibandingkan parameter pada peneilitian ini. Hasil analisa
dengan aktifitas rata rata kreatinin serum kontrol. statistik untuk rasio berat ginjal mengatakan
Aktifitas rata rata kreatinin serum yang diberikan bahwa tidak ada hubungan yang berarti antara
sediaan uji meningkat mungkin dikarenakan besaran dosis yang diberikan. Begitu juga
dosis pemberiaan sediaan uji yang tinggi dan terhadap lama pemberian maupun interaksi antara
pemberiaan sedian uji yang cukup lama sehingga lama pemberian dan besaran dosis. Sedangkan
metabolit metabolit hasil metabolisme sediaan uji pada penelitian sebelumnya dikatakan bahwa
terakumulasi di ginjal dan membuat sel sel epitel peningkatan rasio berat ginjal dipengaruhi oleh
nefron terluka. Kreatinin serum seharusnya tidak faktor lama pemberian. Perbedaan antara hasil
ditemukan pada darah. Kreatinin serum adalah ini kemungkinan karena tidak seragam nya berat
hasil metabolisme pada otot yang seharusnya badan pada penelitian sebelumnya.
dikeluarkan dari luar tubuh. Bila sel sel nefron
ginjal rusak kreatinin serum yang seharusnya KESIMPULAN
dibuang akan masuk kembali kedalam tubuh dan
ikut dalam aliran darah. Tingginya kadar kreatinin Dosis pemberian fraksi etil asetat kulit
serum pada darah merupakan indikasi bahwa buah asam kandis memberikan pengaruh yang
terjadinya penurunan fungsi ginjal pada hewan uji. bermakna terhadap aktivitas SGPT dan terhadap
Pada penelitian uji toksisitas sub akut yang kadar kreatinin serum mencit putih betina. Lama
telah dilaksanakan sebelumnya dikatakan bahwa pemberian tidak memberikan pengaruh yang
aktifitas kreatinin serum berhubungan secara bermakna terhadap peningkatan kadar kreatinin
nyata dengan dosis dan lamanya pemberian. serum mencit putih betina. Lama pemberian dan
Namun pada penilitian uji toksisitas subkronik dosis pemberian tidak memberikan pengaruh
yang telah dilakukan, lama nya pemberian uji tidak bermakna terhadap rasio berat hati dan ginjal
berhubungan dengan peningkatan kadar kreatinin mencit putih betina.
serum. Dapat dikatakan bahwa semakin tinggi
dosis yang diberikan kepada hewan uji, maka akan UCAPAN TERIMA KASIH
meningkatkan kadar kreatinin serum pada hewan
uji. Terima kasih disampaikan terutama kepada
Rasio organ hati juga diamati sebagai parameter Universitas Andalas yang telah mendanai
pada penelitiaan ini hal ini dikarenakan karena penelitian ini melalui Hibah Klaster Riset Guru
organ hati merupakan organ yang juga sensitif Besar No 23/UN.16/HKRGB/LPPM/2016,
terhadap paparan toxican. Dengan menggunakan
uji statistik ANOVA diketahui bahwa tidak adanya DAFTAR PUSTAKA
hubungan antara lama pemberian sediaan uji
1. Perhimpunan Dokter Paru Indonesia. (2003). Pneumonia
terhadap rasio berat hati. Begitu juga terhdap
Komuniti: Pedoman Diagnosa dan Penatalaksanaan di
faktor dosis dan interaksi antar keduanya. Dapat Indonesia, Perhimpunan Dokter Paru Indonesia.
dikatakan bahwa sediaan uji tidak berpengaruh 2. Departemen Kesehatan Republik Indonesia. (2013). Profil
Kesehatan Indonesia 2013. Jakarta: Departemen Kesehatan.
terhadap peningkatan atau penurunan berat dari 3. World Health Organization (WHO). Rational Use of Medicines.
organ hati walaupun bila ditinjau dari kadar SGPT, 2012 . Diakses dari http://www.who.int/medicines/areas/
rational_use/en/
sediaan uji dapat meningkatkan kadar SGPT.
4. Departemen Kesehatan Republik Indonesia. (2012). Profil 14. Darmansjah, I. (2008). Penggunaan Antibiotika pada Pasien
Kesehatan Indonesia 2011. Jakarta: Departemen Kesehatan. Anak. Majalah Kedokteran Indonesia, 58(10).
5. Dinas Kesehatan Provinsi Sumatera Barat. (2013). Profil 15. Priyanto. (2009). Farmakoterapi dan Terminologi Medis.
Kesehatan Provinsi Sumatera Barat Tahun 2012. Padang: Lembaga Studi dan Konsultasi Farmakologi, Jawa Barat.
Dinas Kesehatan. 16. Jawetz, E. (1984). Mikrobiologi Untuk Profesi Kesehatan, Edisi
6. Departemen Kesehatan RI, Direktorat Bina Farmasi Komunitas 16. Jakarta: Penerbit Buku Kedokteran EGC.
dan Klinis. (2005). Pharmaceutical Care Untuk Penyakit Infeksi 17. Anonim. (2010). Efek Samping Obat. Yogyakarta: Farmakologi
Saluran Pernapasan. Jakarta. Klinik Fakultas Kedokteran Universitas Gadjah Mada.
7. Bahry, B. (1989). Kesenjangan Peresepan Pada Anak. 18. Worokarti. (2005). Peran Farmasis Dalam Pengelolaan
Prosiding: Kongres Nasional VII Ikatan Farmakologi Indonesia, Penderita Penyakit Infeksi Untuk Mencegah Timbulnya
Yogyakarta Oktober 1989, Fakultas Kedokteran Universitas Resistensi Antimikroba. In: Naskah Lengkap Simposium
Andalas Padang. Penyakit Infeksi dan Problema Resistensi Antimikroba.
8. Jukemura, E. M., Burattini, M. N., Pereira, C. A., Braga, A. L., & Surabaya: AMRIN Study Group and Infectious Disease Center
Medeiros, E. A. (2007). Control of multi-resistant bacteria and dan FKUA RSU Dr. Soetomo. hal.55-69.
ventilator-associated pneumonia: is it possible with changes 19. Ostapchuk, M., Donna, M.R., Richard, H.M.D., (2004).
in antibiotics?. Brazilian Journal of Infectious Diseases, 11(4), Community-Acquired Pneumoni in Infant and Children, Journal
418-422. of The American Academy of Family Physicians.
9. Huang, K. T., Tseng, C. C., Fang, W. F., & Lin, M. C. (2010). 20. Nugroho, F., Pri I.U., Ika Y. (2011). Evaluasi Penggunaan
An early predictor of the outcome of patients with ventilator- Antibiotika Pada Penyakit Pneumonia Di Rumah Sakit
associated pneumonia. Chang Gung Med J, 33(3), 274-282. Umum Daerah Purbalingga (Skripsi). Purwokerto: Universitas
10. Departemen Kesehatan RI. (2009). Pelayanan Kesehatan Muhammadiyah Purwokerto.
Anak di Rumah Sakit, Pedoman Bagi Rumah Sakit Rujukan 21. Kaparang, P.C., Tjitrosantoso, H., & Yamlean, P.V.Y.
Tingkat Pertama di Kabupaten/Kota, Jakarta. (2014). Evaluasi Kerasionalan Penggunaan Antibiotika Pada
11. Suharjono, Y.T, Sumarno, Semedi J. (2009). Studi penggunaan Pengobatan Pneumonia Anak Di Instalasi Rawat Inap RSUP
antibiotika pada penderita rawat inap pneumonia (penelitian Prof. DR. R. Kandou Manado Periode Januari-Desember 2013.
di sub Departemen Anak Rumkital Dr. Ramelan Surabaya). Pharmacon Jurnal Ilmiah Farmasi, 3(3), 247-254.
Majalah Ilmu Kefarmasian, 6(3), 142-155. 22. Shargel. (1988). Biofarmasetika dan Farmakokinetik Terapan,
12. Advisedly, A.T., Berawi, M.M. (2014). Antibiotic Utilization Of Edisi. 2, Penterjemah Fasich dan Siti Syamsiah. Surabaya:
Pneumonia In Children of 0-59 Month฀s Old in Puskesmas Penerbit Universitas Airlangga.
Kemiling Bandar Lampung Period Januari-October 2013 23. Dipiro, J.T., Robert, L.T., Gary, C.Y., R.M., Barbara, G.W.,
(Skripsi). Lampung; Faculty of Medicine Lampung University. Michael Posey. (2011). Pharmacotheraphy; A Pathophysiology
13. Kementrian Kesehatan Republik Indonesia. (2011). Pedoman approach, Eight Ed. Mc GrawHill Companies.
Umum Penggunaan Antibiotika. Jakarta: Kementrian
Kesehatan.
Haiga Sophia Gunawan
V3720027
Hewan
Penghasil racun : Ikan buntal dari keluarga Tetraodontidae. Tetrodotoksin diproduksi oleh
bakteri laut seperti Vibrio alginolyticus, Shewanella algae, Shewanella putrefaciens dan
Alteromonas tetraodonis yang terdistribusi ke dalam tubuh ikan buntal melalui rantai
makanan. Jenis makanan Ikan Buntal Pisang yang mengandung bakteri penghasil tetrodoksin
yaitu gastropoda Monodonta lineata dan Gibbula umbilicalis, kepiting suku Xanthidae,
bintang laut (Hapsara dkk., 2019).
Nama racun : tetrodotoxin (TTX) dan atau saxitoxins (STXs) (Hapsara dkk., 2019).
Tetrodoksin adalah senyawa yang larut dalam air, tidak berwarna, tidak berbau, stabil oleh
panas dan tidak terdegradasi oleh proses pemasakan (Hapsara dkk., 2019).
Cara kerja racun : Banyak penelitian yang meneliti mekanisme toksisitas ikan buntal dengan
TTX mengungkapkan bahwa ikan buntal tidak melakukan biosintesis TTX tetapi
mengakumulasinya melalui rantai makanan yang dimulai dengan bakteri penghasil TTX.
Akumulasi TTX secara bertahap dihilangkan jika pasokan melalui makanan beracun tidak
dilanjutkan. berbagai percobaan pemberian TTX in vivo dilakukan menggunakan ikan buntal
tidak beracun yang dibesarkan secara artifisial dengan diet tidak beracun, dan menemukan
bahwa kinetika TTX internal pada ikan buntal unik: usus→hati→kulit/indung telur, dan
berubah saat ikan buntal tumbuh dan matang. Tetrodotoksin merupakan racun saraf berat
molekul rendah berbentuk prisma Kristal yang dapat menghambat konduksi saraf dan otot,
dan secara selektif dapat memblokir saluran natrium sehingga mengakibatkan kelumpuhan
pernafasan dan menyebabkan kematian (Zhu et al., 2020)
Target aksi racun : TTX biasanya ditemukan di organ-organ ikan buntal spesies tertentu,
biasanya pada liver, ovarium, and gastrointestinal (GI) tetapi juga kadang-kadang di hati.
TTX juga ditemukan di organisme-organisme lain, dan racun ini dipercaya diproduksi secara
eksogen, mulai dari bakteri yang terakumulasi melalui rantai makanan hingga ikan, kerang,
amfibi, dan gurita. TTX akan menyerang saraf dan otot pada manusia (Reverté et al., 2017)
Efek gejala wujud toksik yang ditimbulkan : Gejala-gejala utama keracunan ikan buntal
adalah dari neuromuscular (paraesthesia bibir dan lidah, kesemutan, dan mati rasa emosional)
hingga gastrointestinal (mual, nyeri abdomen, muntah, dan diare) (Reverté et al., 2017)
Tumbuhan
Penghasil racun : Semua bagian dari tanaman Datura stramonium (DS) atau biasa dikenal
dengan kecubung (Trancă et al., 2017).
Nama racun: tropane alkaloids. Atropin yang terkandung adalah L-hyoscyamine and L-
scopolamine (Trancă et al., 2017).
Cara kerja racun : racun tersebut menyebabkan anticholinergic syndrome yang merupakan
efek dari terhambatnya neurotransmisi muskarinik sentral dan periferal hal ini dapat
menyebabkan kelumpuhan organ. Remaja, terutama mereka yang memiliki riwayat
penyalahgunaan zat polisubstansi, secara sengaja menelan tanaman tersebut untuk mencari
efek halusinogen dan euforia dan munculnya efek antikolinergik. Pengosongan lambung yang
tertunda sehingga menyebabkan gejala keracunan bisa dirasakan hingga 24-48 jam karena
efek antikolinergik (Trancă et al., 2017).
Target aksi racun : Neurotransmitter pada sistem saraf pusat dan perifer (Trancă et al., 2017).
Efek gejala wujud toksik yang ditimbulkan : Gejala khas keracunan DS diwakili oleh kulit
dan mukosa kering, kemerahan, midriasis, sinus takikardia, hiperpireksia, penurunan aktivitas
usus, retensi urin, dan gangguan neurologis dengan ataksia, gangguan memori jangka pendek,
disorientasi, kebingungan, halusinasi (penglihatan dan pendengaran) , psikosis, delirium
agitasi, kejang, dan koma. Gejala-gejala ini menyerupai keracunan atropin. Kegagalan
pernapasan dan kolaps kardiovaskular dilaporkan pada kasus yang parah. Dalam kasus yang
jarang terjadi, rhabdomyolysis dan hepatitis fulminan juga telah dijelaskan. Toksisitas datura
stramonium biasanya terjadi dalam 60 menit setelah konsumsi, dan gejala klinis dapat
bertahan hingga 24-48 jam, karena pengosongan lambung yang tertunda. Penundaan yang
disebabkan oleh efek antikolinergik ini menyebabkan durasi kerja yang lebih lama. Anak-
anak memiliki kerentanan unik terhadap toksisitas atropin, karena jumlah yang lebih kecil
dapat menyebabkan gangguan sistem saraf pusat yang parah (Trancă et al., 2017).
Daftar Pustaka
Hapsara., H., Wijayanti, D.P., dan Redjeki, S. 2019. Korelasi Panjang Berat Ikan Buntal
Pisang Tetraodon lunaris Linnaeus, 1758 (Actinopterygii : Tetraodontidae) di Perairan
Pati, Jawa Tengah. Journal of Marine Research, 8(2): 177-180.
Reverté, Laia, Mònica Campàs, Betsy Jean Yakes, Jonathan R. Deeds, Panagiota Katikou,
Kentaro Kawatsu, Michael Lochhead, Christopher T. Elliott, and Katrina Campbell.
2017. Tetrodotoxin Detection in Puffer Fish by a Sensitive Planar Waveguide
Immunosensor. Sensors and Actuators B: Chemical 253:967–976.
Trancă, S.D., Szabo, R., and Cociş, M. 2017. Acute Poisoning Due to Ingestion of Datura
Stramonium – A Case Report. Romanian Journal of Anaesthesia and Intensive Care,
24(1): 65-68.
Wahyuni, Fatma Sri, Intan Nedia Putri, and Dessy Arisanti. 2017. “Uji Toksisitas Subkronis
Fraksi Etil Asetat Kulit Buah Asam Kandis (Garcinia cowa Roxb.) terhadap Fungsi
Hati dan Ginjal Mencit Putih Betina.” Jurnal Sains Farmasi & Klinis 3(2):202-212.
Zhu, Hongchen, Akinori Yamada, Yui Goto, Linan Horn, Laymithuna Ngy, Minoru Wada,
Hiroyuki Doi, Jong Soo Lee, Tomohiro Takatani, and Osamu Arakawa. 2020.
“Phylogeny and Toxin Profile of Freshwater Pufferfish (Genus Pao) Collected from 2
Different Regions in Cambodia.” Toxins 12(11):689.
Journal of Marine Research Vol.8, No.2 Mei 2019, pp. 177-180
EISSN: 2407-7690
https://ejournal3.undip.ac.id/index.php/jmr
Korelasi Panjang Berat Ikan Buntal Pisang Tetraodon lunaris Linnaeus, 1758
(Actinopterygii : Tetraodontidae) di Perairan Pati, Jawa Tengah
Hudanu Hapsara*, Diah Permata Wijayanti, Sri Redjeki

Departemen Ilmu Kelautan, Fakultas Perikanan dan Ilmu Kelautan, Universitas Diponegoro
Jl. Prof. H. Soedarto S.H, Tembalang,Semarang, Jawa Tengah 50275 Indonesia
*Corresponding author, e-mail: 21hapsara@gmail.com
ABSTRAK : Perairan Pati mempunyai kekayaan sumberdaya jenis ikan yang beragam jenisnya.
Salah satu hasil tangkapannya adalah Ikan Buntal Pisang (Tetraodon lunaris). Ikan Buntal Pisang
memiliki bentuk badan membulat dan ukuran mulut yang kecil dengan moncongnya yang tumpul
dan memiliki racun yang disebut tetrodotoksin (TTX). Namun Ikan Buntal Pisang memiliki
kandungan gizi yang cukup tinggi dan sebagian masyarakat Pati mengolah ikan ini menjadi ikan
asin. Penelitian ini didasarkan pada hasil tangkapan Ikan Buntal Pisang yang didaratkan di PPI
Banyutowo oleh nelayan yang melakukan penangkapan di perairan Pati. Penelitian ini bertujuan
untuk mengetahui nilai hubungan panjang berat Ikan Buntal Pisang yang berada di perairan Pati.
Penelitian ini dilakukan dengan metode penelitian deskriptif, dimana pengambilan sampling
berdasarkan metode pertimbangan (purposive sampling method). Penelitian dilaksanakan bulan
Februari – April 2016 di PPI Banyutowo. Materi yang digunakan adalah 360 sampel Ikan Buntal
Pisang. Sampling Ikan Buntal Pisang dilakukan sebanyak 3 kali yaitu 13 Februari, 12 Maret, dan 9
April 2016. Analisa data berupa analisis hubungan panjang berat Ikan Buntal Pisang. Hasil
penelitian menunjukan pertumbuhan Ikan Buntal Pisang pada bulan Februari – April 2015 bersifat
allometrik positif yang memiliki nilai slope (b) sebesar 3,30.
Kata kunci : Tetraodon lunaris, Hubungan, Panjang, Berat
Long Term Correlation of Banana-Spotted Fish Tetraodon lunaris Linnaeus, 1758

(Actinopterygii: Tetraodontidae) in the Pati Waters, Central Java
ABSTRACT :Pati waters has a rich variety of fish species. One of the catches is Green Rough-
Backed Puffer (Tetraodon lunaris). Green Rough-Backed Puffer have a rounded body shape and
small mouth size with a blunt snout and a poison called tetrodotoxin (TTX). But this fish has a high
nutritional content and some Pati people process this fish into salted fish. This research is based on
the catch of Green Rough-Backed Puffer landed in PPI Banyutowo by fishermen who make arrests
in the waters of Pati. This study aims to determine the value of the long weight relationship of Gre en
Rough-Backed Puffer in the waters of Pati. This research was conducted with descriptive research
method, where sampling was taken based on the method of consideration purposive sampling
method. The study was conducted in February - April 2016 at PPI Banyutowo. The material used
was 360 samples of Green Rough-Backed Puffer. Sampling of Green Rough-Backed Puffer was
carried out 3 times, namely February 13, March 12, and April 9, 2016. Analysis of the data was in
the form of an analysis of the long weight relationship of Green Rough-Backed Puffer. The results
showed that the growth of Green Rough-Backed Puffer in February - April 2015 was positive
allometric which had a slope value (b) of 3,30.
Keywords: Tetraodon lunaris, Relationship, Long, Weight
PENDAHULUAN
Ikan Buntal Pisang (Tetraodon lunaris) memiliki bentuk badan membulat. Ukuran mulut yang
kecil dengan moncongnya yang tumpul (Kottelat et al, 1993). Ikan Buntal Pisang adalah ikan
karnivora dan perenang lambat. Ikan ini hidup hampir di seluruh perairan Indonesia dan dapat
hidup di bebagai ekosistem – laut, muara (Wahyuni et al. 2004).
Diterima : 03-02-2019; Diterbitkan :17-04-2019

