Damai Noviasari and Dr. dr. Umi Kalsum,, M.Kes; and Dr. Husnul Khotimah,, S.Si., M.Kes. (2019) Pengaruh Ekstrak Etanol Pegagan Terhadap Ekspresi Tirosin Hidroksilase, Kadar Dopamin Dan Aktivitas Lokomotor Pada Larva Zebrafish Yang Dipapar Timbal. Magister thesis, Universitas Brawijaya.
Abstract
Timbal merupakan contoh logam berat yang dapat menyebabkan keracunan pada saraf sehingga dapat menyebabkan gangguan integritas, struktural bahkan fungsi syaraf sehingga terjadi penurunan kecerdasan, fungsi kognitif, penurunan minat serta perubahan aktivitas dan gangguan motorik kasar. Kemenkes menyebutkan bahwa 5-10 % balita mengalami keterlambatan perkembangan sedangkan 1-3 % diperkiraka mengalami gangguan perkembangan motorik, bahasa, sosio-emosional dan kognitif. Masuknya timbal ke dalam tubuh akan meningkatkan jumlah radikal bebas sehingga menyebabkan stres oksidatif. Keadaan stres oksidatif dapat menghambat kerja enzim tirosin hidroksilase serta menyebabkan penurunan dopamin sehingga mengakibatkan perubahan aktivitas lokomotor. Pegagan merupakan tumbuhan yang mengandung triterpen yang berfungsi sebagai antioksidan dan quarcetin yang berfungsi sebagai pengikat (chelator) logam berat. Tujuan penelitian ini adalah membuktikan ekstrak etanol pegagan dapat mencegah penurunan ekspresi tirosin hidroksilase, kadar dopamin serta aktivitas lokomotor pada larva zebrafish yang dipapar timbal mulai 2 hpf – 72 hpf. Penelitian ini adalah true experimental menggunakan post test only control group design. Paparan timbal 2.5 ppm dan ekstrak etanol pegagan dengan konsentrasi 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml diberikan secara bersamaan pada larva zebrafish mulai usia 2 hpf sampai dengan 72 hpf (hour post fertilization). Ekspresi tirosin hidroksilase larva zebrafish diukur saat usia 6 dpf dengan menggunakan Reverse Trancription Polymerase Chain Reaction (RT-PCR). Hasil elektroforesis dikuantifikasikan menggunakan Bio-Rad Gel Doc Imaging system, sedangkan kadar dopamin diukur menggunakan Enzyme- Linked Immunosorbent Assay (ELISA). Data dianalisis menggunakan uji One Way Anova. Lokomotor zebrafish diukur dengan menggunakan hasil kuantifikasi pergerakan larva melewati pola kemudian dibuat pattern dengan menggunakan Image J. Hasil penelitian menunjukkan bahwa ekspresi tirosin hidroksilase antar kelompok tidak terdapat perbedaan signifikan (p > 0.05), namun terdapat kecenderungan kelompok kontrol positif memiliki rata-rata terendah bila dibandingkan dengan kontrol negatif. Pemberian ekstrak pegagan dengan konsentrasi berbeda membuktikan bahwa semakin tinggi konsentrasi konsentrasi semakin meningkat ekspresi tirosin hidroksilase. Kadar dopamin pada larva zebrafish mengalami peningkatan pada kelompok yang diberi pegagan dengan konsentrasi berbeda bila dibandingkan dengan kontrol positif. Analisis data menunjukkan terdapat perbedaan signifikan kadar dopamin dengan p < 0.05. Hasil pengamatan yang dilakukan terhadap aktivitas lokomotor larva zebrafish usia 4 – 6 dpf menunjukkan adanya perbedaan signifikan p < 0.05. Pada usia 5 dpf dan 6 dpf timbal menurunkan aktivitas lokomotor secara signifikan dan pemberian ekstrak pegagan meningkatkan aktivitas lokomotor seiring dengan peningkatan konsentrasi. Korelasi Spearman antara ekspresi tirosin hidroksilase dengan kadar dopamin menunjukkan adanya hubungan positif (searah) dan kuat (0.700) artinya semakin tinggi ekspresi tirosin hidroksilase maka semakin tinggi kadar dopamin. Hubungan antara kadar dopamin dengan aktivitas lokomotor menunjukkan hubungan positif (searah) dan sempurna (1.000) yang berarti bahwa peningkatan kadar dopamin akan diikuti dangan peningkatan aktivitas lokomotor. Timbal mampu menurunkan ekspresi tirosin hidroksilase, kadar dopamin serta aktivitas lokomotor pada larva zebrafish. Pemberian ekstrak etanol pegagan mampu meningkatkan ekspresi tirosin hidroksilase, kadar dopamin serta aktivitas lokomotor. Paparan timbal diketahui menyebabkan peningkatan Ca2+ intraseluler sehingga terjadi disfungsi mitokondria. Kerusakan pada mitokondria akan mengakibatkan stress oksidatif serta menghambat kerja dari tirosin hidroksilase bahkan terjadi apoptosis. Tirosin hidroksilase merupakan enzim yang berfungsi untuk biosintesis dopamin. Penurunan ekspresi tirosin hidroksilase akan mengakibatkan penurunan terhadap dopamin dan lokomotor. Ekstrak etanol pegagan memiliki kandungan quercetin yang berfungsi sebagai chelator logam berat serta sebagai scavenger radikal bebas. Asiaticoside pada pegagan berfungsi untuk peningkatan enzim antioksidan endogen, sehingga memberikan efek neuroproteksi pada dopaminergik dan mampu meningkatkan ekspresi tirosin hidroksilase. Hasil penelitian ini didukung oleh penelitian sebelumnya bahwa timbal dengan konsentrasi 50 mg/kg dapat menghambat dopaminergik dan lokomotor pada tikus Kesimpulan dalam penelitian ini adalah ekstrak etanol pegagan dapat meningkatkan aktivitas lokomotor melalui peningkatan kadar dopamine pada larva zebrafish yang dipapar timbal
English Abstract