178
Ikan Buntal Pisang memiliki racun yang disebut tetrodotoksin (TTX). Tetrodoksin adalah
senyawa yang larut dalam air, tidak berwarna, tidak berbau, stabil oleh panas dan tidak
terdegradasi oleh proses pemasakan (Him, 2007). Racun ini merupakan neurotoksin dan belum
ada penawar racunnya (Nieto et al., 2012). Tetrodotoksin merupakan racun saraf berat molekul
rendah berbentuk prisma Kristal yang dapat menghambat konduksi saraf dan otot, dan secara
selektif dapat memblokir saluran natrium sehingga mengakibatkan kelumpuhan pernafasan dan
menyebabkan kematian (Noguchi et al., 2008). Tetrodotoksin diproduksi oleh bakteri laut seperti
Vibrio alginolyticus, Shewanella algae, Shewanella putrefaciens dan Alteromonas tetraodonis yang
terdistribusi ke dalam tubuh ikan buntal melalui rantai makanan (Noguchi et al., 2011). Menurut
(Widodo et al., 2006). Jenis makanan Ikan Buntal Pisang yang mengandung bakteri penghasil
tetrodoksin yaitu gastropoda Monodonta lineata dan Gibbula umbilicalis (Silva et al,. 2012),
kepiting suku Xanthidae (Wahyudi, 2006), bintang laut (Noguchi et al., 2011).
Ikan Buntal Pisang di Indonesia keberadaannya cukup melimpah dan belum dimanfaatkan
secara optimal karena Ikan Buntal Pisang dianggap ikan beracun yang mematikan sehingga Ikan
Buntal Pisang tidak termasuk salah satu ikan ekonomis penting. Tetapi jika dikelola dengan baik
Ikan Buntal Pisang mempunyai kandungan gizi yang tinggi (Yusfiati, 2006). Ikan Buntal
mempunyai nilai gizi dagingnya yaitu, air 78,9%, protein 16,5%, lemak 0,7%, karbohidrat 2,5% dan
abu 1,4% (Saito et al., 1998).
Ikan Buntal Pisang adalah ikan karnivora dan perenang lambat. Di Jepang, ikan ini dikenal
sebagai dokusabafugu, dan itu adalah salah satu spesies ikan buntal yang sering dikonsumsi
selain ikan buntal lain yaitu takifugurubrip. Ikan ini hidup hampir di seluruh perairan Indonesia dan
dapat hidup di bebagai ekosistem – laut, muara, dan air tawar (Wahyuni et al., 2004). Di beberapa
daerah masyarakat pesisir mengkonsumsi Ikan Buntal Pisang yaitu daerah Pelabuhan Ratu,
Sukabumi, dan Tuban. Masyarakat ini mengonsumsi Ikan Buntal Pisang dengan cara
mengolahnya menjadi ikan asin (Yusfiati, 2006).
Sampai saat ini belum banyak keterangan informasi tentang biologi Ikan Buntal Pisang di
Indonesia. Menurut Richter (2007) dan Blackweel et al. (2000), pengukuran panjang dan berat ikan
bertujuan untuk mengetahui variasi berat dan panjang ikan secara individual atau kelompok
individu, sehingga dapat dijadikan petunjuk mengenai tingkat kegemukan, kesehatan, produktifitas,
kondisi fisiologi dan kematangan gonad. Pengungukuran hubungan panjang dan berat perlu
dilakukan untuk mengetahui pola pertumbuhan Ikan Buntal Pisang di perairan Pati. Dengan
adanya pengamatan hubungan panjang berat dapat digunakan sebagai informasi dasar
pemanfaatan dan pengelolaan Ikan Buntal Pisang khususnya di perairan Pati.
MATERI DAN METODE

Penelitian ini merupakan penelitian deskriptif yaitu peneliti yang menelaah secara mendalam
suatu masalah pada waktu dan tempat tertentu, sehingga memberikan gambaran tentang situasi
dan kondisi secara local dan hasilnya tidak dapat digeneralisasikan untuk tempat dan waktu yang
berbeda (Hadi, 1979).
Untuk pengambillan data menggunakan sample survey method, metode yang dilakukan
dengan mencatat sebagian kecil sampel populasi, dengan harapan hasil yang diperoleh dapat
menggambarkan sifat-sifat organisme yang diteliti (Nazir, 1988). Menurut Gay et al. (2009), jumlah
sampel yang digunakan dalam penelitian deskriptif mimal 10 % dari jumlah populasi. Dengan
jumlah sampel tersebut diharapkan hasil yang diperoleh mempunyai probabilitas yang tinggi untuk
menyerupai korelasi dan regresi populasi. Hubungan panjang berat mengikuti hukum kubik, bahwa
berat ikan sebagai pangkat tiga dari panjangnya sesuai dengan persamaan dari (Effendie, 1979).
HASIL DAN PEMBAHASAN
Hasil perhitungan hubungan panjang total dan berat Ikan Buntal Pisang diperoleh nilai b
sebesar 3,30 yang bersifat allometrik positif yang berarti pertambahan panjang ikan lebih lambat
daripada berat tubuh. Nilai R = 0,89 menunjukkan pertumbuhan panjang ikan buntal pisang
mempengaruhi pertumbuhan berat sebanyak 89%.
Studi Hubungan Panjang Berat Ikan Buntal Pisang (H. Hapsara et al.)
179
3,00 y = 3.30x - 1.97

R² = 0.89
Log Berat (gr)

2,50
2,00
1,50
1,00
0,90 1,00 1,10 1,20 1,30 1,40 1,50
Log Panjang (cm)
Gambar 1. Hubungan Panjang dan Berat Ikan Buntal Pisang (Tetraodon lunaris) di PPI
Banyutowo, Pati, Jawa Tengah
Nilai b pada persamaan hubungan pajang berat ikan bersifat relatif, dapat berubah dengan
perubahan waktu. Jika faktor-faktor disekitar organisme seperti kondisi lingkungan perairan dan
ketersediaan makanan berubah makan nilai b yang diperoleh mungkin akan berubah. Apabila
dikaitkan dengan ketersediaan makanan, maka pertumbuhan Ikan Buntal Pisang bersifat
allometrik positif diduga karena makanan Ikan Buntal pisang di perairan Pati melimpah. Menurut
Effendie (2002), Makanan memiliki peran penting bagi organisme diantaranya untuk proses
pertumbuhan dan reproduksi. Makanan yang dimakan oleh ikan akan diproses di dalam tubuh
yang nantinya akan menghasilkan energi yang digunakan untuk aktivitas metabolisme dan
aktivitas fisik serta pertumbuhan.
Faktor luar berupa perubahan lingkungan di habitat ikan tersebut berupa, sifat fisika dan
kimia perairan serta komponen hayati seperti ketersedian makanan dan kompetisi (Devadoss,
1983). Hal yang sama juga dinyatakan oleh Al-Zibdah et al. (2007), perbedaan pola pertumbuhan
dapat disebabkan perbedaan kesuburan perairan yang memiliki hubungan dengan ketersediaan
makanan. Ikan yang didaratkan di PPI Banyutowo memiliki variasi yang sangat beragam
diantaranya Pari, Manyung, Mremang, Bambangan, Cucut, Cumi-cumi, Kuro, Belanak, Tiga waja,
Beloso, Ekor Kuning, Mata besar, Kakap Putih, Kuniran, Kapasan dan ikan hasil tangkapan lain
sebagainya (Dinas Kelautan dan Perikanan Kabupaten Pati, 2013). Banyaknya ikan yang
didaratkan di PPI Banyutowo menunjukkan perairan Pati masih dalam kondisi yang sangat baik.
Diduga dengan banyaknya jenis ikan tersebut variasi jenis makanan Ikan Buntal Pisang sangat
beragam di perairan Pati sehingga menurunkan kompetisi dalam mencari makan.
KESIMPULAN
Hasil penelitian studi hubungan panjang berat Ikan Buntal Pisang (Tetraodon lunaris) di
perairan Pati, Jawa Tengah dapat disimpulkan nilai hubungan panjang berat ikan buntal pisang
pada periode bulan Februari-Maret bersifat allometrik positif, dengan nilai b menunjukkan 3,30
berarti pertambahan berat lebih cepat dari pertambahan panjangnya.
DAFTAR PUSTAKA
Al-Zibdah ,M. & Odat, N. 2007. Fishery Status, Growth, Reproduction Biology And Feeding Habit
of Two Scombrid Fish From The Gulf of aqaba Red Sea. Lebanese. Science Journal. 8(2):3-
16.
Blackweel, B.G., Brown M.L. & Willis D.W. 2000. Relative weight (Wr) status and current use in
fisheries assessment and management. Reviews in Fisheries Science. 8:1-44.
180
Devadoss P. 1983. Further Observations On The Biology Of The Stingray, Dasyatis imbricatus
(Schneidier) at Porto Novo. Matsya 9:129-134.
Dinas Kelautan dan Perikanan Kabupaten Pati. 2013. Iktisar Penangkapan Perikanan Tangkap
Laut. Kabupaten Pati.
Effendie, M.I., 1979. Biologi Perikanan. Yayasan Dewi Sri, Bogor, 112 hlm.
Effendie, M.I., 2002. Biologi Perikanan. Yayasan Pustaka Nusatama, Yogyakarta, 157 hlm.
Gay, L.R., Mills. G.E. & Airasian, P. 2009. Educational Research, Competencies for Analysis
and Application. New Jersey: Pearson Education, Inc.
Hadi, S. 1979. Statistik. Cetakan Ke IV. Yayasan Penerbit Fakultas Psikologi UGM. Yogyakarata.
5 Hlm.
Him, Y.C. 2007. Detection and Biosynthesis of Puffer Fish Toxin From Bacterial Culture For Novel
Medical Application. [Tesis]. Hongkong: The Hongkong Polytechnic University.
Kottelat, M., Whitten, J.N., Kartikasari, S.N. & Wirjoatmodjo, S. 1993. Freshwater Fishes of
Western Indonesia and Sulawesi. Jakarta: CV Jaya Book.
Nazir. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta, 622 Hlm.
Nieto, F.R., Cobos, E.J., Tejada, M.A., Fernandez, C.S., Cano, R.G. & Cendan, C.M. 2012.
Tetrodotoxin (TTX) as a therapeutic agent for pain. Marine Drugs. 10:281-305.
Noguchi, T. & Arakawa, O. 2008. Tetrodotoxin-Distribution And Accumulation In Aquatic
Organisms, And Cases of Human Intoxication. Marine Drugs. 6:220-242.
Noguchi, T., Onuki, K. & Arakawa, O. 2011. Tetrodotoxin Poisoning Due To Pufferfish And
Gastropods, And Their Intoxication Mechanism. Toxicology. 11:1-10.
Richter, T.J. 2007. Development And Evaluation Of Standard Weight Equations Forbridgelip
Sucker And Large Scale Sucker. North American Journal o f Fisheries Management. 27:936-
939.
Saito, M. and Kunisaki, N., 1998. Proximate composition, fatty acid composition, free amino acid
contents, mineral contents, and hardness of muscle from wild and cultured puffer fish Takifugu
rubripes. Nippon Suisan Gakkaishi, 64(1):116-120.
Silva, M., Azevedo, J., Rodriguez, P., Alfonso, A., Botana, L.M. and Vasconcelos, V., 2012. New
gastropod vectors and tetrodotoxin potential expansion in temperate waters of the Atlantic
Ocean. Marine drugs, 10(4):712-726.
Wahyuni, T. and Affandi, R., 2017. Kebiasaan Makanan Ikan Buntal Pisang (Tetraodon Lunaris)
Di Perairan Mayangan, Jawa Barat. Jurnal Iktiologi Indonesia, 4(1):25-30.
Wahyudi, A.J. 2006. Kepiting beracun suku xanthidae: kajian dan hipotesis faktor–faktor
penyebabnya. Oseana. 301(4):31-38.
Widodo, J Dan Suadi. 2006. Pengelolaan Sumberdaya Perikanan Laut. Gajahmada University
Press. Yogyakarta. 252 Hlm.
Yusfiati. 2006. Anatomi Alat Pencernaan Ikan Buntal Pisang (Teraodon lunaris). [Tesis].
Departemen Biologi. Institut Pertanian Bogor.
Accepted Manuscript
Title: Tetrodotoxin Detection in Puffer Fish by a Sensitive

Planar Waveguide Immunosensor
Authors: Laia Reverté, Mònica Campàs, Betsy Jean Yakes,

Jonathan R. Deeds, Panagiota Katikou, Kentaro Kawatsu,
Michael Lochhead, Christopher T. Elliott, Katrina Campbell
PII: S0925-4005(17)31200-5
DOI: http://dx.doi.org/doi:10.1016/j.snb.2017.06.181
Reference: SNB 22652
To appear in: Sensors and Actuators B
Received date: 17-2-2017

Revised date: 25-6-2017
Accepted date: 29-6-2017
Please cite this article as: Laia Reverté, Mònica Campàs, Betsy Jean Yakes,
Jonathan R.Deeds, Panagiota Katikou, Kentaro Kawatsu, Michael Lochhead,
Christopher T.Elliott, Katrina Campbell, Tetrodotoxin Detection in Puffer Fish
by a Sensitive Planar Waveguide Immunosensor, Sensors and Actuators B:
Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.181
This is a PDF file of an unedited manuscript that has been accepted for publication.
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apply to the journal pertain.
Tetrodotoxin Detection in Puffer Fish by a Sensitive Planar Waveguide
Immunosensor
Laia Revertéa,b, Mònica Campàsb, Betsy Jean Yakesc, Jonathan R. Deedsc, Panagiota
Katikoud, Kentaro Kawatsue, Michael Lochheadf, Christopher T. Elliotta and Katrina
Campbella*
a Institute for Global Food Security, School of Biological Sciences, Queen’s University,
David Keir Building, Stranmillis Road, Belfast, UK BT9 5AG.
b IRTA, Ctra. Poble Nou, km 5.5, Sant Carles de la Ràpita, Spain.
c US Food and Drug Administration, Center for Food Safety and Applied Nutrition
(CFSAN), 5001 Campus Drive, College Park, MD 20740, USA.
d National Reference Laboratory on Marine Biotoxins, Ministry of Rural Development and
Food, 54627 Thessaloniki, Greece.
e Division of Bacteriology, Osaka Prefectural Institute of Public Health, 3-69, Nakamichi 1-
chome, Higashinari-ku, Osaka 537-0025, Japan.
f MBio Diagnostics Inc., 5603 Arapahoe Avenue, Boulder, Colorado 80303, USA.
Footnote: Laia Revertéa,b, PhD student at IRTA, conducted this work as Visiting
Researcher at QUB.
Corresponding Author
Dr Katrina Campbell
Institute for Global Food Security,
School of Biological Sciences,
Queen’s University,
David Keir Building,
Stranmillis Road,
Belfast, UK
BT9 5AG.
E-mail: katrina.campbell@qub.ac.uk
1
Highlights
 A sensitive nanoarray planar waveguide immunosensor was developed for

tetrodotoxin.
 Accurate toxin quantifications were obtained in only 10 minutes.
 The immunosensor has been successfully applied to the analysis of puffer fish.
 Good correlations are obtained between the immunosensor and other techniques.
Abstract
A nanoarray planar waveguide biosensor was developed for the detection of tetrodotoxin
(TTX). This technique, specifically designed for the first time for TTX, provided a compact
versatile user friendly device that obtained a test result in ten minutes. The device consisted
of nanoprinted toxin-conjugate arrays constructed in the manner of an indirect competitive
immunoassay, for the analysis of puffer fish samples under high flow conditions. The
applicability to natural samples was investigated through the study of matrix effects and toxin
recovery. The biosensor enabled the detection of TTX from 0.4 to 3.29 µg g-1 puffer fish
tissue. The sensitivity attained demonstrates this assay as a rapid and sensitive screening
method to detect TTX in different species of puffer fish, well below the Japanese maximum
permitted level (2 µg g-1) and the estimated safety level used in the EU and the US (0.8 µg
g-1). Assay repeatability and reproducibility were assessed at 0.4 and 0.8 µg g-1, showing
relative standard deviation (RSD) values below 15% and toxin recovery within 85-115%.
The appropriate correlation of data obtained from the biosensor compared to that reported
by ELISA, RBA, SPR biosensor and LC-MS/MS for the analysis of 12 puffer fish samples,
proved the feasibility and reliability of this immunosensor to support monitoring programs
and research activities.
Keywords: nanoarray; planar waveguide immunosensor; tetrodotoxin; puffer fish
2
1. Introduction
Tetrodotoxin (TTX) is one of the most hazardous low-molecular-weight marine neurotoxins
[1], whereby poisoning following consumption has a notorious lethal incidence. TTX is
commonly found in the organs of select puffer fish species, typically the liver, ovaries, and
gastrointestinal (GI) tract but also occasionally in the meat. TTX is also found in other
organisms, and this toxin is believed to be exogenously produced, starting from bacteria
which accumulate up through the food chain into fish, bivalve shellfish, amphibians and
octopus [2]. Human fatalities from Puffer Fish Poisoning (PFP) are mostly caused by the
ingestion of puffer fish (“fugu”) in Japan [3], but the presence of TTX has also been reported
in bivalve shellfish, harvested in several parts of Europe [4-6] and other countries [7, 8]. The
main symptoms of PFP range from neuromuscular (lips and tongue paraesthesia, tingling
and numbness) to gastrointestinal (nausea, abdominal pain, vomiting and diarrhea) [9].
More severe symptoms consist of uncoordinated movements, muscle spasms, and
progressive muscle paralysis that can result in respiratory failure in severe intoxication
cases. TTX toxicity (LD50) is 2-10 µg kg-1 intravenously and 10-14 µg kg-1 subcutaneously
in mammals as well as 334 µg kg-1 orally in mice [10].
TTX and its related analogues are hydrophilic, heat-stable, heterocyclic compounds [11, 12],
able to selectively block sodium channels [13, 14]. To date, 29 analogues of TTX have been
described, which when classified according to their structure are: hemylactal, lactone and
4,9 anhydro types. To the best of our knowledge, little is known about the role that these
analogues may play in terms of toxicity, with the exception of the study performed by Yotsu-
Yamashita and co-workers in rat membranes where 5,6,1-trideoxy TTX showed less toxicity
than other analogues [15]. To protect consumers against PFP in Japan, the regulatory limit
for TTX in food is 2 µg TTX equivalents g-1 [16]. In the US, no guidance level has been
established, and TTX intoxications from puffer fish are predominantly limited through
importation restrictions which only allow the puffer fish species Takifugu rubripes from Japan
3
to be brought into the country with additional strict processing guidelines [17]. All other
imported puffer fish products are subject to automatic detention without physical
examination by the US Food and Drug Administration (FDA) [18]. Despite these strict import
restrictions, several cases of PFP have been reported in recent years in the US due to the
unapproved importation of puffer species [19, 20]. Additionally, no regulatory limits have
been specifically set for TTX in Europe. However, in both Europe and the US, safety limits
have been set at 0.8 µg saxitoxin (STX) equivalents g-1 shellfish meat for the
pharmacologically similar paralytic shellfish poisoning (PSP) toxins. This concentration can
be used as a guide in determining the role of TTX in incidents of illness. The increasing
incidence of seafood TTX poisoning episodes in Europe and in other non-regulated regions
might have relevant consequences to food safety and economic fisheries. Therefore, there
is a real need for the development of methods suitable to regulate this toxin in food by means
of routine monitoring, thus, providing sufficient protection to human health and fishery
products [21].
In Europe, the shift for PSP toxins screening in bivalves and seafood from the mouse
bioassay (MBA) which inadvertently detected TTX toxins, to the method of Lawrence using
high-performance liquid chromatography with fluorescence detector (HPLC-FLD) [22],
created a gap in the detection for other important paralytic toxins such as TTX that are
relevant to food safety and that are not detected by the latter technique. Current methods
for TTX detection are mainly those based on analytical chemistry, such as HPLC and liquid
chromatography tandem-mass spectrometry (LC-MS/MS) [23]. Although useful techniques,
their performance is occasionally hampered by the lack of commercially available analytical
standards for some TTX analogues and by the requirement of skilled personnel and
expensive equipment. TTX detection has also been achieved by biochemical methods,
including immunoassays [24-30] and electrochemical [25, 31] and optical immunosensors
[32-37], some of these having shown promise not only as research activities but also as
4
screening tools in monitoring programs. Biosensors are increasingly being investigated due
to the advantages they offer in terms of sensitivity, portability, ease of use and robustness.
To date, surface plasmon resonance (SPR) immunoassay has been the most widely applied
biosensor-based method for TTX, though the cost of instrumentation has prevented this
method’s broader uptake. More recently, another optical biosensor, based on planar
waveguide technology, is gaining attention, and it has been successfully developed for other
marine toxins, such as okadaic acid (OA), domoic acid (DA) and saxitoxin (STX) [38], but
not yet explored for TTX detection. Accordingly, an immunosensor based on the planar
waveguide technology with fluorescence detection for the determination of TTX is described
herein. In this technique, waveguide cartridges are previously spotted with picoliter drops of
the desired toxin-conjugate, and the extent of the molecular interaction upon addition of the
antibody is determined through a fluorescence reader. In the assay, when toxin is not
present in a sample, nearly all the primary antibody is bound to the toxin conjugate attached
on the surface, and a high fluorescence signal is obtained from the secondary antibody.
When toxin is present in the sample, the antibody binds to this toxin in solution, thereby
decreasing the amount of primary antibody available to bind to the surface and thus the
fluorescence signal decreases proportionally to the toxin concentration. The signal itself is
generated from light that is transmitted from the bottom of the waveguide cartridges, leading
to the excitation of the fluorophores on the labeled-secondary antibody, thus enabling the
sensitive recording of binding events on the surface (Figure 1). The aim of this work was to
investigate this planar waveguide biosensor for the rapid detection of TTX in puffer fish
samples using a simple extraction method. After development, this biosensor was applied
to the analysis of samples, and the results were compared to other methods of analysis
including the MBA, receptor binding assay (RBA), ELISA, SPR biosensor and LC-MS/MS
analysis.
5
2. Material and methods
2.1 Reagents
Bovine serum albumin (BSA), casein from bovine milk, phosphate buffered saline (PBS)
tablets, sodium acetate anhydrous, sodium chloride, sodium phosphate dibasic, sodium
phosphate monobasic and Tween 20 were purchased from Sigma-Aldrich (Dorset, UK).
Alexa Flour 647 goat anti-mouse IgG (GaM) antibody and BSA-Alexa Fluor 647 conjugate
were obtained from Invitrogen Ltd. (Paisley, Scotland). TTX standard was procured from
Biomol/Affiniti Research Products (Exeter, UK).
2.2 Sample collection
Seven puffer fish samples (Lessepsian migrant puffer fish Lagocephalus sceleratus) were
obtained from the National Reference Laboratory for Marine Biotoxins (NRLMB) of Greece.
Five puffer fish muscle samples (Lagocephalus lunaris), which were previously associated
with several outbreaks of illness in the US in 2007 [19], were from collaborators at the FDA.
TTX-free puffer fish muscle from Takifugu rubripes was also provided by FDA and used as
negative tissue for matrix effects and toxin recovery experiments.
2.3 Sample extraction method
The extraction method used was previously described by Bates and co-workers [39] for the
SPR analysis of PSP toxins in shellfish [40]. Briefly, 1 g portions of puffer fish homogenate
were weighed into 20-mL disposable Sterilin centrifuge tubes, and 4 mL of sodium acetate
buffer pH 5.0 was added. Each tube was vigorously vortex mixed for 10 s followed by roller
mixing (STUART SRT9, Bibby Scientific Ltd, UK) at 33 rpm for 30 min. After mixing, samples
were centrifuged at 3,600×g (SORVALL Legend RT, DJB Labcare Ltd, UK) for 10 min at
room temperature. The supernatant was decanted, collected, and 40-fold diluted in MBio
6
assay buffer at pH 7.4 (25 μL extract to 975 μL buffer). MBio assay buffer consisted of 1%
BSA, 0.05% Tween 20, one PBS tablet and 200 mL of distilled water.
2.4 Synthesis of the immunological reagents
TTX conjugate, TTX-jeffamine-keyhole limpet hemocyanin (TTX-jeff-KLH), was produced by
combining different methods and with some modifications [24, 41-44]. In brief, jeffamine was
conjugated initially to KLH via amine coupling in the same manner as that previously
described for BSA [41]. One mg of TTX and 50 µL of 37% formaldehyde were then added
to 5 mg of jeff-KLH protein. This mixture was allowed to react for 72 h at room temperature
followed by dialysis in 0.15 M saline solution (3 x 4 L) over 24 h. The production of
monoclonal antibody against TTX (mAb) was previously described [24]. Moreover, the mAb
has been further characterized and used in published works [27, 40].
2.5 Cartridge spotting and standardized building process
Microarrays were printed using a sciFLEXARRAYER S5 spotter (Scienion, Germany)
equipped with a Bio-Jet print head capable of dispensing 25-nL droplets with spot diameters
of approximately 0.5 mm on the slides in a 2×22-array format. TTX conjugate diluted in
sterile filtered printing buffer (100 mM sodium phosphate, 50 mM sodium chloride, 100 µg
mL-1 BSA, 0.005% Tween 20, pH 8.0) was spotted at four concentrations (10, 25, 50, and
100 μg mL-1) in replicates of two. Upon optimization, 50 and 100 µg mL -1 TTX conjugate
concentrations were selected and spotted in replicates of four per cartridge for the following
experiments. In addition, fluorescently labeled BSA conjugate was spotted at 4 µg mL-1 at
each of the four corners of the array to enable the proper alignment within the SnapESi
reader. Printing buffer drops were spotted as negative controls between BSA and TTX
conjugate spots. All printing procedures were performed at room temperature and 65%
humidity. Printed waveguide arrays were then left at 25 °C and 35% humidity overnight.
7
After this, the waveguide arrays were blocked with casein solution for 5 min, rinsed with
deionized water and dried by 1,000 rpm centrifugation (SORVALL Legend RT, DJB Labcare
Ltd, UK) for 5 min. To fit the waveguides on the MBio reader, waveguides were inserted into
cartridge housing that provided a flow channel of 5-mm-wide, 50-mm-long and 0.145-mm-
high. The building process was completed with the addition of an adsorbent pad and a top
cover. Cartridge components and the SnapEsi LS system were supplied by MBio
Diagnostics Inc. (Boulder, Colorado, US).
2.6 Immunosensor protocol
MBio cartridges were placed on a cartridge rack at a special angle to enhance fluidic flow.
The protocol was similar to that described by McGrath and co-workers [45]. The Ttotal assay
time was 10 min. First, 150 µL of 1/500 mAb dilution:standard TTX or sample (1:1, v:v) were
applied to the cartridge for 5 min, followed by 150 µL of 10 µg mL -1 Alexa Fluor 647 GaM
IgG solution for 5 min. Each cartridge was then read on the MBio assay reader. Initially,
different time exposures were collected, ranging from 25 to 100 ms of exposure. After this
evaluation, readings were recorded at the fixed time point 75 ms after sample addition and
were exported into Microsoft Excel for data analysis.
2.6.1 Preparation of buffer calibration curves and data analysis
A stock solution of 1,000 µg L-1 was used to prepare TTX calibrants at concentrations of 0,
0.5, 1, 2.5, 5, 10 and 100 µg L-1 in MBio assay buffer. These working standards were
equivalent to 0, 0.08, 0.16, 0.4, 0.8, 1.6 and 16 μg TTX g -1 puffer fish. A calibration curve
was prepared for each conjugate concentration. Two cartridges (n=2) were used for each
TTX working standard. Each cartridge consisted of a total of 16 spots, 8 spots per conjugate
concentration.
8
Curves were normalized with respect to the controls without TTX (0 µg L -1) and fitted to a
sigmoidal logistic four-parameter equation using SigmaPlot software 12.0 (Systat Software
Inc., California, US). The concentrations of TTX in the samples were then interpolated from
these curves. In order to compare the response obtained with both TTX conjugate
concentrations, data was first tested for normality using the Shapiro-Wilk test. For data
following a normal distribution, t-test was performed; otherwise, Mann-Whitney Rank Sum
Test was done. The level of significance was set at p<0.05. Finally, correlation between both
TTX conjugate concentrations was evaluated using the linear regression model. All statistics
were carried out using Sigmastat 3.1 software (Systat Software Inc., California, US).
The sensitivity (IC50) and dynamic range (IC20-IC80) of the assay were determined from the
calibration curves obtained. The LOD for the present work was defined as the 20% inhibitory
concentration (IC20), which corresponds to the concentration of toxin required to decrease
the response by 20% compared to the response when no toxin is present in the system
(100% mAb binding).
2.7 Evaluation of puffer fish matrix effects and toxin recovery
For puffer fish matrix calibration curves, aliquots of 1 g of negative puffer fish muscle tissue
homogenate were fortified with 20 µL of several concentrations of TTX and then extracted.
Additionally, negative puffer tissue was extracted and then fortified with TTX (50 µL of TTX
solution to 950 µL MBio assay buffer). With this procedure, puffer fish tissue and puffer fish
extracts were fortified with TTX at 0, 0.08, 0.16, 0.4, 0.8, 1.6 and 16 μg TTX g-1 puffer fish.
The spiking protocol was designed for the regulatory limit for PSP of 0.8 μg STX equiv. g -1
tissue to fall within the middle of the calibration curves pre and post-extraction, thus ensuring
the capability of the present method to screen TTX effectively at this concentration. Both,
TTX fortified-puffer fish tissue and extracts were 40-fold diluted in MBio assay buffer and
then run on the immunosensor for subsequent analysis. Curves were analyzed and
9
compared to that prepared in buffer in order to evaluate the puffer fish matrix effects and to
measure the TTX recovery under experimental conditions. Sensitivity (IC 50) and dynamic
range (IC20-IC80) of the assay for the three curves were obtained by fitting the data to
sigmoidal, logistic, four-parameter equations. The LOD, defined previously as the 20%
inhibitory concentration (IC20), was calculated from an average of two replicates of
calibration curves. In order to calculate the toxin recovery, mAb binding percentages
obtained at TTX fortification levels of 0.4 and 0.8 µg g-1 of puffer fish tissue were compared
to the fortification levels.
2.8 Repeatability studies
The performance of the method was assessed using the PSP toxin maximum permitted level
of 0.8 µg STX equivalents (equiv.) g-1 shellfish, since no regulatory limit is currently set within
EC legislation for TTX. This repeatability study was carried out at two levels of TTX
fortification (0.8 and 0.4 µg g-1) and by the analysis of 5 replicates (n=5) of a single puffer
fish sample. Each replicate was extracted and 40-fold diluted in MBio assay buffer for
analysis. Then, toxin recoveries as well as relative standard deviation (RSD) at each
fortification level were calculated using the response value obtained from each individual
analysis. Additionally, the same extracts were reanalyzed the following day, and toxin
recoveries and RSD values at each fortification level were calculated.
2.9 Puffer fish sample analysis and comparison with analytical, biological and
biochemical assays, and biosensors
Different tissues and species of puffer fish (n=12) were analyzed (16 replicates each) by the
present method using the extraction method previously detailed. Once extracted, samples
were 40-fold diluted in MBio assay buffer. Specifically, 2 muscle samples, 2 liver samples,
10
2 skin samples and 1 GI tract from 3 individuals of L. sceleratus, and 5 muscle samples from
5 individuals of L. lunaris were analyzed, and TTX contents expressed in µg g-1 were
calculated and compared with other analytical techniques. Puffer fish tissues were split into
two groups, named as set 1 and set 2, according to the other techniques used to analyze
the samples. Therefore, set 1 includes samples 1-5 (L. lunaris samples), which were
analyzed by LC-MS/MS, RBA, ELISA and SPR biosensor. Analysis methods and data of
this first set of samples was previously described in the work performed by Cohen and co-
workers [19] and Yakes and colleagues [46]. Set 2 comprises three different fish (L.
sceleratus samples) with different tissues: 6 (muscle), 7.A (GI tract), 7.B (liver), 7.C (skin),
and 8.A (liver), 8.B (skin) and8.C (muscle), previously analyzed by LC-MS/MS and MBA.
Analysis methods and data of the second set were described in the work performed by
Katikou and co-workers [47] and Rodríguez and colleagues [48], respectively. Statistics to
compare data obtained with different methods was performed following the same procedure
used to compare data from calibration curves of both TTX conjugate concentrations. TTX
concentrations in these samples was achieved by taking into account the mAb binding
inhibitory concentration of the sample (within the dynamic range IC20-IC80) from the
associated calibration curve. Those samples that showed responses above 80% were
further diluted to bring their fluorescence signals within the dynamic range of the calibration
curve.
11
3. Results and discussion
Given the increasing concern about the prevalence of TTXs in fish and shellfish products in
European waters and the high potency of these toxins, increased monitoring through the
development of rapid, sensitive and robust methods is needed to ensure human health
protection. With the aim of replacing animal use methods such as the MBA for TTX
detection, in the present work the development of a sensitive planar waveguide-based
biosensor able to detect TTX in multiple puffer fish tissues is described.
3.1. Immunosensor protocol optimization
Following the improved MBio procedure described by McGrath and co-workers [45], TTX-
jeff-KLH conjugate concentration and mAb dilution were firstly optimized. Of the four spotted
TTX conjugate concentrations (10, 25, 50 and 100 µg mL -1), those that gave a fluorescence
value below 150 or above 500 when no TTX was present, were discarded. Following this
criteria, 10 and 25 µg mL -1 TTX conjugate concentrations were no longer used. As such,
given that the spotting of TTX conjugate at 50 and 100 µg mL-1 provided similar responses,
both concentrations were selected for comparison in subsequent experiments. The capacity
of the immobilized TTX conjugates to inhibit the mAb binding response was then assessed
using 1/250 and 1/500 mAb dilutions at 100 µg L-1 of TTX standard. At both antibody
dilutions, this TTX concentration was able to completely inhibit the mAb binding. However,
as a lower antibody concentration generally results in a higher sensitivity, the 1/500 mAb
dilution was selected for the assay. With the experimental parameters set at TTX conjugate
spotting concentrations of 50 and 100 µg mL -1, mAb at 1/500 dilution and fluorescently-
labeled GaM IgG antibody at 10 µg mL-1 (concentration set through checkerboard titration
in the work performed by Meneely and colleagues [49]), the whole calibration curves were
performed in buffer.
12
3.2 Assay sensitivity determined from buffer calibration curves
Calibration curves obtained for both TTX conjugate spotting concentrations in MBio assay
buffer are shown in Figure 2 a and b, and, the midpoint (IC50), limit of detection (LOD) and
dynamic range determined are shown in Table 1. The IC50 and LOD values were fairly
similar at both spotting concentrations (P=0.797; T=0.275) with a correlation of y=1.45x-
1.72. A narrower dynamic range was obtained with 100 µg mL-1 of TTX conjugate, likely due
to the binding kinetics effects of reduced competition in binding with additional coating
antigen on the arrayed surface. IC50 and LOD values provided by this new immunosensor
(6.0 and 2.5 µg L-1, respectively), compared well with a previous indirect SPR immunosensor
that used the same antibody (6.6 and 2.4 µg L-1, respectively) [32], as well as with an SPR
biosensor that employed a commercial antibody (approximately 6 and 0.3 µg L -1,
respectively) [33]. Further, the IC50 values of the sensor developed herein were better than
those recently reported for other indirect SPR immunosensors for TTX on a commercial
instrument ((IC50 of 28.9 and IC20 of 7.8 µg L-1) [46] and from the average of three
laboratories (IC50 of 74 and IC20 of 14 µg L-1) [35]). As such, the sensitivity achieved in buffer
was determined to be suitable for further assay development and the evaluation of matrix
effects.
3.3. Evaluation of puffer fish matrix effects and toxin recovery
In order to demonstrate the applicability of the method to the analysis of real samples, matrix
effects of puffer fish and of the extraction protocol procedure were investigated through
spiking TTX into puffer fish extracts and into puffer fish fish tissue homogenates,
respectively. Calibration curves obtained for both TTX conjugate spotting concentrations in
buffer and in puffer fish matrix fortified pre and post-extraction are shown in figure 2. As the
curves essentially overlap in the dynamic range of the assay, it can be concluded that there
were little matrix effects from the puffer fish tissue and extraction procedure when these are
13
diluted and run on the immunosensor. Furthermore, TTX concentrations and toxin
recoveries obtained pre and post-extraction are shown in Table 2.
TTX concentrations determined in fortified-puffer fish extracts were similar to those obtained
in buffer (P=1.000; T=5.000). Statistically, no significant differences were found between the
50 or 100 µg mL-1 TTX conjugate surfaces for both fortification levels (P=0.667; T=6.000;
y=0.98x+0.01). These similar results demonstrate that there are negligible puffer fish matrix
effects on the system.
With regard to TTX concentrations determined when spiking TTX in puffer fish tissues,
appropriate recovery values were obtained, and these concentrations were similar to the
TTX fortification levels (P=1.000; T=5.000). These accurate recovery values demonstrated
the good efficiency of the extraction protocol and that there were limited toxin losses during
the extraction process. Again, no significant differences were found between using 50 or
100 µg mL-1 of TTX conjugate (P=0.667; T=5.500; y=1.03x-0.03). TTX concentrations
obtained in fortified-puffer fish tissues were also similar to that in fortified-puffer fish extracts
(P=1.000; T=5.000), reaffirming the negligible matrix effects on the biosensor and that
accurate results from natural samples could be obtained.
3.4. Repeatability studies
The five homogenate-tissue samples fortified at 0.8 as well as five at 0.4 µg g-1 extracted
and analyzed over two days, showed acceptable toxin recovery values. Specifically, results
expressed in percentage values with respect to the TTX-fortified concentration (0.8 or 0.4
µg g-1) and determined from the calibration curve in buffer were 89.1 and 78.5% for TTX-
conjugate spotted at 50 µg mL-1 and 106.3 and 106.8% for 100 µg mL-1 of conjugate,
respectively, on day 1. Similarly, the same extracts reanalyzed on day 2 provided toxin
recoveries of 81.5 and 97.6% for 50 µg mL-1 of conjugate and 96.6 and 107.3% for 100 µg
14
mL-1 of conjugate. To assess the repeatability of the present sensor, precision was
expressed as the RSD of the results , according to the 2012 AOAC International Guidelines
[50]. Hence, on day 1, RSD values with respect to 50 µg mL-1 of TTX conjugate were 14.6%
for 0.8 µg g-1 and 10.8% for 0.4 µg g-1, while values were 9.9% for the 0.8 µg g-1 and 2.1%
for the 0.4 µg g-1 for TTX conjugate 100 µg mL-1. On day 2, RSD values were 12.5 and 8.3%
for 50 µg mL-1 of TTX conjugate and, 10.4 and 13.1 for 100 µg mL-1 of TTX conjugate at 0.8
and 0.4 µg g -1, respectively. Considering the expected precision as a function of analyte
concentration, RSD values for 0.8 and 0.4 µg toxin g-1 puffer fish tissue should be lower than
15%, and all the obtained RSD values for this assay were below this value. It should be
noted that this was a comparison of extracts between days and not re-extraction of the fish
due to limitations in toxin availability. Nevertheless, this results illustrate the degree of
repeatability in analysis. Additionally, the toxin recovery values obtained in same extracts
on day 2, demonstrate that samples containing TTX were stable at least for 1 day. All these
results demonstrated that the new planar waveguide biosensor allowed for suitable precision
under repeatability conditions within independent tests and that already extracted samples
could be reanalyzed on the following day with the same precision. Additionally, differences
between values obtained with both TTX conjugate concentrations were statistically not
significant. The good agreement of the results provided by this repeatability study at 0.8 and
0.4 µg TTX g-1 fish tissue confirmed the feasibility and reliability of the planar waveguide
biosensor for being applied to the analysis of real samples.
3.5. Puffer fish samples analysis and comparison with other methods
Table 3 shows the TTX concentrations determined per g of puffer fish tissue by the
nanoarray-based biosensor as well as by other analytical and biochemical methods. In
15
general, TTX contents determined in the samples provided by the present biosensor
correlated well with the data reported by the other methods.
Among the 12 puffer fish samples analyzed, 4 samples (1 and 5 muscle tissues from
L. lunaris, and 8.B (skin) and 8.C (muscle) from L. sceleratus) were found to contain TTX
contents below the regulated Japanese level of 2 µg TTX g-1 fish tissue by all the techniques.
With respect to the other samples, within the L. lunaris samples (Set 1, Table 3a), the trend
from the most to the lowest toxic muscle tissue was: 4 > 3 > 2 > 1 > 5 for all the techniques
employed. The high concentrations of TTX found in muscle tissue extracts 3 and 4 from L.
lunaris individuals were in accordance with the high contents of TTX found in the meat of
this species of puffer fish, which is a causative source of food poisonings worldwide [51]. On
the contrary, the distribution of TTX found among the L. sceleratus tissues from the most to
the lowest toxicity was: GI-tract (7.A) > liver (7.B, 8.A) > skin (7.C, 8.B) ≥ muscle (6, 8.C).
Again, the toxin distribution found within tissues agrees well with that reported for L.
sceleratus species [3, 52], as the gastrointestinal tract is expected to be the most toxic,
followed by the liver and finally by the skin and muscle.
In the analysis of the first set of samples (1-5), excellent correlation was obtained between
the TTX contents determined by the planar-waveguide biosensor at both conjugate
concentrations (y=1.06x-0.11; R²=0.996; P=1.000; T=27.500). Because of this similarity
(differences statistically not significant) and to simplify the results, the comparison with other
methods was only performed with reference to the 100 µg mL -1 conjugate surface. When
comparing the quantifications provided by the planar waveguide biosensor with those LC-
MS/MS values reported previously in Cohen and co-workers [19] and Yakes and colleagues
[46], higher TTX contents were reported by the biosensor with a correlation of y=1.70x-0.08
(R²=0.987) obtained, although the differences were statistically not significant (P=0.517;
T=0.677). The higher TTX concentrations in the L. lunaris muscle samples determined by
the biosensor could be attributed to the fact that the previous LC-MS/MS quantifications for
16
these samples were determined solely for TTX while the mAb used herein may react with
multiple TTX analogues potentially present in the puffer fish samples. Rodriguez and co-
workers [48] found multiple TTX analogs in the closely related species L. sceleratus, several
of which were in higher concentration than TTX itself. Most of these analogs are less potent
than TTX, as evidenced by the poor correlation to MBA data in that study. It is not currently
known if these same TTX analogs are also present in L. lunaris and what their contributions
are to total toxicity. This possibility was supported by the correlation obtained by RBA and
the planar-waveguide biosensor (y=1.32x-0.36; R²=0.994; P=0.757; T=0.320). The high
TTX concentration achieved by the biosensor in this case could also be due to the different
affinity between the two biorecognition molecules (mAb for the waveguide and sodium-
channel receptor for the RBA) as well as the final reporting element (fluorescently labeled
secondary antibody for the waveguide and H3-STX for the RBA).
The same trend was observed when comparing the waveguide assay data to that obtained
by ELISA (y=1.12x+0.36; R²=0.933; P=0.489; T=0.725) and SPR immunosensor
(y=1.70x+0.02; R²=0.986; P=0.510; T=0.690). Although data obtained by the biosensor was
similar to that obtained by ELISA and SPR immunosensor, small differences in determining
TTX content can similarly be attributed to differences in biorecognition elements and
transduction techniques. Specifically, in the SPR biosensor the TTX was chemically
immobilized on dextran chips, and the signal was measured in real time. In ELISA and the
waveguide sensor, TTX-conjugate was coated on wells and on nanoarrays, respectively,
which likely resulted in the slightly different responses. Furthermore, although both ELISA
and the biosensor are indirect strategies that use a secondary labeled antibody, the final
output is given by the absorbance of an enzyme in ELISA and by fluorescence in the
biosensor. Although TTX contents determined by the planar waveguide biosensor were
slightly higher than those reported by the other techniques, they were still in good
17
agreement. Within this set of samples, the trend from higher to lower TTX concentrations
was the same by all techniques.
With regard to the second set of samples, excellent correlation was obtained between data
provided by the present biosensor for both conjugate concentrations (y=1.07x-0.39;
R²=0.998), with no significant statistical difference (P=0.902; T=51.00). As for the first set of
samples, data provided by the biosensor using 100 µg mL-1 of conjugate was used for further
comparison. As observed in other works [27], TTX concentrations determined by the planar
waveguide biosensor were generally slightly lower than those reported by MBA, but the data
were still in good agreement (y=0.74x+0.42; R²=0.987), with the differences statistically not
significant (P=1.000; T=27.000). The overestimation of the TTX contents provided by MBA
is attributed to the different principle of the techniques; specifically, MBA reports global
toxicity using animal models and could detect co-existing toxins other than TTXs. Moreover,
since no cross-reactivity data of the TTX analogues is available for this biosensor and
antibody and toxin equivalency factors (TEFs) are not well-established, the biosensor
response was not necessarily expected to fully match the MBA data.
Different scenarios were assumed when comparing the TTX contents obtained with the
current biosensor to those determined by LC-MS/MS analysis. If only individual TTX is
evaluated, the data correlated quite well (R2=0.945) but much higher TTX contents were
determined by the biosensor (y=3.16x+0.76). These differences in the concentrations,
although statistically not significant (P=0.310; T=1.109), seem to indicate that some of the
TTX analogues detected in the LC-MS/MS were also recognized by the antibody. In the
second scenario, TTX analogues were considered to have the same mAb affinity as TTX,
thus with a cross-reactivity factor (CRF) of 1. Unfortunately, worse correlation (R2=0.702)
and higher overestimation (y=8.83x+28.91) were obtained compared to LC-MS/MS analysis
using this scenario. This underestimation of LC-MS/MS analysis with respect to the planar
waveguide biosensor suggests that not all the TTX analogues were recognized by the
18
antibody in the MBio biosensor, or they are recognized with low affinity. Looking at the LC-
MS/MS individual concentrations, it seems evident that the analogues 5,6,11-trideoxy-TTX
(1 and 2) cannot be recognized by the antibody at the same extent as TTX (i.e., much higher
quantifications would have been obtained with the planar waveguide biosensor for 7.A (GI
tract) and 7.B (liver) samples). Moreover, this analogue has been considered almost non-
toxic by other authors [52].
As an intermediate approach, only 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX and 11-
deoxyTTX were theorized to be recognized by the antibody with same affinity than TTX. In
this case, excellent correlations (R2=0.954) and similar quantifications (y=1.07x+0.68) were
obtained between MBio biosensor and LC-MS/MS analysis (P=0.690; T=30.000). Of course,
this criterion was used only to demonstrate that results can change to a high extent
depending on the analogues considered. Ideally, and according to what has been previously
published [27], to better compare the concentrations obtained by the biosensor to that
provided by LC-MS/MS, CRFs of the antibody towards co-existing TTX analogues should
be established and applied to the individual measurements determined by LC-MS/MS.
Unfortunately, TTX analogue standards were not available to investigate this issue in detail.
As for the general results, the biosensor, MBA and LC-MS/MS were in agreement in
determining the gastrointestinal tract as the most toxic tissue, followed by the liver tissue of
L. sceleratus species. Due to the low TTX content, it was more difficult to observe a trend in
muscle and skin tissues by the different methods used herein.
Notably, the present biosensor was proven to be useful as screening tool and was also
capable of rapidly (in 10 min) detecting trace TTX contents, thereby meeting the
requirements in terms of sensitivity not only of the Japanese regulation but also of the similar
mode-of-action PSP toxins legislation.
19
4. Conclusions
The present work reported the development of a rapid and specific nanoarray planar
waveguide biosensor for the determination of TTX concentration in puffer fish samples. The
biosensor attained good sensitivity (LOD of 2.5 µg L-1), thus being capable of detecting TTXs
as low as 0.4 µg g-1 puffer fish. This concentration is below the EU and US regulatory limit
of 0.8 µg STX equiv. g-1 shellfish meat for the similar PSP toxins and can be used as a guide.
Additionally, the negligible effects of puffer fish matrix on the biosensor and the excellent
toxin recoveries obtained reinforce the reliability and applicability of the innovative method.
This is further evidenced by the general agreement found between the TTX concentrations
determined by the biosensor and the other methods (LC-MS/MS, RBA, ELISA, MBA and
SPR biosensor) in the analysis of naturally-contaminated puffer fish samples of different
species.
This biosensor configuration presents multiple benefits, including simplicity and rapidity of
the assay, design versatility, small reagent volumes, good reproducibility, and accurate and
precise toxin quantifications. In addition to these advantages, this nanoarrayed configuration
could be used together with other related toxins for simultaneous multi-toxin detection, which
represents a breakthrough in the development of compact, multiplexed devices. Despite the
planar waveguide technology not being new per se, this is the first immunosensor using this
technique that has been specifically developed for TTX. Thus, this new approach can be
considered as a proof of concept, being not only applicable to TTX but also to other emerging
toxins of seafood safety and environmental surveillance fields.
On the whole, the biosensor described herein has been proven to be a promising high-
throughput sample screening tool for implementation in research and monitoring programs,
being able to analyze multiple samples with toxin contents below the permitted levels of
concern. Furthermore, taking into consideration the presence of TTX recently reported in
20
European shellfish mollusks of England, Greece and Netherlands, the biosensor developed
herein could have suitable sensitivity, at lower proposed action levels, to be applicable to
shellfish by only slightly modifying the sample preparation.
Acknowledgements
This research was funded through the Advanced ASSET project, partly funded through
InvestNI and the European Sustainable Programme 2007-2013 under the European
Regional Development Fund. The authors would like to acknowledge funding received from
the CERCA Programme/Generalitat de Catalunya. The authors would like to thank Sara
McNamee for technical support and training in the use of equipment, and Stacey DeGrasse
and Kevin White for their previous work on the analytical detection of tetrodotoxin in
samples.
21
References
[1] EG Moczydlowski, The molecular mystique of tetrodotoxin, Toxicon, 63(2013) 165-83.