Lead is a heavy metal that is poisonous to the nerves system, resulting in integrity, structural even nerve disorders which eventually lead to decreased intelligence, cognitive skill, enthusiasm, and also lead to changes in activities and gross motor disorders. The Ministry of Health reported 5-10% of toddlers experienced developmental delays, in which it was estimated that 1-3% suffered from motor, language, socio- emotional and cognitive developmental disorders. The level of lead content in the body increases the level of free radicals causing oxidative stress. This oxidative stress inhibits tyrosine hydroxylase enzyme functions and causes decreases in dopamine level, resulting in locomotor activity disorders. Centella asiatica is a plant that contains triterpenes which functions as an antioxidant and quarcetin which functions as a heavy metal chelator. This research was conducted to confirm the potentials of Centella asiatica ethanol extract in preventing decreases in tyrosine hydroxylase expression, dopamine levels and locomotor activity in zebrafish larvae exposed to lead at 2 hpf - 72 hpf. This true experimental research employed post test only control group design. Lead exposure at 2.5 ppm and 1.25 μg / ml, 2.5 μg / ml, 5 μg / of ethanol extract from Centella asiatica were administered simultaneously to zebrafish larvae from the age of 2 hpf to 72 hpf (hour post fertilization). The expression of tyrosine hydroxylase zebrafish larvae was measured at 6 dpf using a Reverse Trancription Polymerase Chain Reaction (RT-PCR). The electrophoresis results were quantified using the Bio-Rad Gel Doc Imaging system, while dopamine levels were measured using Enzyme-Linked Immunosorbent Assay (ELISA). The obtained data were then analyzed using One Way Anova test. The zebrafish locomotor was calculated based on the results of larvae movement calculation through certain pattern to form a pattern using Image J. The results of this research showed no significant differences in the tyrosine hydroxylase expression between groups (p> 0.05). However, the positive control group was found to have a tendency of having the lowest average compared to the negative control. The administration of Centella asiatica extract at concentrations proved that higher concentration increased tyrosine hydroxylase expression. Dopamine levels in zebrafish larvae were found to increase in groups which were given Centella asiatica extract at different concentrations compared to positive controls. The results of the data analysis indicated significant differences in dopamine levels at p <0.05. Meanwhile, observations showed that the locomotor activity of 4 - 6 dpf zebrafish larvae showed a significant difference p <0.05. At the age of 5 dpf and 6 dpf, the level of lead content significantly reduced locomotor activity and the administration of higher concentration of Centella asiatica extract improved the locomotor activity. A positive (direct) and perfect correlation were also found between dopamine levels and locomotor activity at (1,000), indicating that an increase in dopamine levels would be followed by an increase in locomotor activity. Lead content could reduce tyrosine hydroxylase expression, dopamine levels and locomotor activity in zebrafish larvae. The use of Centella asiatica ethanol extract successfully increased tyrosine hydroxylase expression, dopamine levels and locomotor activity. Lead exposure which has been known to cause increases in intracellular Ca2+ would also cause mitochondrial dysfunction. Damages to the mitochondria trigger oxidative stress which inhibits tyrosine hydroxylase from functioning, even causes apoptosis. Tyrosine hydroxylase is an important enzyme in dopamine biosynthesis. Lower tyrosine hydroxylase expression will result in decreases in dopamine level and locomotor activity. The ethanol extract of Centella asiatica plant contains quercetin which functions as a heavy metal chelator as well as a free radical scavenger. Asiaticoside content found in the plant increases endogenous antioxidant enzymes, providing a neuroprotective effects on dopaminergic and increasing the tyrosine hydroxylase expression. The findings of this research support the ones of previous research stating that lead content at a concentration of 50 mg / kg can inhibit dopaminergic and locomotor in mice It is concluded from the results of this research that ethanol extract from Centella asiatica plant has been able to improve the locomotor activity through increases in dopamine levels in zebrafish larvae exposed to lead
Other obstract
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| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/615.323 849/FK/p/2019/041902805 |
| Subjects: | 600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.3 Organics drugs > 615.32 Drugs derived from plants and mikroorganisms |
| Divisions: | S2/S3 > Magister Kebidanan, Fakultas Kedokteran |
| Depositing User: | Nur Cholis |
| Date Deposited: | 23 Aug 2022 03:45 |
| Last Modified: | 23 Aug 2022 03:45 |
| URI: | http://repository.ub.ac.id/id/eprint/193439 |
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