[2] V Pratheepa, V Vasconcelos, Microbial diversity associated with tetrodotoxin production in
marine organisms, Environ. Toxicol. Pharmacol., 36(2013) 1046-54.
[3] T Noguchi, O Arakawa, Tetrodotoxin - Distribution and accumulation in aquatic organisms,
and cases of human intoxication, Mar. Drugs, 6(2008) 220-42.
[4] P Rodriguez, A Alfonso, C Vale, C Alfonso, P Vale, A Tellez, et al., First toxicity report of
tetrodotoxin and 5,6,11-trideoxyTTX in the trumpet shell Charonia lampas lampas in Europe,
Anal. Chem., 80(2008) 5622-9.
[5] AD Turner, A Powell, A Schofield, DN Lees, C Baker-Austin, Detection of the pufferfish toxin
tetrodotoxin in European bivalves, England, 2013 to 2014, Eurosurveill., 20(2015a) 2-8.
[6] A Vlamis, P Katikou, I Rodriguez, V Rey, A Alfonso, A Papazachariou, et al., First Detection of
Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the
Dinoflagellate Prorocentrum minimum, Toxins, 7(2015) 1779-807.
[7] Y Bentur, J Ashkar, Y Lurie, Y Levy, ZS Azzam, M Litmanovich, et al., Lessepsian migration
and tetrodotoxin poisoning due to Lagocephalus sceleratus in the eastern Mediterranean,
Toxicon, 52(2008) 964-8.
[8] PS McNabb, DI Taylor, SC Ogilvie, L Wilkinson, A Anderson, D Hamon, et al., First Detection
of Tetrodotoxin in the Bivalve Paphies australis by Liquid Chromatography Coupled to Triple
Quadrupole Mass Spectrometry With and Without Precolumn Reaction, J. AOAC Int., 97(2014)
325-33.
[9] T Noguchi, K Onuki, O Arakawa, Tetrodotoxin poisoning due to pufferfish and gastropods,
and their intoxication mechanism, ISRN toxicol., 2011(2011) article ID 276939, 10 pages.
doi:10.5402/2011/276939
[10] A Alcaraz, RE Whipple, HR Gregg, BD Andresen, PM Grant, Analysis of tetrodotoxin,
Forensic Sci. Int. , 99(1999) 35-45.
[11] K Tsuda, R Tachikawa, C Tamura, S Ikuma, M Kawamura, K Sakai, et al., On structure of
tetrodotoxin, Chem. Pharm. Bull., 12(1964) 1357-74.
[12] RB Woodward, JZ Gougouta, Structure of tetrodotoxin, J. Am. Chem. Soc., 86(1964) 5030-
5030. doi: 10.1021/ja01076a076
[13] RJ French, D Yoshikami, MF Sheets, BM Olivera, The Tetrodotoxin Receptor of Voltage-
Gated Sodium Channels-Perspectives from Interactions with mu-Conotoxins, Mar. Drugs,
8(2010) 2153-61.
[14] CH Lee, PC Ruben, Interaction between voltage-gated sodium channels and the
neurotoxin, tetrodotoxin, Channels, 2(2008) 407-12.
[15] M Yotsu-Yamashita, A Sugimoto, A Takai, T Yasumoto, Effects of specific modifications of
several hydroxyls of tetrodotoxin on its affinity to rat brain membrane, J. Pharmacol. Exp.
Ther., 289(1999) 1688-96.
[16] T Kawabata, The manual for the methods of food sanitation tests . Bureau of
Environmental Health, Ministry of Health and Welfare, Japan Food Hygienic Association,
Tokyo, Japan, 1978, pp. 232-40.
[17] US FDA, Exchange of letters between Japan and US Food and Drug Administration
regarding puffer fish. In International cooperative agreements manual. p. 57-73. Available at:
http://www.fda.gov/InternationalPrograms/Agreements/MemorandaofUnderstanding/ucm10
7601.htm. Accessed 9 October 2016, 1989.
[18] US FDA, Import Alert #16-20: Detention without physical examination of puffer fish and
foods that contain puffer fish. http://www.accessdata.fda.gov/cms_ia/importalert_37.html
Accessed by October 2016, 2015.
[19] NJ Cohen, JR Deeds, ES Wong, RH Hanner, HF Yancy, KD White, et al., Public Health
Response to Puffer Fish (Tetrodotoxin) Poisoning from Mislabeled Product, J. Food Prot.,
72(2009) 810-7.
22
[20] JB Cole, WG Heegaard, JR Deeds, SC McGrath, SM Handy, Tetrodotoxin Poisoning
Outbreak from Imported Dried Puffer - Fish Minneapolis, Minnesota, 2014, Mmwr-Morbidity
and Mortality Weekly Report, 63(2015) 1222-5.
[21] AD Turner, C Higgins, W Higman, J Hungerford, Potential Threats Posed by Tetrodotoxins
in UK Waters: Examination of Detection Methodology Used in Their Control, Mar. Drugs,
13(2015b) 7357-76.
[22] JF Lawrence, B Niedzwiadek, C Menard, Quantitative determination of paralytic shellfish
poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography
with fluorescence detection: Collaborative study, J. AOAC Int., 88(2005) 1714-32.
[23] M Asakawa, Shida, Y., Miyazawa, K., Noguchi, T., Chromatography - The Most Versatile
Method of Chemical Analysis. Instrumental Analysis of Tetrodotoxin., InTech Open Science,
Rijeka, Croatia, 2012, p. 245.
[24] K Kawatsu, Y Hamano, T Yoda, Y Terano, T Shibata, Rapid and highly sensitive enzyme
immunoassay for quantitative determination of tetrodotoxin, Jpn. J. Med. Sci. Biol. , 50(1997)
133-50.
[25] D Neagu, L Micheli, G Palleschi, Study of a toxin-alkaline phosphatase conjugate for the
development of an immunosensor for tetrodotoxin determination, Anal. Bioanal. Chem.,
385(2006) 1068-74.
[26] TJG Raybould, GS Bignami, LK Inouye, SB Simpson, JB Byrnes, PG Grothaus, et al., A
monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples, J.
Clin. Lab. Anal., 6(1992) 65-72.
[27] L Reverté, P de la Iglesia, V del Río, K Campbell, CT Elliott, K Kawatsu, et al., Detection of
Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and
Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay (vol 87, pg
10839, 2015), Anal. Chem., 88(2015) 2511-.
[28] AN Stokes, BL Williams, SS French, An improved competitive inhibition enzymatic
immunoassay method for tetrodotoxin quantification, Biol. Proced. Online 14(2012) 3.
[29] R Wang, A Huang, L Liu, S Xiang, X Li, S Ling, et al., Construction of a single chain variable
fragment antibody (scFv) against tetrodotoxin (TTX) and its interaction with TTX, Toxicon,
83(2014) 22-34.
[30] Y Zhou, YS Li, FG Pan, ZS Liu, Z Wang, Identification of tetrodotoxin antigens and a
monoclonal antibody, Food Chem., 112(2009) 582-6.
[31] MP Kreuzer, M Pravda, CK O'Sullivan, GG Guilbault, Novel electrochemical immunosensors
for seafood toxin analysis, Toxicon, 40(2002) 1267-74.
[32] K Campbell, P Barnes, SA Haughey, C Higgins, K Kawatsu, V Vasconcelos, et al.,
Development and single laboratory validation of an optical biosensor assay for tetrodotoxin
detection as a tool to combat emerging risks in European seafood, Anal. Bioanal. Chem.,
405(2013) 7753-63.
[33] AD Taylor, J Ladd, S Etheridge, J Deeds, S Hall, SY Jiang, Quantitative detection of
tetrodotoxin (TTX) by a surface plasmon resonance (SPR) sensor, Sens. Actuat. B-Chem.,
130(2008) 120-8.
[34] AD Taylor, H Vaisocherova, J Deeds, S DeGrasse, S Jiang, Tetrodotoxin Detection by a
Surface Plasmon Resonance Sensor in Pufferfish Matrices and Urine, Journal of Sensors,
(2011).
[35] H Vaisocherova, AD Taylor, S Jiang, K Hegnerova, M Vala, J Homola, et al., Surface Plasmon
Resonance Biosensor for Determination of Tetrodotoxin: Prevalidation Study, J. AOAC Int.,
94(2011) 596-604.
[36] BJ Yakes, J Deeds, K White, SL DeGrasse, Evaluation of Surface Plasmon Resonance
Biosensors for Detection of Tetrodotoxin in Food Matrices and Comparison to Analytical
Methods, J. Agric. Food Chem., 59(2011) 839-46.
[37] BJ Yakes, KM Kanyuck, SL DeGrasse, First Report of a Direct Surface Flasmon Resonance
Immunosensor for a Small Molecule Seafood Toxin, Anal. Chem., 86(2014) 9251-5.
23
[38] SE McNamee, CT Elliott, B Greer, M Lochhead, K Campbell, Development of a Planar
Waveguide Microarray for the Monitoring and Early Detection of Five Harmful Algal Toxins in
Water and Cultures, Environ. Sci. Technol., 48(2014) 13340-9.
[39] HA Bates, R Kostriken, H Rapoport, Chemical assay for saxitoxin - improvements and
modifications, J. Agric. Food Chem., 26(1978) 252-4.
[40] K Campbell, SA Haughey, H van den Top, H van Egmond, N Vilarino, LM Botana, et al.,
Single Laboratory Validation of a Surface Plasmon Resonance Biosensor Screening method for
Paralytic Shellfish Poisoning Toxins, Anal. Chem., 82(2010) 2977-88.
[41] K Campbell, LD Stewart, GJ Doucette, TL Fodey, SA Haughey, N Vilarino, et al., Assessment
of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical
biosensor technology, Anal. Chem., 79(2007) 5906-14.
[42] RI Huot, DL Armstrong, TC Chanh, Protection against nerve toxicity by monoclonal-
antibodies to the sodium-channel blocker, J. Clin. Invest., 83(1989) 1821-6.
[43] HM Johnson, JE Campbell, R Angelotti, KH Lewis, PA Frey, Haptenic properties of paralytic
shellifsh poison conjugated to proteins by formaldehyde treatment, Proc. Soc. Exp. Biol. Med. ,
117(1964) 425-&.
[44] J Tao, WJ Wei, L Nan, LH Lei, HC Hui, GX Fen, et al., Development of competitive indirect
ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the
tissues of wild puffer fish in the waters of south-east China, Food Addit. Contam. Part A
27(2010) 1589-97.
[45] TF McGrath, L McClintock, JS Dunn, GM Husar, MJ Lochhead, RW Sarver, et al.,
Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in
raw milk, Anal. Bioanal. Chem., 407(2015) 4459-72.
[46] BJ Yakes, J Deeds, K White, SL DeGrasse, Evaluation of Surface Plasmon Resonance
Biosensors for Detection of Tetrodotoxin in Food Matrices and Comparison to Analytical
Methods, J. Agric. Food Chem., 59(2011) 839-46.
[47] P Katikou, D Georgantelis, N Sinouris, A Petsi, T Fotaras, First report on toxicity assessment
of the Lessepsian migrant pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European
waters (Aegean Sea, Greece), Toxicon, 54(2009) 50-5.
[48] P Rodriguez, A Alfonso, P Otero, P Katikou, D Georgantelis, LM Botana, Liquid
chromatography-mass spectrometry method to detect Tetrodotoxin and Its analogues in the
puffer fish Lagocephalus sceleratus (Gmelin, 1789) from European waters, Food Chem.,
132(2012) 1103-11.
[49] JP Meneely, K Campbell, C Greef, MJ Lochhead, CT Elliott, Development and validation of
an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic
shellfish toxins in marine algae, Biosens. Bioelectron., 41(2013) 691-7.
[50] AOAC, Guidelines for single laboratory validation of chemical methods for dietary
supplements and botanicals. http://www.aoac.org/Official_Methods/slv_guidelines.pdf.
Accessed by October 2016., 2012.
[51] CC Yang, SC Liao, JF Deng, Tetrodotoxin poisoning in Taiwan: An analysis of poison center
data, Vet. Hum. Toxicol., 38(1996) 282-6.
[52] J Jang, M Yotsu-Yamashita, Distribution of tetrodotoxin, saxitoxin, and their analogs
among tissues of the puffer fish Fugu pardalis, Toxicon, 48(2006) 980-7.
24
Biographies for publication
Laia Reverté
Laia Reverté is a PhD student at IRTA in collaboration with the department of Biotechnology of
Universitat Autonoma de Barcelona. Her thesis focuses on the development of cytotoxicity
assays, immunoassays and biosensors for the detection of emerging marine toxins in seafood
samples. She obtained her degree in Biotechnology from the Universitat Rovira i Virgili,
Tarragona and acquired a master degree in Immunology in 2010 from the Universitat Autonoma
de Barcelona.
Mònica Campàs
Dr. Mònica Campàs is researcher at IRTA, where she leads a research line on biosensors for the
detection of toxins, toxic phytoplankton and other analytes. Mònica obtained a BSc in Chemistry
in 1996 and a PhD in Chemical Engineering in 2002 from the Universitat Rovira i Virgili. Her
research interests lie in the development of colorimetric assays and electrochemical biosensors
using new biorecognition elements, immobilisation techniques and supports, transduction
schemes and signal amplification strategies. Presently, her work focuses on the development
and applicability of bioanalytical systems for the detection of emerging marine toxins (e.g.
tetrodotoxins), toxic microalgae (e.g. Karlodinium, Ostreopsis and Gambierdiscus) and virus (e.g.
oyster herpes virus). She has published 52 articles and 10 book chapters. She has been the
principal investigator of 10 projects. She has directed 1 PhD thesis, 3 more being in progress.
Betsy Jean Yakes

Betsy Jean Yakes received her PhD degree from the Department of Chemistry at Iowa State
University in 2007. She is currently a research chemist in the Office of Regulatory Science at the
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition. Her research
interests include developing accurate biosensor detection for toxins and pathogens as well as
spectroscopy methods for improved authentication and adulteration evaluation for foods,
dietary supplements, and cosmetics.
Jonathan R. Deeds
Jonathan R. Deeds received a Master’s of Science degree in Environmental Toxicology from the
University of Louisiana at Lafayette in 1997 and his PhD in Marine Estuarine and Environmental
Science from the University of Maryland in 2003. He is currently a research biologist in the Office
of Regulatory Science at the U.S. Food and Drug Administration, Center for Food Safety and
Applied Nutrition. He serves as a subject matter expert in seafood safety and authenticity with
specialties in marine biotoxin detection and the species identification of seafood.
Panagiota Katikou
Panagiota Katikou is a veterinarian employed by the Greek Ministry of Rural Development and
Food and has been the Head of the National Reference Laboratory of Marine Biotoxins since
2006. Her main research interests are the analysis and risk assessment of marine biotoxins, with
a particular interest in emerging toxins and especially tetrodotoxins.
Kentaro Kawatsu
Dr Kentaro Kawatsu is the Manager of the Bacteriology Section at the Division of Microbiology
at Osaka Institute of Public Health in Japan. Kentaro obtained a BVSc from Faculty of Agriculture,
Tottori University, Japan and PhD in Science and Technology from Nagasaki University, Japan.
His research focuses on the development of rapid, simple and sensitive immunoassays for the
detection of marine toxins and bacterial pathogens within the entire food supply chain from
“environment to farm to fork” as early warning prevention strategies to ensure food security.
25
Michael Lochhead
Michael Lochhead, Ph.D. is Chief Technology Officer at MBio Diagnostics, Inc., where he is
responsible for overall technical operations, new product R&D, and commercial partnerships.
The company develops portable technologies for rapid testing in fields ranging from
environmental monitoring to clinical diagnostics. Dr. Lochhead earned his Ph.D. at the University
of Wisconsin, Madison.
Christopher T. Elliott
Prof Christopher Elliott is Pro Vice Chancellor for the Faculty of Medicine, Health & Life Sciences,
Professor of Food Safety and founder of the Institute for Global Food Security at Queen’s
University Belfast. He has published more 300 peer review articles, many of them relating to the
detection and control of agriculture, food and environmental related contaminants. One key
research interest is in the development of innovative techniques to provide early warning
systems of toxin threats across complex food supply systems.
Katrina Campbell
Dr Katrina Campbell is a Lecturer in Bioanalytical Systems and heads the Biosensors Strand
within the Centre for ASsured, SafE and Traceable food (ASSET) at Queen’s University Belfast.
Katrina is a member of the Royal Society of Chemistry and the Royal Institute of Biology. Katrina
obtained a BSc (Hons) in Biochemistry; a MMedSc in Ultrastructural Anatomy and Pathology and
PhD in Pharmacy from Queen's University Belfast. Her research focuses on the development of
innovative screening and (bio)analytical methods for the detection, identification and
recognition of known and emerging chemical, toxin and pathogenic threats within the entire
food supply chain from “environment to farm to fork” as early warning prevention strategies to
ensure food security with the publication of more than 80 articles.
26
Figure 1. Schematic representation of the planar waveguide immunosensor for
TTX.
27
Figure 2. Calibration curves in buffer and fortified-puffer fish tissues and extracts
obtained for a) 50 µg mL-1 and b) 100 µg mL-1 TTX-conjugate. Curves normalized
to mAb signal when no TTX is present. Standard deviations for 16 replicates are
represented by the error bars.
120
a)
100
80
% mAb binding
60
40
buffer
fortified-puffer fish tissue
20 fortified-puffer fish extract
0
0,001 0,010 0,100 1,000 10,000
[TTX] g g-1
120
b)
100
80
% mAb binding
60
40
buffer
fortified-puffer fish tissue
20 fortified-puffer fish extract
0
0,001 0,01 0,1 1 10
[TTX] g g-1
28
Table 1. Midpoint of the curve (IC50), LOD (IC20) and dynamic range (IC20−IC80)
values ± SD provided by the planar waveguide biosensor in buffer for both TTX
[TTX conjugate] Midpoint (IC50) µg L-1 LOD (IC20) µg L-1 Dynamic range (IC20-IC80) µg L-1
50 µg mL-1 6.1 ± 0.5 2.5 ± 0.1 2.5 (± 0.1)-20.5 (± 2.2)

100 µg mL-1 5.8 ± 0.3 2.6 ± 0.1 2.6 (± 0.1)-15.2 (± 1.7)
conjugate concentrations and for 16 replicates.
Table 2. TTX concentration ± standard deviation for 16 replicates in fortified-
puffer fish tissue and extracts determined by the planar waveguide biosensor.
Toxin recovery is expressed in percentages (%) and calculated with reference
to the TTX fortification levels of 0.4 and 0.8 µg TTX g-1 puffer fish.
TTX fortification level

[TTX conjugate] Matrix
0.8 µg g-1 0.4 µg g-1
fortified-puffer fish tissue 0.82 ± 0.03 (102.5%) 0.35 ± 0.02 (87.5%)

50 µg mL-1
fortified-puffer fish extract 0.78 ± 0.06 (97.5%) 0.40 ± 0.09 (100.0%)
fortified-puffer fish tissue 0.81 ± 0.03 (101.3%) 0.35 ± 0.05 (87.5%)

100 µg mL-1
fortified-puffer fish extract 0.77 ± 0.06 (96.3%) 0.38 ± 0.01 (95.0%)
29
Table 3. TTX concentration (µg TTX g-1 puffer fish tissue) determined in a) 5 sample
tissues from L. lunaris and b) 7 sample tissues from L. sceleratus by the MBio biosensor
and comparison with other methods [46-48].

a)
µg TTX g-1 puffer fish tissue
MBio biosensor LC-MS/MS RBA ELISA SPR

Sample code
TTX conjugate 50 µg mL -1
TTX conjugate 100 µg mL -1 (TTX) [46] [46] [46] [46]
1 0.27 ± 0.07 0.31 ± 0.04 0.10 0.18 0.04 0.07
2 3.38 ± 0.08 3.23 ± 0.15 2.14 3.52 2.68 2.47
3 13.26 ± 0.64 13.49 ± 0.64 8.76 10.50 14.12 7.09
4 19.02 ± 5.19 17.50 ± 5.03 9.61 13.24 12.48 10.74
5 0.24 ± 0.03 0.24 ± 0.07 0.10 0.16 0.02 0.07
b)
µg TTX g-1 puffer fish tissue
MBio biosensor LC-MS/MS [48]
Sam MB
11- 11-
TTX TTX A 4- 4,9- 5- 11- norTT norTT 5,6,11- 5.6.11-
ple conjug conjug
[47] TTX epiT anhydro deoxyT deoxyT X- X- trideoxy trideoxy
ate 50 ate 100
TX TTX TX TX 6(R)- 6(S)- TTX (1) TTX (2)
code µg mL-1 µg mL-1
ol ol
2.71 ± 2.73 ± 1.6 <LO <LO

6 <LOD <LOD <LOQ <LOQ <LOQ 1.20 2.30
0.79 0.54 9 Q D
41.07 ± 43.72 ± 56. 12.0

7.A 2.96 1.97 3.67 17.45 3.53 16.50 109.75 220.75
8.49 4.88 78 5
10.18 ± 9.08 ± 16.

7.B 2.39 0.47 0.52 0.76 2.55 0.36 4.38 94.00 192.25
1.64 0.99 12
2.99 ± 3.07 ± 2.4 <LO

7.C 0.65 <LOQ <LOQ 0.56 1.08 0.83 23.55 56.50
0.49 0.33 2 D
6.64 ± 6.73 ± 10.

8.A 4.20 0.46 0.63 1.62 5.50 3.16 8.70 0.71 4.07
0.72 0.84 84
1.34 ± 1.34 ± <1. <LO <LO <LOD

8.B <LOD <LOQ <LOQ <LOQ <LOQ <LOQ
0.45 0.24 10 Q D
1.41 ± 1.41 ± <1. <LO

8.C n.d. <LOD <LOQ 0.54 0.47 0.48 <LOQ <LOQ
0.23 0.07 10 Q
LOQ: 0.32 µg g-1; LOD: 0.08 µg g-1; n.d.: not detected
30
Acute poisoning due to ingestion of Datura stramonium
Romanian Journal of Anaesthesia and Intensive Care 2017 Vol 24 No 1, 65-68
CASE REPORT DOI: http://dx.doi.org/10.21454/rjaic.7518.241.szb
Acute poisoning due to ingestion of Datura stramonium

– a case report
Sebastian Daniel Trancă1, Robert Szabo2, Mihaela Cociş1
1
Department of Anaesthesiology and Intensive Care 2, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
2
Emergency County Hospital Cluj, Romania
Abstract
Datura stramonium (DS) is a widespread annual plant, containing atropine, hyoscyamine, and scopolamine,
which can produce poisoning with a severe anticholinergic syndrome. Teenagers ingest the roots, seeds or
the entire plant to obtain its hallucinogenic and euphoric effects. We presented the case of a 22 year old
male who was admitted to the Emergency Room in a coma after consuming Datura stramonium, 2 hours
earlier. The patient presented with fever, tachycardia with right bundle branch block, and urinary retention.
Rapid sequence induction and intubation was performed immediately, with sedation and assisted-control
mechanical ventilation, after being transferred to the Intensive Care Unit. The patient received activated
charcoal, in repeated doses, external and internal cooling was applied, and an infusion of neostigmine was
started. The biological assessment revealed rhabdomyolysis and prevention of renal failure was initiated.
After a proper neurological evaluation, 36 hours after using Datura stramonium, the patient was extubated
and transferred to the Psychiatric ward for further assessment and care.
Keywords: Datura stramonium, anticholinergic syndrome, toxic delirium, coma
Received: 28 November 2016 / Accepted: 12 March 2017 Rom J Anaesth Int Care 2017; 24: 65-68
vary between leaves, plants and from one season to

another. The highest levels of toxins are found in the
seeds approximating 0.1 mg of atropine per seed or
36 mg / 50100 seeds (Fig. 1).
Introduction
Datura stramonium (DS), also known as Jimson
Weed, Locoweed, Angel’s Trumpet, Thorn Apple,
Devil’s Trumpet is a hallucinogenic plant found in the
urban and rural areas, along roadsides, in cornfields
and pastures [1, 2-5]. The range of toxicity of Datura
stramonium is highly variable and unpredictable. It
occurs when ingested, smoked and absorbed topically,
in particular through mucous membranes. Toxicity may
Address for correspondence: dr. Sebastian Daniel Trancă

“Iuliu Hatieganu” Univ. of Medicine
and Pharmacy Cluj-Napoca
Anaesthesia and Intensive Care Dpt II
Str. Clinicilor 3-5
400006 Cluj-Napoca, Romania
E-mail: tranca.sebastian@umfcluj.ro Fig. 1. Datura stramonium
66 Trancă et al.
The classic anticholinergic poisoning occurs by assisted mode being considered unsafe at that moment.
consumption of the tropane alkaloid-containing plant External cooling blankets were used together with
[2]. Tropane alkaloids include hyoscyamine contained antipyretics (metamizole and paracetamol) to lower
in leaves, roots, seeds; hyoscine, atropine (dl-hyo- body temperature. Neostigmine, 0.5 mg, was also ad-
scyamine) and scopolamine (l-hyoscine) found in roots. ministered in a slow perfusion. The dose of activated
They act as competitive antagonists to peripheral and charcoal was repeated 12 hours after the ingestion.
central muscarinic acetylcholine receptors leading to Re-evaluation of blood tests revealed rising levels
a general paralysis of the parasympathetic innervated of creatine-kinase (1140 U/L). Because of technical
organs. Acute psychosis or delirium can occur due to problems, serum myoglobin levels and myoglobinuria
its effect on the central nervous system as tertiary were not assessed. Prevention of renal failure induced
amines can inhibit CNS receptors [6]. Coma and sei- by acute rhabdomyolysis was performed with intra-
zures are rare findings but raise concerns of extreme venous infusion of sodium bicarbonate, diuretics and
gravity [7]. Teenagers with intentional ingestion of the an increased volume of crystalloids.
plant represent most cases of Datura stramonium 24 hours after the admission, propofol and midazo-
poisoning reported in the literature, as they seek for its lam infusions were stopped to evaluate the neurological
hallucinogenic and euphoric effects. status. The patient presented severe agitation, and
therefore sedation was continued. After 36 hours he
became calm and was extubated without any signs of
Case report respiratory or circulatory impairment.
He was transferred to the internal medicine unit
A 22 year old male patient was brought to the
and later to the psychiatric ward.
Emergency Department by the ambulance services
with a history of deliberate ingestion of Datura
stramonium 2 hours before presentation. Discussion
At arrival in the Emergency Room the patient
presented with Glasgow Coma Scale 7 points (eye Datura stramonium (DS), known as Jimson weed
opening response: 1; motor response: 5, verbal is a wild-growing herb. The entire plant especially the
response: 1), mydriatic and areactive pupils and without foliage and seeds, is toxic due to its content of tropane
clinical signs of meningeal irritation. He was agitated, alkaloids. The contained atropine, L-hyoscyamine and
with a body temperature of 38.0°C and dry skin and L-scopolamine cause anticholinergic syndrome, which
mucosa, with tachycardia (heart rate – 111 beats per results from the inhibition of central and peripheral
minute), and a blood pressure of 135/61 mmHg. The muscarinic neurotransmission [2, 6, 8].
electrocardiogram revealed sinus rhythm with right Teenagers, especially those with a history of
bundle branch block, without any changes of the ST polysubstance abuse, voluntary ingest the plant seeking
segment or T wave. Ultrasound showed severe urinary its hallucinogenic and euphoric effects, and represent
retention. Rapid sequence intubation using propofol 120 most of the cases reported in the literature. Intoxication
mg and succinylcholine 70 mg was performed to with Datura stramonium has also been described in
prevent aspiration and was followed by mechanical children [2, 3]. Our case presented an intentional
ventilation in assisted-controlled mode, under conti- ingestion of DS seeds for its hallucinogenic effects,
nuous sedation with midazolam. After the gastric lavage and similar to those reported by Diker et al. [9] was
that revealed Datura seeds, activated charcoal admitted with coma, the central element that carries a
(Carbomix 75 g) was administered. A urinary catheter bad prognosis as is related to a higher morbidity [10].
was inserted, and the toxicology screening was Typical symptoms of DS poisoning are represented
performed but did not show the presence of other by dry skin and mucosa, flushing, mydriasis, sinus
substances. Blood tests showed rhabdomyolysis with tachycardia, hyperpyrexia, decreased bowel activity,
creatine-kinase levels of 518 U/L. The blood pH was urinary retention, and neurological disorders with ataxia,
normal with a lactate level of 1.5 mmol/L, and normal impaired short-term memory, disorientation, confusion,
values of serum electrolytes (sodium: 138 mmol/L, hallucinations (visual and auditory), psychosis, agitated
potassium 3.9 mmol/L) were recorded. Fluid balance delirium, seizures, and coma. These symptoms re-
was achieved using crystalloid solution and the CT scan semble atropine intoxication [6]. Respiratory failure
excluded any cerebral lesions. and cardiovascular collapse were reported in severe
Transferred to the ICU, the patient was agitated, cases [2, 3]. In rare cases, rhabdomyolysis and fulmi-
and the sedation was achieved with propofol and nant hepatitis have also been described [11]. Datura
midazolam infusion. Because of the heavy sedation stramonium toxicity usually occurs within 60 min after
he was ventilated in an assisted-controlled mode, an ingestion, and the clinical symptoms may persist up to
Acute poisoning due to ingestion of Datura stramonium 67
24 to 48 h, due to delayed gastric emptying. This delay proper management applied. Fatalities due to Datura
caused by the anticholinergic effect results in a species poisoning are rare, but adverse effects are
prolonged duration of action. Children have a unique common [6].
susceptibility to atropine toxicity, as smaller amounts We reported this case as the first published case of
may cause profound central nervous system distur- severe Datura stramonium voluntary intoxication in
bances [12, 13]. Romania, with coma and life-threatening manifestations
Management is mainly supportive. It consists of and we reviewed the leading clues for its diagnostic
gastric decontamination with activated charcoal admi- and treatment.
nistered by mouth or tube, sedation with benzodiaze- In conclusion, every acute anticholinergic syn-
pines to control agitation, and the hyperpyrexia control drome presenting with coma/seizures, in young people
(fluids administration and internal and external cooling lacking other objective findings, can suggest poisoning
methods) [6]. Gastric emptying and decontamination with Datura stramonium, and early decontamination/
are necessary managing tools if they are initiated early. management should be initiated.
In our case, the ingestion was done 2 hours before
admission, and the early gastric emptying and decon- Conflict of interest
tamination, with activated charcoal through a gastric
Nothing to declare
tube proved to be safe and efficient, as long as the
airway was already secured, and the maneuver was
applied before the toxins were totally absorbed.
Decreased gastrointestinal motility may have raised References
the efficacy of the activated charcoal.
1. Bouziri A, Hamdi A, Borgi A, Hadj SB, Fitouri Z, Menif K, et al.
Tachycardia is usually responsive to crystalloids Datura stramonium L. poisoning in a geophagous child: a case
[11]. report. Int J Emerg Med 2011; 4: 31. DOI: 10.1186/1865-
The antidote for anticholinergic toxicity is physo- 1380-4-31
stigmine. It led to controversies, despite the recent 2. Kurzbaum A, Simsolo C, Kvasha L, Blum A. Toxic delirium due
reports of its safe use [14]. Relative contraindication to Datura stramonium. Isr Med Assoc J 2001; 3: 538-539
of physostigmine is cardiac conduction defects (AV 3. Arouko H, Matray M-D, Bragança C, Mpaka JP, Chinello L,
Castaing F, et al. Voluntary poisoning by ingestion of Datura
block) [15]. The patient presented with right bundle
stramonium. Another cause of hospitalization in youth seeking
branch block, a cardiac conduction defect that has been
strong sensations. Ann Med Interne (Paris) 2003; 154: S46-50
proved to carry a reduced cardiovascular risk in healthy 4. Spina SP, Taddei A, Forrester MB. Teenagers with Jimson weed
people [16]. The clinical background of our patient (Datura stramonium) poisoning. CJEM 2007; 9: 467-469. DOI:
required its administration, in a reduced dose (0.5 mg), 10.1017/S1481803500015530
because of the severity of the neurological symptoms. 5. Gaire BP, Subedi L. A review on the pharmacological and
Physostigmine is recommended in severe cases of toxicological aspects of Datura stramonium L. J Integr Med
agitation or psychosis, intractable seizures/coma or 2013; 11: 73-79. DOI: 10.3736/jintegrmed2013016
6. Krenzelok EP. Aspects of Datura poisoning and treatment. Clin
tachycardia/dysrhythmias with hemodynamic com-
Toxicol (Phila) 2010; 48: 104-110. DOI: 10.3109/
promise [2, 6]. In our patient the administration of 15563651003630672
physostigmine resulted in no harm and ameliorated the 7. Vanderhoff BT, Mosser KH. Jimson weed toxicity: Management
neurological syndrome. of anticholinergic plant ingestion. Am Fam Physician 1992;
Sedation pauses and serial neurological monitoring 46: 526-530
allowed for an early extubation and prevention of 8. Freye E. Toxicity of Datura Stramonium. In: Freye E, Levy JV.
complications related to intubation and mechanical Pharmacology and Abuse of Cocaine, Amphetamines, Ecstasy
and Related Designer Drugs: A comprehensive review on their
ventilation.
mode of action, treatment of abuse and intoxication. Dordrecht:
Another particularity of the case was the rhabdo- Springer Netherlands; 2010. p. 217-218. DOI: 10.1007/978-
myolysis. Acute kidney injury is a frequent finding (10- 90-481-2448-0
50%), as a result of the direct toxicity of the alkaloids, 9. Diker D, Markovitz D, Rothman M, Sendovski U. Coma as a
and secondary to myoglobinuria that results from presenting sign of Datura stramonium seed tea poisoning. Eur J
myocyte destruction, due to the severe agitation [17]. Intern Med 2007; 18: 336-338. DOI: 10.1016/j.ejim.2006.
We prevented this complication by adequate fluid re- 09.035
10. Mowry JB, Spyker DA, Brooks DE, McMillan N, Schauben JL.
placement, urine alkalinization, and diuretics.
2014 Annual Report of the American Association of Poison
Most of the cases described in the literature had a
Control Centers’ National Poison Data System (NPDS): 32 nd
good prognosis after supportive treatment. Annual report. Clin Toxicol (Phila) 2015; 53: 962-1147. DOI:
Despite its severity the favorable evolution of this 10.3109/15563650.2015.1102927
patient was probably due to the quick diagnosis of the 11. Ertekin V, Selimođlu MA, Altinkaynak S. A combination of
major anticholinergic syndrome and to the early and unusual presentations of Datura stramonium intoxication in a
68 Trancă et al.
child: Rhabdomyolysis and fulminant hepatitis. J Emerg Med 15. Kearney TE. Chapter 223: Physostigmine and Neostigmine. In:
2005; 28: 227-228. DOI: 10.1016/j.jemermed.2004.11.006 Olson KR, editor. Poisoning & Drug overdose. 6 th ed. San
12. Al-Shaikh AM, Sablay ZM. Hallucinogenic plant poisoning in Francisco: McGraw-Hill; 2012. p. xxx
children. Saudi Med J 2005; 26: 118-121 16. Fahy GJ, Pinski SL, Miller DP, McCabe N, Pye C, Walsh MJ, et
13. Rodgers GC Jr, Von Kanel RL. Conservative treatment of al. Natural history of isolated bundle branch block. Am J Cardiol
jimsonweed ingestion. Vet Hum Toxicol 1993; 35: 32-33 1996; 77: 1185-1190. DOI: 10.1016/S0002-9149(96)00160-9
14. Frascogna N. Physostigmine: is there a role for this antidote in 17. Holt SG, Moore KP. Pathogenesis and treatment of renal
pediatric poisonings? Curr Opin Pediatr 2007; 19: 201-205. dysfunction in rhabdomyolysis. Intensive Care Med 2001; 27:
DOI: 10.1097/MOP.0b013e32802c7be1 803-811
toxins
Article
Phylogeny and Toxin Profile of Freshwater Pufferfish
(Genus Pao) Collected from 2 Different Regions
in Cambodia
Hongchen Zhu 1 , Akinori Yamada 1 , Yui Goto 2 , Linan Horn 3 , Laymithuna Ngy 3 ,
Minoru Wada 1 , Hiroyuki Doi 4 , Jong Soo Lee 5 , Tomohiro Takatani 1 and Osamu Arakawa 1, *
1 Graduate School of Fisheries and Environmental Sciences, Nagasaki University, 1-14, Bunkyo-machi,
Nagasaki 852-8521, Japan; zhc957286316@hotmail.com (H.Z.); ayamada@nagasaki-u.ac.jp (A.Y.);
miwada@nagasaki-u.ac.jp (M.W.); taka@nagasaki-u.ac.jp (T.T.)
2 Faculty of Fisheries, Nagasaki University. 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan;
gtoui555@gmail.com
3 Faculty of Agronomy, University of Kratie, National Road No. 7, Orussey District, Kratie City,
Kratie Province, Cambodia; hornlinan189@gmail.com (L.H.); ngy_mithuna@yahoo.com (L.N.)
4 Nifrel, Osaka Aquarium Kaiyukan. 2-1, Senribanpakukoen, Suita, Osaka 565-0826, Japan; doi@kaiyukan.com
5 College of Marine Science, Gyeongsang National University, 2, Tongyeonghaean-ro, Tongyeong,
Kyungnam 53064, Korea; leejs@gnu.ac.kr
* Correspondence: arakawa@nagasaki-u.ac.jp; Tel.: +81-95-819-2844

Received: 15 October 2020; Accepted: 28 October 2020; Published: 30 October 2020
Abstract: The species classification of Cambodian freshwater pufferfish is incomplete and confusing,
and scientific information on their toxicity and toxin profile is limited. In the present study,
to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to
contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic
and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie
(designated PNH and KTI, respectively). Phylogenetic analysis of partial sequences of three
mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for
each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and
Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2)
gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile
revealed that both Pao sp. A and Pao sp. B possess saxitoxins (STXs), comprising STX as the main
component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding
the regulatory limit for STXs set by the Codex Committee, whereas in Pao sp. B, only the skin
contained high toxin concentrations. The difference in the STX accumulation ability between the two
species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in
freshwater pufferfish.
Keywords: Cambodia; pufferfish; Pao; phylogenetic analysis; tributyltin-binding protein type 2 (TBT-bp2);
saxitoxin (STX)
Key Contribution: We revealed a gene-based phylogeny linked to the toxicity profile of the Cambodian
freshwater pufferfish, and suggest for the first time that tributyltin-binding protein type 2 is involved
in saxitoxin accumulation in freshwater pufferfish.
1. Introduction
Pufferfish of the family Tetraodontidae contain tetrodotoxin (TTX) and/or saxitoxins (STXs), but the
toxin ratio differs depending on the genus or species. Marine pufferfish of the genus Takifugu inhabiting
Toxins 2020, 12, 689; doi:10.3390/toxins12110689 www.mdpi.com/journal/toxins

Toxins 2020, 12, 689 2 of 15
the coastal waters of Japan and Dichtomyctere (formerly known as Tetraodon) living in the brackish
waters of Thailand have TTX as their main toxin component [1–3], whereas freshwater pufferfish of the
genera Pao and Leiodon (both formerly known as Tetraodon), which inhabit Southeast Asian countries,
generally have only STXs [4–7]. On the other hand, Sphoeroides pufferfish from Florida, and Arothron
and Canthigaster pufferfish from the Philippines, Japanese coastal waters, or the Caribbean Sea have
both TTX and STXs [8–12]. The toxin ratio varies depending on the species, but STXs are dominant in
many cases. Marine pufferfish of the genus Takifugu may have small amounts of STXs in addition to
TTX [13–15].
Many studies examining the toxification mechanism of pufferfish with TTX revealed that pufferfish
do not biosynthesize TTX, but accumulate it through the food chain that starts with TTX-producing
bacteria [1]. The accumulated TTX is gradually eliminated if supply via toxic food is not continued [16].
We recently conducted various in vivo TTX administration experiments using nontoxic pufferfish
artificially raised with nontoxic diets, and found that the internal TTX kinetics in pufferfish are
unique: intestine → liver → skin/ovary, and change as the pufferfish grows and matures [17–22].
The pufferfish STX- and TTX-binding protein (PSTBP) isolated from the plasma of Takifugu pardalis by
Yotsu-Yamashita et al. [23] is thought to be involved in the internal TTX kinetics, i.e., the absorption,
transportation, and accumulation of TTX inside the pufferfish body [24]. PSTBP homologous proteins
are widely distributed in TTX-bearing toxic pufferfish of the genera Takifugu and Arothron, but have
not been detected in common fish or in nontoxic species of pufferfish [25–27].
The accumulation mechanism of STXs in pufferfish, however, remains unclear, but is presumed to
be exogenous via the food chain that starts with STX-producing dinoflagellates in marine environments
and STX-producing cyanobacteria in freshwater environments [6,9,11,28]. In a recent in vivo TTX/STX
administration experiment using nontoxic cultured pufferfish, Gao et al. [29] found that T. pardalis,
which naturally contains TTX, selectively accumulates TTX, and Pao suvattii, which naturally
contains STXs, selectively accumulates STXs. In other words, the ratio of TTX/STX in pufferfish
appears to depend more strongly on the inherent TTX/STX selectivity of the pufferfish than the
prevalence of TTX/STX in prey organisms, but the molecular mechanisms underlying the toxin
selectivity remain to be elucidated. Our ongoing genome and transcriptome analyses indicate that the
three freshwater pufferfish species, Pao abei, P. suvattii, and Leiodon cutcutia, do not have the PSTBP
gene (unpublished data), suggesting that PSTBP is not involved in STX accumulation or selectivity of
the genus Pao.
Several species of freshwater pufferfish inhabit the rivers and lakes of Cambodia. These pufferfish
are considered delicious and there have been many cases of poisoning due to the ingestion of pufferfish
caught with other generally edible freshwater fish. According to the Ministry of Health in Cambodia,
at least seven poisoning incidents occurred due to freshwater pufferfish consumption from 2017
to 2019, with more than 40 people poisoned and 5 deaths. The Codex Committee on Fish and
Fishery Products has set the regulatory limit for STXs as 0.8 mg STX·diHCl eq/kg edible tissue [30],
but there is no strict food safety standard for the edible use of freshwater pufferfish in Cambodia.
This may be partly due to the fact that the species classification of Cambodian indigenous pufferfish
is incomplete and quite confusing, and the limited scientific information on the toxicity and toxin
profile. In the present study, to accumulate genetic information on the taxonomy of freshwater
pufferfish and the TTX/STX distribution profiles in pufferfish of the family Tetraodontidae, and to
contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and
toxin analyses using freshwater pufferfish specimens collected from two different regions in Cambodia.
Moreover, to explore candidate molecules other than PSTBP involved in the absorption, transportation,
and accumulation of STXs in freshwater pufferfish, a partial sequence of a putative origin of the
molecular evolution of PSTBP [31,32], tributyltin-binding protein type 2 (TBT-bp2), and four commonly
used genes (cytochrome b [CYTB], 16S rRNA, cytochrome c oxidase subunit I [COI], and rhodopsin)
were used as targets in the phylogenetic analysis.
Toxins 2020,
Toxins 12, x689
2020, 12, FOR PEER REVIEW 33of
of15
15
2. Results
Toxins 2020, 12, x FOR PEER REVIEW 3 of 15
2. Results
2.1. 2. Results
2.1. Phylogeny
Phylogeny
2.1. Phylogeny
A
A total
total of
of1616freshwater
freshwater pufferfish
pufferfish were
were sampled
sampled from from small
small lakes
lakes near
near Phnom
Phnom Penh Penh (PNH)
(PNH) and and
Kratie
Kratie (KTI)
(KTI) (Figures
A total
(Figures 11 and
and 2,
of 16 freshwater and
and Table
2, pufferfish
Table A1).
were
A1). Partial
sampled
Partial sequences
from small lakes
sequences of
of the
the three
near Phnom
three mitochondrial
Penh (PNH) and
mitochondrial genes,
genes,
CYTB,
CYTB, 16S
Kratie rRNA,
16S (KTI)
rRNA, and
and COI,
(Figures 1 and
COI, were
2, and
were determined
Table A1). for
determined for each
Partial
each sample
sequences
sample of and
andthephylogenetically
three mitochondrial
phylogenetically analyzed
genes, for
analyzed for
taxonomic
CYTB, identification.
16S rRNA, and Phylogenetic
COI, were analysis
determined of 870-bp
for each CYTB
sample sequences
and
taxonomic identification. Phylogenetic analysis of 870-bp CYTB sequences showed that the PNH showed
phylogenetically that the
analyzed PNHfor and
KTI taxonomic eachidentification. aPhylogenetic analysis of 870-bp CYTB sequences showed that the PNH and
andsequences
KTI sequences formed
each formed tight cluster
a tight together with Pao
cluster together cochinchinensis
with (AB741980.1)
Pao cochinchinensis or Pao
(AB741980.1) baileyi
or
KTI sequences
(AB741978.1) each3).
(Figure formed
Similara tight clusterwere
results together with Pao
obtained cochinchinensis
from analyses (AB741980.1)
of concatenated or Paodatasets
baileyi of
Pao baileyi (AB741978.1) (Figure 3). Similar results were obtained from analyses of concatenated
(AB741978.1)
CYTB+16S (Figure 3).and
Similar results were
(621obtained from analyses
A1 and of concatenated datasets of
datasets of rRNA
CYTB+16SCYTB+16S (555 rRNA
rRNA (555
bp)
bp) (555
CYTB+COI
bp) and CYTB+COI
and CYTB+COI
bp) (Figures
(621 bp)
(621 bp) (Figures A1(Figures
A2).
and A2).A1
Considering
and the genetic
A2). Considering
Considering the genetic the
variation
genetic of P. suvattii
variation in Figure
P. suvattii
ofsuvattii 3, each
in Figure cluster
3, each seems
cluster seems to represent
to represent a single
a single species,
species, though
though the
variation of P. in Figure 3, each cluster seems to represent a single species, though the the
intraspecific
intraspecific variation
variation in the
in theingenus genus Pao
Pao might might be unexpectedly large
Pao(see (see
leiurus Pao leiurus and
P. cochinchinensis P.
intraspecific variation the genus Pao be unexpectedly
might large (see
be unexpectedly large Paoandleiurus and P.
cochinchinensis
in Figure in Figures
3, Figure
cochinchinensis A1,
in and
Figures3, Figure
A1,
3, A1,and
andA2).
A2).A2).Hereafter,
Hereafter,
Hereafter,we we thus
wethus
thus refer
refer
refer toto
to the
thethe samples
samples
samples from
from
from PNH
PNH PNH and
and and
KTI KTI
KTI as
as Pao sp.
as Pao
Pao sp. A and
sp. A
A and Pao
and
Pao sp.
sp.Pao B,
B,sp. respectively.
B, respectively.
respectively.
Figure 1. Map showing the pufferfish sampling locations in Cambodia.

Figure 2. Typical pufferfish collected from Phnom Penh (A, PNH-1) and Kratie (B, KTI-2).
Figure 2. Typical pufferfish collected from Phnom Penh (A, PNH-1) and Kratie (B, KTI-2).
A 492 to 778 bp region of the nuclear rhodopsin gene was directly sequenced from the polymerase
chain reaction (PCR) product of each sample. As no double peaks were observed in the Sanger sequence
electropherograms, the sequenced
Figure 2. Typical regionsfrom
pufferfish collected werePhnom
considered toPNH-1)
Penh (A, be homogeneous inKTI-2).
and Kratie (B, all the samples.
Furthermore, there was no sequence variation between any pair of samples. Compared with sequences
of other Pao species in the database, all the mutations found were synonymous, and the amino acid
sequence was identical among the Pao species examined.
Toxins 2020, 12, 689 4 of 15
KTI-1, 2, 4, 5
Pao sp. B
KTI-3
97/96 Pao b aileyi (AB741978.1)
69/58
Pao ab ei (AB741977.1)
90/89
Pao ab ei (LC586270.1)
98/99
Pao leiurus (KF667490.1)
Pao leiurus (GU057266.1)
Pao leiurus (AB741985.1)
100/99
Pao camb odgiensis (JQ681921 1)
83/94 Pao suvattii (AB741991.1)
100/100 93/98
Pao suvattii (LC586271.1)
Pao suvattii (JQ681934.1)
77/65 Pao cochinchinensis (JQ681922.1)
Pao palembangensis (JQ681932.1)
100/100 Pao turgidus (AB741992.1)
Pao leiurus (JQ681927.1)
54/55 61/93 Pao cochinchinensis (AP011925.1)
PNH-1, 2, 4, 5, 6, 8, 9, 10, 11
100/100 PNH-7 Pao sp. A
PNH-3
86/67 Pao cochinchinensis (AB741980.1)
Pao palembangensis (AP011920.1)
100/100 Arothron manilensis (AP011929.1)
Arothron hispidus (AP011930.1)
0.200 0.150 0.100 0.050 0.000
Figure 3. Phylogenetic tree of Pao species constructed by Bayesian inference (BI) and maximum
Figure 3. Phylogenetic tree of Pao species constructed by Bayesian inference (BI) and maximum
likelihood (ML) based on the CYTB gene. The BI tree is shown. Node numbers are Bayesian posterior
likelihood (ML) based on the CYTB gene. The BI tree is shown. Node numbers are Bayesian posterior
probabilities (left) and ML bootstrap values (right).
probabilities (left) and ML bootstrap values (right).
A 298 to 536 bp region of the nuclear TBT-bp2 gene was determined for all the samples except
A 492 to 778 bp region of the nuclear rhodopsin gene was directly sequenced from the
KTI-3polymerase
by direct sequencing
chain reactionof(PCR)
the PCR products.
product We observed
of each sample. doublepeaks
As no double peakswereat several
observednucleotide
in the
positions in the Sanger sequence electropherograms of almost all the samples,
Sanger sequence electropherograms, the sequenced regions were considered to be homogeneous indicating that
in the
TBT-bp2
all gene had multiple
the samples. alleles (Table
Furthermore, there A2).
was Allele types were
no sequence apparently
variation betweensimilar between
any pair samples of
of samples.
Pao sp. A and Pao
Compared sp. sequences
with B. Compared withPao
of other thespecies
allele types
in thefound in Pao
database, allsp. and Pao sp.
theAmutations B, there
found were was
synonymous, and the amino acid sequence was identical among the Pao species examined.
no sample in which the allele types could be represented as a hybridization of Pao sp. A and Pao
sp. B. An AML 298phylogenetic
to 536 bp region of the nuclear
analysis clearlyTBT-bp2
separatedgene
thewas determined
samples into for
PNHall the
andsamples except Pao
KTI, namely
KTI-3 by direct sequencing of the PCR products. We observed double peaks at several
sp. A and Pao sp. B, as in the case of the mitochondrial genes (Figure 4). Amino acid sequences also nucleotide
positions in the Sanger sequence electropherograms of almost all the samples, indicating that the
differed between Pao sp. A and Pao sp. B. Note that although we tried to obtain longer sequences,
TBT-bp2 gene had multiple alleles (Table A2). Allele types were apparently similar between samples
PCR amplifications were unsuccessful, probably due to the presence of microsatellite-like sequences in
of Pao sp. A and Pao sp. B. Compared with the allele types found in Pao sp. A and Pao sp. B, there was
neighboring
no sampleintrons.
in which the allele types could be represented as a hybridization of Pao sp. A and Pao sp.
B. An ML phylogenetic analysis clearly separated the samples into PNH and KTI, namely Pao sp. A
2.2. Toxin Profile
and Pao sp. B, as in the case of the mitochondrial genes (Figure 4). Amino acid sequences also differed
between
Toxins Pao extracted
were sp. A and from
Pao sp.theB. skin,
Note muscle,
that although
liver,we
andtried to obtain
gonads longer
of each sequences,pufferfish.
freshwater PCR
amplifications were unsuccessful, probably due to the presence of microsatellite-like
Analysis of the extracts by high-performance liquid chromatography with post-column fluorescence sequences in
neighboring introns.
derivatization (HPLC-FLD) for STXs [12,33] revealed that the tissues of each individual, except for
one or two tissues of KTI-1 and KTI-2, contained STXs comprising STX as the main component and
decarbamoylSTX (dcSTX) as a minor component (typical chromatograms are shown in Figure A3).
NeoSTX was also contained in some tissues as a minor component, but no other known STX components,
such as C toxins and gonyautoxins (GTXs), were detected. When the same extracts were analyzed for
TTX by liquid chromatography tandem mass spectrometry (LC-MS/MS) [12,34], no TTX was detected
at all.
Toxins 2020, 12, 689 5 of 15
99 KTI-5
KTI-4
Pao sp. B
KTI-2
86
KTI-1
PNH-1
PNH-9
PNH-2, 3, 10
83 PNH-5, 6 Pao sp. A
82
PNH-7
PNH-4
59
PNH-8
0.0200 0.0150 0.0100 0.0050 0.0000
Figure4.4.Inter-sample
Figure relationships
Inter-sample of the
relationships of TBT-bp2 gene using
the TBT-bp2 gene maximum likelihood
using maximum (ML). A midpoint
likelihood (ML). A
rooted tree is shown. Node numbers are ML bootstrap values. KTI-3 and PNH-11 were not included
midpoint rooted tree is shown. Node numbers are ML bootstrap values. KTI-3 and PNH-11 were not
due to their shorter sequences.
included due to their shorter sequences.
The toxin concentration (converted value to mg STX·diHCl eq/kg) in each tissue from the pufferfish
2.2. Toxin Profile
is shown in Figure 5 and Table A1. The toxin concentrations of Pao sp. A were generally high and far
aboveToxins were extracted
the regulatory limit for from themg
STX (0.8 skin, muscle, eq/kg)
STX·diHCl liver, and
in allgonads
tissues.of Theeach
mean freshwater pufferfish.
toxin concentration
Analysis of the extracts by high-performance liquid chromatography with
in the ovary (58.7 mg STX·diHCl eq/kg) was the highest, followed by the skin (40.8 mg STX·diHCl eq/kg). post-column fluorescence
derivatization
The mean toxin(HPLC-FLD)
concentrationfor in STXs [12,33]
the liver (23.1revealed that theeq/kg)
mg STX·diHCl tissues wasof lower
each individual,
than that inexcept for
the skin,
one or two
although tissuesindividuals,
in some of KTI-1 and theKTI-2, contained STXs
toxin concentration comprising
in the STX asthat
liver exceeded the inmainthe component
skin. The mean and
decarbamoylSTX
toxin concentration(dcSTX) as a minor
in the muscle (22.3component
mg STX·diHCl (typical
eq/kg)chromatograms
was the lowest, arebutshown in Figure
still almost A3).
28-fold
NeoSTX
higher thanwasthe also contained
regulatory limit.inInsome tissues
contrast, as a concentrations
the toxin minor component, of Pao but
sp. Bno were other knownmuch
generally STX
components,
lower than those suchofasPao C toxins
sp. A.and Thegonyautoxins (GTXs), were detected.
mean toxin concentration in the skin When
(6.0 the
mg same extracts
STX·diHCl were
eq/kg)
analyzed
was for TTXfollowed
the highest, by liquidbychromatography
the ovary (1.3 mg tandem mass spectrometry
STX·diHCl eq/kg). The mean (LC-MS/MS) [12,34], no TTX
toxin concentrations in
wastestis,
the detected
liver,atand
all. muscle were below the regulatory limit (0.5–0.6 mg STX·diHCl eq/kg), but some
The toxin
individuals had concentration
concentrations (converted
that slightlyvalue
exceeded to mg theSTX·diHCl
regulatory limit.eq/kg) in each tissue from the
pufferfish
The totalis shown
amount in of
Figure
toxin5per andindividual
Table A1. is The toxinin
shown concentrations
Figure 6. As of Pao sp. A
described were the
above, generally
toxin
high and far above
concentration in each the regulatory
tissue was much limithigher
for STX in (0.8
Pao mg
sp. STX·diHCl
A than in Pao eq/kg)
sp. B,in but
all tissues.
due to the Thelarger
mean
individual size of Pao sp. B over Pao sp. A (Table A1), the total toxin amount of KTI-3–5 (172–417skin
toxin concentration in the ovary (58.7 mg STX·diHCl eq/kg) was the highest, followed by the µg
(40.8 mg STX·diHCl
STX·diHCL eq/kg).was
eq/individual) The generally
mean toxin concentration
comparable in the
to that inliver
Pao sp.(23.1Amg STX·diHCl
individuals eq/kg) was
(122–463 µg
lower than that
STX·diHCL in the skin, although
eq/individual) other than in some
PNH-1, individuals,
in whichthe thetoxin
toxinconcentration
concentration in the
in liver exceeded
the skin was
that in the skin.
exceptionally low.TheThe mean toxin concentration
toxin distribution profile ininthe the muscle
body, (22.3 differed
however, mg STX·diHClbetweeneq/kg) Pao sp.was the
A and
lowest,
Pao but
sp. B. In still
Pao almost
sp. A, in 28-fold
addition higher than
to the thethe
skin, regulatory
amount of limit.
toxinInincontrast,
the muscle the andtoxinovary
concentrations
was high,
of Pao sp.
whereas inBPaoweresp. generally
B, most ofmuch lower
the toxin than those
amount in theofbody
Pao sp.
wasA. The meanfor
accounted toxin
by the concentration
skin, irrespectivein the
skin
of the(6.0
sex.mg STX·diHCl eq/kg) was the highest, followed by the ovary (1.3 mg STX·diHCl eq/kg). The
mean toxin
The concentrations
toxin compositionininthe testis,
each liver,
tissue perand muscle
species were below
is shown the regulatory
in Figure 7. In Pao limitsp. B,(0.5–0.6
the toxinmg
STX·diHCl eq/kg), but some individuals had concentrations that slightly
concentrations in the muscle, liver, and gonads were very low, and neoSTX concentrations were below exceeded the regulatory
limit.
the limit of quantification. Other than this, however, there was no considerable difference between Pao
sp. A and Pao sp. B. In each tissue, most of the toxins were accounted for by STX. In the whole body,
both Pao sp. A and Pao sp. B contained 6% to 7% neoSTX and 3% to 4% dcSTX as the minor components,
in addition to the main component STX (~90%).
Toxins 2020, 12, 689 6 of 15
Figure 5. Toxin concentration in each tissue in each pufferfish collected from Phnom Penh (PNH, Pao
sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.
The total amount of toxin per individual is shown in Figure 6. As described above, the toxin
concentration in each tissue was much higher in Pao sp. A than in Pao sp. B, but due to the larger
individual size of Pao sp. B over Pao sp. A (Table A1), the total toxin amount of KTI-3–5 (172–417 µg
STX·diHCL eq/individual) was generally comparable to that in Pao sp. A individuals (122–463 µg
STX·diHCL eq/individual) other than PNH-1, in which the toxin concentration in the skin was
exceptionally low. The toxin distribution profile in the body, however, differed between Pao sp. A
and Pao sp. B. In Pao sp. A, in addition to the skin, the amount of toxin in the muscle and ovary was
Figure 5. Toxin concentration in each tissue in each pufferfish collected from Phnom Penh (PNH,
high, whereas
Figure 5.in Paoconcentration
Toxin sp. B, mostin of eachthe toxin
tissue amount
in each in collected
pufferfish the body was
from accounted
Phnom forPao
Penh (PNH, by the skin,
Pao sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.
irrespective of the sex.
sp. A) and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.
The total amount of toxin per individual is shown in Figure 6. As described above, the toxin
concentration in each tissue was much higher in Pao sp. A than in Pao sp. B, but due to the larger
individual size of Pao sp. B over Pao sp. A (Table A1), the total toxin amount of KTI-3–5 (172–417 µg
STX·diHCL eq/individual) was generally comparable to that in Pao sp. A individuals (122–463 µg
STX·diHCL eq/individual) other than PNH-1, in which the toxin concentration in the skin was
exceptionally low. The toxin distribution profile in the body, however, differed between Pao sp. A
and Pao sp. B. In Pao sp. A, in addition to the skin, the amount of toxin in the muscle and ovary was
high, whereas in Pao sp. B, most of the toxin amount in the body was accounted for by the skin,
irrespective of the sex.
Figure 6. Total toxin amount per individual pufferfish collected from Phnom Penh (PNH, Pao sp. A)
and Kratie (KTI, Pao sp. B). * The ovary was too small to analyze.
concentrations in the muscle, liver, and gonads were very low, and neoSTX concentrations were
below the limit of quantification. Other than this, however, there was no considerable difference
between Pao sp. A and Pao sp. B. In each tissue, most of the toxins were accounted for by STX. In the
whole body, both Pao sp. A and Pao sp. B contained 6% to 7% neoSTX and 3% to 4% dcSTX as the
minor
Toxins components,
2020, 12, 689 in addition to the main component STX (~90%). 7 of 15
Figure 7. Toxin composition in each tissue in Pao sp. A (left) and Pao sp. B (right). S = skin, M = muscle,
= liver,
LFigure 7. G = gonads,
Toxin composition
and WBin=each tissue
whole in Pao sp. A (left) and Pao sp. B (right). S = skin, M = muscle,
body.
L = liver, G = gonads, and WB = whole body.
3. Discussion
3. Discussion
The present study revealed that freshwater pufferfish collected from two regions of Cambodia,
Phnom ThePenh andstudy
present Kratie, are different
revealed species belonging
that freshwater pufferfish to collected
the genusfrom Pao two
(Paoregions
sp. A and Pao sp. B,
of Cambodia,
respectively), and both possess STXs, with Pao
Phnom Penh and Kratie, are different species belonging to the genus Pao (Pao sp. A and B
sp. A having high toxicity and Pao sp. having
Pao sp. B,
low toxicity.
respectively), and both possess STXs, with Pao sp. A having high toxicity and Pao sp. B having low
The CYTB analysis suggests that Pao sp. A is the same species as P. cochinchinensis (AB741980.1),
toxicity.
whereas
TheCYTB
CYTBsequences
analysis suggests P. cochinchinensis
of other that Pao sp. A is the specimens
same species (AP011925.1, JQ681922.1)(AB741980.1),
as P. cochinchinensis are distantly
related
whereastoCYTBPao sp. A (Figure
sequences 3), which
of other makes it difficult
P. cochinchinensis specimens to identify species
(AP011925.1, with DNAare
JQ681922.1) barcoding
distantly
methods. According to Igarashi et al. [35], at least one of the CYTB
related to Pao sp. A (Figure 3), which makes it difficult to identify species with DNA barcoding sequences is likely an error
resulting
methods.from the misidentification
According to Igarashi et of al. specimens.
[35], at least Likewise,
one of the PaoCYTB
sp. B is closely related
sequences to P.
is likely anbaileyi
error
as well as to P. abei and P. leiurus (KF667490.1, GU057266.1) (Figure 3), and
resulting from the misidentification of specimens. Likewise, Pao sp. B is closely related to P. baileyi as could be the same
species as P.
well as to oneabeiofand
these. The (KF667490.1,
P. leiurus phylogeneticGU057266.1) P. leiurus,
positions of (Figure however,
3), and could belargely differ
the same among
species as
specimens (Figure 3). As discussed in Igarashi et al. [35], these situations
one of these. The phylogenetic positions of P. leiurus, however, largely differ among specimens are most likely due to
similarities
(Figure 3). inAsthe morphologic
discussed characteristics,
in Igarashi et al. [35],geographic distribution,
these situations are most andlikely
ecology
dueoftothe Pao species.
similarities in
Our mitochondrial gene analyses indicated the presence of actual and/or
the morphologic characteristics, geographic distribution, and ecology of the Pao species. Our potential genetic boundaries
of species (i.e., gene
mitochondrial species-level
analyses clusters
indicatedinthe thepresence
phylogenetic
of actual trees), suggesting
and/or potentialthat an accumulation
genetic boundaries of
of genetic information could contribute to resolve the taxonomic status
species (i.e., species-level clusters in the phylogenetic trees), suggesting that an accumulation and relationships among of
Pao species.
genetic On the other
information couldhand, if crossbreeding
contribute to resolve occurred
the taxonomic Pao species,
in thestatus an introgression
and relationships among of the
Pao
mitochondrial
species. On the genome
other might
hand,have caused such discordance
if crossbreeding occurred inbetween the Paomorphologic
species, an species identification
introgression of the
mitochondrial genome might have caused such discordance between morphologic Takifugu
and mitochondrial DNA phylogeny. Crossbreeding is frequently observed in the marine species
pufferfish species
identification and(e.g., Takahashi etDNA
mitochondrial al., 2017 [36]), andCrossbreeding
phylogeny. an introgressionisoffrequently
the mitochondrial
observedgenomein the
from the freshwater Rhinogobius goby species was reported [37]. As demonstrated
marine Takifugu pufferfish species (e.g., Takahashi et al., 2017 [36]), and an introgression by the analysis of of the
the
TBT-bp2 gene, this
mitochondrial possibility
genome fromshould be carefullyRhinogobius
the freshwater considered in parallel
goby to taxonomic
species reexamination,
was reported [37]. As
though no evidence
demonstrated by thewas foundoffor
analysis thethe presence
TBT-bp2 of crossbreeding
gene, this possibility shouldPao
between Pao sp. B. in
sp. A andconsidered
be carefully
The toxin concentration in each tissue differed greatly between Pao
parallel to taxonomic reexamination, though no evidence was found for the presence of crossbreeding sp. A and Pao sp. B: it was
generally
between Pao much sp. higher
A and Paoin Pao B. A than in Pao sp. B. Ngy et al. [7] examined the toxicity of two
sp. sp.
Cambodian freshwater pufferfish species, Pao turgidus and Pao sp., during the rainy and dry seasons,
and reported that only the skin and ovary of P. turgidus were toxic in both seasons. The highest toxicity
scores of the skin and ovary were 37 mouse unit (MU)/g (5.5 mg STX·diHCl eq/kg) and 27 MU/g
(4.0 mg STX·diHCl eq/kg), respectively, which indicate low levels of toxicity, similar to Pao sp. B.
Ngy et al. [7] did not conduct a genetic analysis, and while there is some uncertainty in species
identification, it is assumed that several species of low-toxicity freshwater pufferfish inhabit Cambodia.
The Bangladeshi L. cutcutia (highest toxicity score of the most toxic tissue, the skin, 20 MU/g (3.0 mg
Toxins 2020, 12, 689 8 of 15
STX·diHCl eq/kg)) [6] could also be considered a low-toxicity species, similar to Pao sp. B. According to
Kungsuwan et al. [4], the Thai freshwater pufferfish P. leiurus and P. suvattii are both highly toxic, with
the highest mean toxicity scores of the skin, muscle, liver, and ovary ranging from 61 to 109 MU/g
(9.1–16 mg STX·diHCL eq/kg) in P. leiurus and 42 to 200 MU/g (6.3–30 mg STX·diHCL eq/kg) in P. suvattii.
The toxin concentration of Pao sp. A is comparable to or even higher than that of P. leiurus/P. suvattii,
and this species is one of the most highly toxic species among freshwater pufferfish.
The total toxin amount per fish was generally similar between Pao sp. A and Pao sp. B, with the
highest score exceeding 400 µg STX·diHCL eq/individual in both species. As the minimum lethal dose
for humans is approximately 400–1000 µg STX·diHCL eq [38,39], ingestion of one–two whole bodies of
these freshwater pufferfish can cause death by poisoning. Therefore, both species should be considered
extremely dangerous to eat. In Pao sp. B, however, the majority of the harbored toxin was distributed
in the skin, and the toxin concentrations of the other tissues were below or only slightly above the
regulatory limit. Therefore, at least with the individuals examined in this study, removing the skin
may decrease the possibility of poisoning. In addition to individual differences, regional and seasonal
variations are observed in pufferfish toxicity [1,40]. Careful accumulation of data on the phylogeny
and toxin profile of freshwater pufferfish, with such considerations in mind, may help to establish a
taxonomic system linked with toxicity profiles and the presentation of species that can be consumed
by removing toxic parts and other treatments.
In both Pao sp. A and Pao sp. B, the main toxin was STXs comprising STX as the main component,
and neoSTX and dcSTX as the minor components, and TTX was not detected at all. The toxin of
P. turgidus from Cambodia and Pao freshwater pufferfish from Thailand is similarly composed of
STX-dominated STXs and does not contain TTX [4,5,7]. There are some exceptional cases, such as
the case of the Bangladeshi L. cutcutia in which a unique STX derivative (carbamoyl-N-methylSTX)
is the main component [6,41], but even in the case of marine pufferfish that simultaneously possess
STXs and TTX, the main component of STXs is often STX [9,11,13–15]. The origins of STXs in Floridian
Sphoeroides and Philippine Arothron marine pufferfish are considered to be STX-producing dinoflagellates
Pyrodinium bahamense and P. bahamense var. compressum, respectively, based on similarities in the
toxin composition [8,9]. Although the origin of STXs in freshwater pufferfish is presumed to be
STX-producing cyanobacteria [6], little is known about the toxicity of cyanobacteria in the freshwater
environments of Cambodia, and the origin of STXs in Cambodian freshwater pufferfish remains to
be elucidated.
The STX distribution profiles clearly differed between Pao sp. A and Pao sp. B. In contrast to TTX,
of which the absorption, transportation, and accumulation in marine pufferfish have been studied
with respect to candidate molecules involved in these processes (i.e., PSTBP), such molecules for STXs
remain unknown regardless of the marine or freshwater pufferfish species. PSTBP genes in marine
pufferfish obviously evolved as a result of the fusion of two TBT-bp2 genes at the sixth and first exons,
and the sequences remain quite similar to TBT-bp2 genes on the same genome (Hashiguchi et al.
2015 [32]; Yamada et al., unpublished). TBT-bp2 was originally identified as a protein that binds the
environmental toxin TBT in the fish body [31]. These findings together suggest that the TBT-bp2 gene
could be one of the molecules involved in the kinetics of STX in the pufferfish body. As expected from
the observed difference in the STX distribution profiles between Pao sp. A and Pao sp. B, we found
different amino acid sequences of TBT-bp2 between Pao sp. A and Pao sp. B, which could result in
interspecific differences in the structure and function of TBT-bp2. Given the identical amino acid
sequence of the rhodopsin gene in Pao species, including Pao sp. A and Pao sp. B, the TBT-bp2 gene
might be one of the genes that determines the physiologic and ecologic characteristics of the host.
A more comprehensive analysis of the expression, distribution, and function of PSTBP/TBT-bp2
isoforms coupled with TTX/STX distribution profiles could provide a better understanding of the
molecular mechanisms and evolutionary processes of TTX/STX accumulation in Tetraodontidae.
Toxins 2020, 12, 689 9 of 15
4. Materials and Methods
4.1. Pufferfish Specimens

In November 2019, 11 (PNH-1–11) and 5 (KTI-1–5) freshwater pufferfish were sampled from a
small lake connected by a channel to the Mekong River near Phnom Penh and Kratie, Cambodia,
respectively (Figures 1 and 2, and Table A1). The specimens were transported alive to the laboratory at
the University of Kratie, where PNH-1–3 were dissected to obtain the skin (including the fins), muscle,
liver, and gonads. Each tissue was placed in a plastic bag, frozen, and transported to the laboratory
at Nagasaki University. The other specimens were frozen as whole bodies and transported to the
laboratory at Nagasaki University, where they were similarly dissected. Each tissue was stored at
−80 ◦ C until DNA extraction or toxin quantification.
4.2. DNA Extraction, PCR Amplification, and Sequencing

Total DNA was extracted from an approximately 5 × 5 mm piece of fin using a standard DNA
lysis solution containing proteinase K. The 3 mitochondrial genes, CYTB, 16S rRNA, and COI, and 2
nuclear genes, rhodopsin and TBT-bp2, were PCR-amplified using the following primers: cytball-Fl
and cytball-Rl for CYTB [35], 16sar-L and 16sar-H for 16S rRNA [42], FF2d and FR1d for COI [43],
Rh193, Rh545, and Rh1039r for rhodopsin [44], and TBTBP2EX1F: 50 -AAC CAG CGC TKC TSC TGC
TG-30 and TBTBP2EX4R: 50 -TTC TCC TCT GTC AGG ACT CC-30 for TBT-bp2 (this study). The PCR
amplification was carried out in a reaction volume of 25 µL containing 12.5 µL of 2×Ampirect Plus,
0.125 µL of BIOTAQ DNA Polymerase (TaKaRa Bio Inc., Kusatsu, Japan), 500 nM each of forward
primer and reverse primer, 10 µL of 10-fold diluted DNA, and the remaining volume made up by
nuclease-free water. The PCR conditions were as follows: initial denaturation at 94 ◦ C for 2 min,
35 cycles of amplification with each cycle containing 94 ◦ C for 30 s, 52 ◦ C for 40 s (16S rRNA, COI)
or 50 ◦ C for 40 s (CYTB, rhodopsin) or 60 ◦ C for 40 s (TBT-bp2), 72 ◦ C for 1 min (16S rRNA, COI),
2 min (CYTB, rhodopsin, TBT-bp2), and a final extension at 72 ◦ C for 10 min. The amplicons were
purified using ExoSAP-IT (Thermo Fisher Scientific Inc., Waltham, USA) and directly sequenced
by outsourcing (FASMAC Co., Ltd., Atsugi, Japan). GenBank/EMBL accession numbers are from
LC581801 to LC581879.
4.3. Phylogenetic Reconstructions

The sequences determined were checked and trimmed manually. In the case of the 2 nuclear genes,
the Sanger sequence electropherograms were carefully examined for clear double peaks with
comparable height, which reflects allelic heterogeneity at the locus. These sites were coded with
IUPAC ambiguity codes in the sequence alignments. For the mitochondrial genes, a couple of previous
studies [35,45] published CYTB sequences of closely related species with 16S rRNA or COI sequences.
Phylogenetic reconstructions using Bayesian inference (BI) and maximum likelihood (ML) methods
were conducted for CYTB and the 2 concatenated datasets, CYTB+16S rRNA, and CYTB+COI.
Best fit models of nucleotide substitution for BI analyses by MrBayes 3.2.7 [46] were inferred using
Kakusan 4 [47], and the GTR+I+G model was used for ML analyses by FastTree 2 [48]. In the
BI analyses, 4 runs of 5 million generations were performed with 4 chains each, trees were sampled
at 1000-generation intervals, and the first 25% of the trees were discarded as burn-in. In the ML
analyses, bootstrap values were calculated using 1000 trees generated by SEQBOOT in the PHYLIP
Package 3.698 [49], and the consensus tree was obtained by CompareToBootstrap.pl in FastTree 2 [50].
For TBT-bp 2, as the MrBayes analysis did not provide ambiguity codes, only ML analysis was
conducted as described above, but with the JC+CAT model.
4.4. Toxin Quantification

Each tissue of the pufferfish was extracted with 0.1 M HCl, passed through an HLC-DISK membrane
filter (0.45 µm, Kanto Chemical Co., Inc., Tokyo, Japan), and subjected to HPLC-FLD for STXs and
Toxins 2020, 12, 689 10 of 15
LC-MS/MS for TTX according to the previously reported methods [12]. The reference materials of C1, C2,
GTX1-4, and decarbamoylGTX2,3 were provided by the Japan Fisheries Research and Education Agency,
and neoSTX, dcSTX, and STX purified from the toxic crab Zosimus aeneus [51] and crystalline TTX
(Nacalai Tesque, Inc., Kyoto, Japan) were used as external standards. The limit of detection and limit
of quantification for STXs were 0.001–0.007 nmol/mL (S/N = 3) and 0.003–0.02 nmol/mL (S/N = 10),
and for TTX, 0.0009 nmol/mL (S/N = 3) and 0.003 nmol/mL (S/N = 10), respectively.
Based on the concentrations of STX, neoSTX, and dcSTX in each tissue (s, n, and d nmol/g,
respectively), the toxin concentration in each tissue (c mg STX·diHCl eq/kg) was calculated with the
toxicity equivalence factors (STX = 1, neoSTX = 2, and dcSTX = 0.5) [30] and molecular weight of
STX·diHCl (372.211) using the following equation; c = 372.211 × (1s + 2n + 0.5d) × 10−3 .
Author Contributions: Conceptualization, A.Y., M.W., and O.A.; funding acquisition, M.W., J.S.L., and O.A.;
investigation, H.Z., A.Y., Y.G., J.S.L., and T.T.; resources, L.H., L.N., and H.D.; writing—original draft, H.Z. and
A.Y.; writing—review and editing, A.Y. and O.A. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Japan Society for the Promotion of Science, 19H03051, and Nagasaki
University Grant for New Developments in Research, and supported by the Gyeongsang National University
Fund for Professors on Sabbatical Leave, 2020.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Specification and toxin concentration in each tissue from freshwater pufferfish.
Toxin Concentration (mg STXdi·HCL eq/kg)

Species Sample Sex TL (cm) 1 BW (g) 2
Skin Muscle Liver 3 Ovary 3,4 Testis
Pao sp. A PNH-1 ♀ 9.7 26.1 2.1 1.2 1.5 22.5
2 ♀ 8.0 15.1 70.5 13.7 2.7 19.3
3 ♀ 9.1 22.7 63.0 14.4 73.4 96.3
4 ♀ 7.0 14.4 43.1 32.1 15.8 78.5
5 ♀ 8.0 15.9 37.5 17.0 18.1 28.2
6 ♀ 6.5 12.8 39.7 45.3 63.4 63.5
7 ♀ 7.5 21.1 33.2 21.2 18.3 –
8 ♀ 7.5 22.2 60.8 45.4 34.7 97.3
9 ♀ 7.6 17.2 37.2 22.7 8.3 –
10 ♀ 8.0 17.0 33.4 16.7 14.8 –
11 ♀ 8.2 22.0 28.4 16.0 2.7 63.7
Mean ± SD 7.9 ± 0.9 18.8 ± 4.2 40.8 ± 18.9 22.3 ± 13.6 23.1 ± 24.4 58.7 ± 31.9
Pao sp. B KTI-1 ♀ 15.0 85.0 0.3 0.1 0 0
2 ♀ 16.0 90.0 0.8 0.3 0 1.2
3 ♀ 15.7 108 15.8 1.1 1.0 2.8
4 ♂ 15.5 105 6.5 0.5 1.5 0.9
5 ♂ 16.0 109 6.3 0.4 0.2 0.3
Mean ± SD 15.6 ± 0.4 99.4 ± 10.5 6.0 ± 6.2 0.5 ± 0.4 0.6 ± 0.7 1.3 ± 1.4 0.6
1 2
TL: total length. BW: body weight. 3 0: less than the limit of quantification, which was considered to be 0.
4 –: not examined due to tissue scarcity.
Toxins 2020, 12, 689
Toxins 2020, 12, x FOR PEER REVIEW
11 of 15
11 of 15
KTI-3
Pao baileyi (AB741961.1, AB741978.1)
KTI-3
94/96 KTI-1, 2, 4, 5
Pao baileyi (AB741961.1, AB741978.1)
71/89
95/92
94/96 Pao abei
KTI-1, (AB741960.1,
2, 4, 5 AB741977.1)
71/89
Pao leiurus (KF667490.1)
abei (AB741960.1, AB741977.1)
95/92 100/99
Paoleiurus
Pao abei (LC586270.1)
(KF667490.1)
100/99
100/100 Pao
Pao leiurus (AB741968.1, AB741985.1)
abei (LC586270.1)
100/100
85/67 PNH-3
Pao leiurus (AB741968.1, AB741985.1)
100/100
85/67 Pao cochinchinensis (AB741963.1, AB741980.1)
PNH-3
100/100 PNH-
Pao 1, 2, 4, 5, 6, 8, 9,
cochinchinensis 10, 11
(AB741963.1, AB741980.1)
PNH-7
PNH- 1, 2, 4, 5, 6, 8, 9, 10, 11
99/100 Pao suvattii (AB741974.1, AB741991.1)
PNH-7
99/100 Paosuvattii
Pao suvattii(AB741974.1,
(LC586271.1)AB741991.1)
78/67
Pao
Paoturgidus (AB741975.1, AB741992.1)
suvattii (LC586271.1)
100/100
78/67
Pao
Paocochinchinensis (AP011925.1)
turgidus (AB741975.1, AB741992.1)
100/100
Pao cochinchinensis (AP011925.1)
100/100 Arothron manilensis
(AP011920.1)
100/100 Arothron
Arothron hispidus(AP011929.1)
manilensis (AP011930.1)
0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000
0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000
Figure A1. Phylogenetic tree of the Pao species constructed by Bayesian inference (BI) and maximum
likelihood
Figure (ML) based on
A1. Phylogenetic mitochondrial
tree 16S rRNA
of the Pao species and CYTB
constructed genes. inference
by Bayesian The BI tree
(BI)isand
shown. Node
maximum
likelihood (ML) based on mitochondrial 16S rRNA and CYTB genes. The BI tree is shown. Node
numbers are
likelihood Bayesian
(ML) based posterior probabilities
on mitochondrial 16S(left)
rRNA and MLCYTB
and bootstrap values
genes. The (right).
BI tree is shown. Node
numbers are Bayesian posterior probabilities (left) and ML bootstrap values (right).
numbers are Bayesian posterior probabilities (left) and ML bootstrap values (right).
KTI-3
57/82 KTI-3
KTI-4, 5
57/82
99/99 KTI-4,
KTI-1, 52
98/95
98/95 98/97
99/99 KTI-1,
Pao 2 (KF667490.1)
leiurus
98/97
Pao
Paoleiurus (KF667490.1)
abei (LC586270.1)
Pao
Pao abei (LC586270.1)
cambodgiensis (JQ681828.1, JQ681921.1)
Pao cambodgiensis
Pao leiurus (JQ681828.1, JQ681921.1)
(JQ681834.1, JQ681927.1)
100/90
Pao
Pao leiurus (JQ681834.1,
palembangensis JQ681927.1)
(JQ681839.1, JQ681932.1)
100/90
palembangensis (AP011925.1)
Pao cochinchinensis (JQ681839.1, JQ681932.1)
100/100
PaoPao
99/99 cochinchinensis (AP011925.1)
suvattii (JQ681841.1, JQ681934.1)
83/60
100/100
99/99 Pao
Paosuvattii
suvattii(JQ681841.1,
(LC586271.1)JQ681934.1)
83/60
Paocochinchinensis
Pao suvattii (LC586271.1)
(JQ681829.1, JQ681922.1)
PaoPNH-3
cochinchinensis (JQ681829.1, JQ681922.1)
100/100
PNH-3
PNH-10
100/100
PNH-10
PNH- 1, 2, 4, 5, 6, 8, 9, 11
PNH-
PNH-71, 2, 4, 5, 6, 8, 9, 11
PNH-7
100/100 Pao palembangensis (AP011920.1)
Arothron manilensis (AP011929.1)
100/100 Arothron hispidus
Arothron manilensis (AP011929.1)
(AP011930.1)
0.200 0.150 0.100 0.050 0.000
0.200 0.150 0.100 0.050 0.000

likelihood (ML)(ML)
likelihood based on mitochondrial
based ontree
mitochondrial COICOIand
andCYTB
CYTBgenes.
genes. TheBI
The BItree
treeisisshown.
shown. Node numbers
Figure A2. Phylogenetic of the Pao species constructed by Bayesian inference (BI) Node numbers
and maximum
are Bayesian posterior
are Bayesian
likelihood (ML) probabilities
posterior
based on (left)
probabilities
mitochondrial and
(left) ML
and
COI MLbootstrap
and bootstrap values
values
CYTB genes. (right).
The(right).
BI tree is shown. Node numbers
are Bayesian posterior probabilities (left) and ML bootstrap values (right).
Table A2. Sequence variation at 29 nucleotide sites for TBT-bp2 gene sequences.
Sample 7 23 24 33 138 169 193 225 228 231 232 286 367 429 432 448 499 505 524
PNH-1
Toxins 2020, 12,A689 T G C C T C C T G C G A G C C T T A of 15
12
PNH-2 . . . Y1 . . . . W1 R1 M1 . . . S1 Y1 . . .
PNH-3 . C . Y . . . . W R M . . . S Y . . .
PNH-4 . Table Y A2.. Sequence
Y M variation
. . at 29. nucleotide
W R sites
M for. TBT-bp2
. .gene Ssequences.
. . . R
PNH-5 . . . Y . . . M W R M . . . S . . . .
Sample 7 23 24 33 138 169 193 225 228 231 232 286 367 429 432 448 499 505 524
PNH-6 . . . Y . . . M W R M . . . S . . . .
PNH-1 A T G C C T C C T G C G A G C C T T A
PNH-7 . .
PNH-2 . Y . . Y 1Y . M . . . . .. W1 RR 1 MM 1 . . . . . . S S 1 . Y 1 . .
W . . G.
PNH-3
PNH-8 . nd 1 C nd . nd Y nd . M . . . . .M W
W RR MM . . . . . . S S . Y . . . . R .
PNH-4 . Y . Y M . . . W R M . . . S . . . R
PNH-9 . nd . nd . nd Y nd . .
PNH-5 . . . . M. .
W RR MM . . . . . . . S . . . . . . . .
PNH-6
PNH-10 . nd . nd . nd Y nd . . .
. .
. M
. W
W RR MM . . . . . . S S Y . . . . . . .
PNH-7 . Y . Y M . . . W R M . . . S . . . G
PNH-11nd nd ndnd ndnd nd nd M nd . nd . nd Mnd W
PNH-8 1 nd nd R ndM . . . . . . . S . . . . . . . R
PNH-9 nd nd nd nd . . . . . R M . . . . . . . .
KTI-1 nd nd ndC nd. nd .
PNH-10 .
. .
G . Y .. W
. .R . M A . W . K 1. G S . Y C . G. G.
KTI-2 nd G ndC nd. nd . nd . nd G nd Y nd.
PNH-11 .
nd .nd . nd A . W . T . G . . . C . G. G.
KTI-1 nd C . . . G Y . . . . A W K1 G . C G G
KTI-3 G nd C nd . nd . nd . nd G nd Y nd . nd nd
KTI-2 . nd. nd. ndA ndW ndT ndG nd. ndC ndG ndG
KTI-3
KTI-4 nd G nd C nd R nd . nd . nd G nd Y nd . nd
. .nd . nd R nd W nd T nd G nd . nd C nd G nd G nd
KTI-4 G C R . . G Y . . . . R W T G . C G G
KTI-5 nd nd C C R R . .
KTI-5 . . GG . . .. .. .. . . AA . . T T GG . . CC GG GG
1 1YY==C
Cor W==AAor
orT,T,W orT,T,RR==AAor
orG,
G,MM== A
A or C,SS== C
orC, C or
or G, K ==
G, K nd == not determined.
T, nd
G or T, determined.
Figure A3. HPLC-FLD chromatograms of the ovary extract from PNH-4 (left), skin extract from
KTI-3Figure A3. HPLC-FLD
(middle), and of the chromatograms of decarbamoylSTX
saxitoxin (STX), the ovary extract from PNH-4and
(dcSTX), (left), skin extract
neoSTX from KTI-
standards (right).
3 (middle), and of the saxitoxin (STX), decarbamoylSTX (dcSTX), and neoSTX standards (right).
References
References
1. Noguchi, T.; Arakawa, O. Tetrodotoxin—Distribution and accumulation in aquatic organisms, and cases of
1. Noguchi, T.; Arakawa, O. Tetrodotoxin—Distribution and accumulation in aquatic organisms, and cases
human intoxication. Mar. Drugs 2008, 6, 220–242. [CrossRef] [PubMed]
of human intoxication. Mar. Drugs 2008, 6, 220–242.
2. Mahmud, Y.; Yamamori, K.; Noguchi, T. Occurrence of TTX in a brackish water puffer ‘midorifugu’,
2. Mahmud, Y.; Yamamori, K.; Noguchi, T. Occurrence of TTX in a brackish water puffer ‘midorifugu’,
Tetraodon nigroviridis,
Tetraodon collected
nigroviridis, from
collected Thailand.J.J.Food
Thailand.
from Food Hyg. Soc.Jpn.
Hyg. Soc. Jpn.1999, 40,40,
1999, 363–367. [CrossRef]
363–367.
3. Mahmud,
3. Y.; Yamamori,
Mahmud, Y.; Yamamori, K.;K.;Noguchi,
Noguchi, T. T.Toxicity
Toxicity andand tetrodotoxin
tetrodotoxin as theas theprinciple
toxic toxic principle of awater
of a brackish brackish
waterpuffer,
puffer, Tetraodon
Tetraodon steindachneri,
steindachneri, collectedcollected fromJ. Food
from Thailand. Thailand. J. Food
Hyg. Soc. Jpn. 1999, Hyg. Soc. Jpn. 1999,
40, 391–395.
40, 391–395. [CrossRef]
4. Kungsuwan, A.; Arakawa, O.; Promdet, M.; Onoue, Y. Occurrence of paralytic shellfish poisons in Thai
freshwater puffers. Toxicon 1997, 35, 1341–1346. [CrossRef]
5. Sato, S.; Kodama, M.; Ogata, T.; Saitanu, K.; Furuya, M.; Hirayama, K.; Kamimura, K. Saxitoxin as a toxic
principle of a freshwater puffer, Tetraodon fangi, in Thailand. Toxicon 1997, 35, 137–140. [CrossRef]
6. Zaman, L.; Arakawa, O.; Shimosu, A.; Onoue, Y. Occurrence of paralytic shellfish poison in Bangladeshi
freshwater puffers. Toxicon 1997, 35, 423–431. [CrossRef]
Toxins 2020, 12, 689 13 of 15
7. Ngy, L.; Tada, K.; Yu, C.F.; Takatani, T.; Arakawa, O. Occurrence of paralytic shellfish toxins in
Cambodian Mekong pufferfish Tetraodon turgidus: Selective toxin accumulation in the skin. Toxicon
2008, 51, 280–288. [CrossRef]
8. Landsberg, J.H.; Hall, S.; Johannessen, J.N.; White, K.D.; Conrad, S.M. Saxitoxin puffer fish poisoning in the
United States, with the first report of Pyrodinium bahamense as the putative toxin source. Environ. Health Perspect.
2006, 114, 1502–1507. [CrossRef]
9. Sato, S.; Ogata, T.; Borja, V.; Gonzales, C.; Fukuyo, Y.; Kodama, M. Frequent occurrence of paralytic
shellfish poisoning toxins as dominant toxins in marine puffer from tropical water. Toxicon 2000,
38, 1101–1109. [CrossRef]
10. Nakashima, K.; Arakawa, O.; Taniyama, S.; Nonaka, M.; Takatani, T.; Yamamori, K.; Fuchi, Y.; Noguchi, T.
Occurrence of saxitoxins as a major toxin in the ovary of a marine puffer Arothron firmamentum. Toxicon 2004,
43, 207–212. [CrossRef]
11. Barrientos, R.G.; Hernández-Mora, G.; Alegre, F.; Field, T.; Flewelling, L.; McGrath, S.; Deeds, J.; Chacón, Y.S.;
Arrieta, K.R.; Vargas, E.C.; et al. Saxitoxin poisoning in green turtles (Chelonia mydas) linked to scavenging
on mass mortality of Caribbean sharpnose puffer fish (Canthigaster rostrata-Tetraodontidae). Front. Vet. Sci.
2019, 6, 466. [CrossRef] [PubMed]
12. Zhu, H.; Sonoyama, T.; Yamada, M.; Gao, W.; Tatsuno, R.; Takatani, T.; Arakawa, O. Co-occurrence of
tetrodotoxin and saxitoxins and their intra-body distribution in the pufferfish Canthigaster valentini. Toxins
2020, 12, 436. [CrossRef]
13. Kodama, M.; Ogata, T.; Noguchi, T.; Maruyama, J.; Hashimoto, K. Occurrence of saxitoxin and other toxins
in the liver of pufferfish Takifugu Pardalis. Toxicon 1983, 21, 897–900. [CrossRef]
14. Nakamura, M.; Oshima, Y.; Yasumoto, T. Occurrence of saxitoxin in puffer fish. Toxicon 1984,
22, 381–385. [CrossRef]
15. Jang, J.; Yotsu-Yamashita, M. Distribution of tetrodotoxin, saxitoxin, and their analogs among tissues of the
puffer fish Fugu pardalis. Toxicon 2006, 48, 980–987. [CrossRef]
16. Zhang, X.; Zong, J.; Chen, S.; Li, M.; Lu, Y.; Wang, R.; Xu, H. Accumulation and elimination of tetrodotoxin in
the pufferfish Takifugu obscurus by dietary administration of the wild toxic gastropod Nassarius semiplicata.
Toxins 2020, 12, 278. [CrossRef]
17. Honda, S.; Arakawa, O.; Takatani, T.; Tachibana, K.; Yagi, M.; Tanigawa, A.; Noguchi, T. Toxification of
cultured puffer fish Takifugu rubripes by feeding on tetrodotoxin-containing diet. Nippon Suisan Gakkaishi
2005, 71, 815–820. [CrossRef]
18. Ikeda, K.; Murakami, Y.; Emoto, Y.; Ngy, L.; Taniyama, S.; Yagi, M.; Takatani, T.; Arakawa, O. Transfer profile of
intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes.
Toxicon 2009, 53, 99–103. [CrossRef]
19. Wang, J.; Araki, T.; Tatsuno, R.; Nina, S.; Ikeda, K.; Hamasaki, M.; Sakakura, Y.; Takatani, T.; Arakawa, O.
Transfer profile of intramuscularly administered tetrodotoxin to artificial hybrid specimens of pufferfish,
Takifugu rubripes and Takifugu niphobles. Toxicon 2011, 58, 565–569. [CrossRef]
20. Wang, J.; Araki, T.; Tatsuno, R.; Nina, S.; Ikeda, K.; Takatani, T.; Arakawa, O. Transfer profile of orally and
intramuscularly administered tetrodotoxin to artificial hybrid specimens of the pufferfish Takifugu rubripes
and Takifugu porphyreus. J. Food Hyg. Soc. Jpn. 2012, 55, 33–38. [CrossRef]
21. Tatsuno, R.; Shikina, M.; Shirai, Y.; Wang, J.; Soyano, K.; Nishihara, G.N.; Takatani, T.; Arakawa, O. Change in
the transfer profile of orally administered tetrodotoxin to non-toxic cultured pufferfish Takifugu rubripes
depending of its development stage. Toxicon 2013, 65, 76–80. [CrossRef] [PubMed]
22. Tatsuno, R.; Gao, W.; Ibi, K.; Mine, T.; Okita, K.; Nishihara, G.N.; Takatani, T.; Arakawa, O. Profile differences
in tetrodotoxin transfer to skin and liver in the pufferfish Takifugu rubripes. Toxicon 2017, 130, 73–78.
[CrossRef] [PubMed]
23. Yotsu-Yamashita, M.; Sugimoto, A.; Terakawa, T.; Shoji, Y.; Miyazawa, T.; Yasumoto, T. Purification,
characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from
plasma of the puffer fish, Fugu pardalis. Eur. J. Biochem. 2001, 268, 5937–5946. [CrossRef]
24. Yotsu-Yamashita, M.; Okoshi, N.; Watanabe, K.; Araki, N.; Yamaki, H.; Shoji, Y.; Terakawa, T. Localization of
pufferfish saxitoxin and tetrodotoxin binding protein (PSPBP) in the tissues of the pufferfish, Takifugu pardalis,
analyzed by immunohistochemical staining. Toxicon 2013, 72, 23–28. [CrossRef]
Toxins 2020, 12, 689 14 of 15
25. Yotsu-Yamashita, M.; Yamaki, H.; Okoshi, N.; Araki, N. Distribution of homologous proteins to puffer fish
saxitoxin and tetrodotoxin binding protein in the plasma of puffer fish and among the tissues of Fugu pardalis
examined by Western blot analysis. Toxicon 2010, 55, 1119–1124. [CrossRef]
26. Tatsuno, R.; Yamaguchi, K.; Takatani, T.; Arakawa, O. RT-PCR- and MALDI-TOF mass spectrometry-based
identification and discrimination of isoforms homologous to pufferfish saxitoxin- and tetrodotoxin-binding
protein in the plasma of non-toxic cultured pufferfish (Takifugu rubripes). Biosci. Biotechnol. Biochem. 2013,
77, 208–212. [CrossRef]
27. Yotsu-Yamashita, M.; Nagaoka, Y.; Muramoto, K.; Cho, Y.; Konoki, K. Pufferfish saxitoxin and tetrodotoxin
binding protein (PSPBP) analogues in the blood plasma of the pufferfish Arothron nigropunctatus, A. hispidus,
A. manilensis, and Chelonodon patoca. Mar. Drugs 2018, 16, 224. [CrossRef]
28. Cusick, K.D.; Sayler, G.S. An overview on the marine neurotoxin, saxitoxin: Genetics, molecular targets,
methods of detection and ecological functions. Mar. Drugs 2013, 11, 991–1018. [CrossRef] [PubMed]
29. Gao, W.; Kanahara, Y.; Yamada, M.; Tatsuno, R.; Yoshikawa, H.; Doi, H.; Takatani, T.; Arakawa, O.
Contrasting toxin selectivity between the marine pufferfish Takifugu pardalis and the freshwater pufferfish
Pao suvattii. Toxins 2019, 11, 470. [CrossRef]
30. World Health Organization & Food and Agriculture Organization of the United Nations. Toxicity Equivalence
Factors for Marine Biotoxins Associated with Bivalve Molluscs; World Health Organization: Geneva, Switzerland,
2016.
31. Oba, Y.; Shimasaki, Y.; Oshima, Y.; Satone, H.; Kitano, T.; Nakao, M.; Kawabata, S.; Honjo, T. Purification and
characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus.
J. Biochem. 2007, 142, 229–238. [CrossRef]
32. Hashiguchi, Y.; Lee, J.M.; Shiraishi, M.; Komatsu, S.; Miki, S.; Shimasaki, Y.; Mochioka, N.; Kusakabe, T.;
Oshima, Y. Characterization and evolutionary analysis of tributyltin-binding protein and pufferfish saxitoxin
and tetrodotoxin-binding protein genes in toxic and nontoxic pufferfishes. J. Evol. Biol. 2015, 28, 1103–1118.
[CrossRef] [PubMed]
33. Oshima, Y. Post-column derivatization HPLC method for analysis of PSP. J. AOAC Inter. 1995,
78, 528–532. [CrossRef]
34. Gao, W.; Kanahara, Y.; Tatsuno, R.; Soyano, K.; Nishihara, G.N.; Urata, C.; Takatani, T.; Arakawa, O.
Maturation-associated changes in internal distribution and intra-ovarian microdistribution of tetrodotoxin
in the pufferfish Takifugu pardalis. Fish. Sci. 2018, 84, 723–732. [CrossRef]
35. Igarashi, Y.; Doi, H.; Yamanoue, Y.; Kinoshita, S.; Ishibashi, T.; Ushio, H.; Asakawa, S.; Nishida, M.; Watabe, S.
Molecular phylogenetic relationship of Tetraodon pufferfish based on mitochondrial DNA analysis. Fish. Sci.
2013, 79, 243–250. [CrossRef]
36. Takahashi, H.; Toyoda, A.; Yamazaki, T.; Narita, S.; Mashiko, T.; Yamazaki, Y. Asymmetric hybridization and
introgression between sibling species of the pufferfish Takifugu that have undergone explosive speciation.
Mar. Biol. 2017, 164, 90. [CrossRef]
37. Yamasaki, Y.Y.; Nishida, M.; Suzuki, T.; Mukai, T.; Watanabe, K. Phylogeny, hybridization, and life
history evolution of Rhinogobius gobies in Japan, inferred from multiple nuclear gene sequences.
Mol. Phylogenetics Evol. 2015, 90, 20–33. [CrossRef]
38. Quayle, D.B. Paralytic shellfish poisoning in British Columbia. Fish. Res. Bd. Can. Bull. 1969, 158, 1–68.
39. Etheridge, S.M. Paralytic shellfish poisoning: Seafood safety and human health perspective. Toxicon 2010,
56, 108–122. [CrossRef]
40. Ikeda, K.; Emoto, Y.; Tatsuno, R.; Wang, J.-J.; Ngy, L.; Taniyama, S.; Takatani, T.; Arakawa, O.
Maturation-associated change in toxicity of the pufferfish Takifugu poecilonotus. Toxicon 2010,
55, 289–297. [CrossRef]
41. Zaman, L.; Arakawa, O.; Shimosu, A.; Shida, Y.; Onoue, Y. Occurrence of a methyl derivative of saxitoxin in
Bangladeshi freshwater puffers. Toxicon 1988, 36, 627–630. [CrossRef]
42. Palumbi, S.; Martin, A.; Romaro, S.; McMillan, W.O.; Stice, L.; Grabowski, G. The Simple Fool’s Guide to PCR.
Version 2.0; University of Hawaii: Honolulu, HI, USA, 2002; pp. 1–45.
43. Ivanova, N.V.; Zemlak, T.S.; Hanner, R.H.; Hebert, P.D.N. Universal primer cocktails for fish DNA barcoding.
Mol. Ecol. Notes 2007, 7, 544–548. [CrossRef]
Toxins 2020, 12, 689 15 of 15
44. Chen, W.J.; Bonillo, C.; Lecointre, G. Repeatability of clades as a criterion of reliability: A case study for
molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa. Mol. Phylogenetics Evol.
2003, 26, 262–288. [CrossRef]
45. Santini, F.; Nguyen, M.T.T.; Sorenson, L.; Waltzek, T.B.; Lynch Alfaro, J.W.; Eastman, J.M.; Alfaro, M.E.
Do habitat shifts drive diversification in teleost fishes? An example from the pufferfishes (Tetraodontidae).
J. Evol. Biol. 2013, 26, 1003–1018. [CrossRef]
46. Ronquist, F.; Teslenko, M.; Van Der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.;
Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice
across a large model space. Syst. Biol. 2012, 61, 539–542. [CrossRef] [PubMed]
47. Tanabe, A.S. Kakusan4 and Aminosan: Two programs for comparing nonpartitioned, proportional and
separate models for combined molecular phylogenetic analyses of multilocus sequence data. Mol. Ecol. Resour.
2011, 11, 914–921. [CrossRef] [PubMed]
48. Price, M.N.; Dehal, P.S.; Arkin, A.P. FastTree 2—Approximately maximum-likelihood trees for
large alignments. PLoS ONE 2010, 5, e9490. [CrossRef]
49. Felsenstein, J. PHYLIP (Phylogeny Interference Package) Version 3.6. 2005. Available online: https:
//evolution.genetics.washington.edu/phylip.html (accessed on 20 August 2020).
50. Price, M.N. Fast Tree-Comparison Tools. Available online: http://www.microbesonline.org/fasttree/treecmp.
html (accessed on 20 August 2020).
51. Arakawa, O.; Noguchi, T.; Shida, Y.; Onoue, Y. Occurrence of carbamoyl-N-hydroxy derivatives of saxitoxin
and neosaxitoxin in a xanthid crab Zosimus aeneus. Toxicon 1994, 32, 175–183. [CrossRef]
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Diterima : 03-02-2019; Diterbitkan :17-04-2019