AIP Conference Proceedings 2231, 040036 (2020); https://doi.org/10.1063/5.0002606 2231, 040036

© 2020 Author(s).

Ethanolic extract of Centella asiaticaincreased IGF-1 and IRS expression inzebrafish (Danio rerio) rotenone-inducedCite as: AIP Conference Proceedings 2231, 040036 (2020); https://doi.org/10.1063/5.0002606Published Online: 22 April 2020

Fitra Arsy Nur Cory’ah, Vanda Primaditya, Linda Ika Puspita Ariati, Zakiah, Dyah Woro Kartiko KusumoWardani, Dianita Primihastuti, Yuningsih, Husnul Khotimah, Mohammad Muljohadi Ali, and Nurdiana

ARTICLES YOU MAY BE INTERESTED IN

Low dose nicotine lower MDA level and does not affect wall to lumen ratio in surgicallymenopausal ratsAIP Conference Proceedings 2231, 040054 (2020); https://doi.org/10.1063/5.0003164

Photos: International Conference on Life Science and Technology (ICoLiST)AIP Conference Proceedings 2231, 010003 (2020); https://doi.org/10.1063/12.0000514

Identification of damage and key species of coral reefs on Gili-Noko Bawean IslandAIP Conference Proceedings 2231, 040033 (2020); https://doi.org/10.1063/5.0002579

Ethanolic Extract of Centella asiatica Increased IGF-1 and IRS Expression in Zebrafish (Danio rerio) Rotenone-induced

Fitra Arsy Nur Cory’ah1,2 , Vanda Primaditya1,3, Linda Ika Puspita Ariati1,4, Zakiah1,5, Dyah Woro Kartiko Kusumo Wardani1,6, Dianita Primihastuti1,7 and Yuningsih1,8, Husnul Khotimah9,*, Mohammad Muljohadi Ali9, and Nurdiana9

1Master Program of Midwifery, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia 2 Poltekkes Kemenkes Mataram, Midwifery Academy, NTB, Indonesia

3 Midwifery Academy of Berlian Nusantara, Magetan, East Java, Indonesia 4Midwifery Academy of Jember, Jember, East Java, Indonesia

5University of Darul Ulum Jombang High Islamic Boarding School, Jombang, East Java, Indonesia 6Institute of Health Science Sint Carolus, Jakarta, Indonesia

7Institute of Health Science William Booth, Surabaya, East Java, Indonesia 8Institute of Health Science Dr. Soebandi, Jember, East Java, Indonesia

9Laboratory of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia

*Corresponding author: husnul_farmako.fk@ub.ac.id

Abstract. IRS (insulin receptor substrate) is responsible for signal transduction of Insulin-like growth factor-1 (IGF-1) for cell differentiation, proliferation, and anti-apoptosis. Centella asiatica (CA) contains triterpenes as antioxidant and anti-inflammatory, macro and micronutrients. This study observed the effect of CA extract on the expression of IGF-1, IRS, and the linear growth of rotenone-induced zebrafish larvae. The zebrafish embryos were divided into five groups: control, Rotenone 12 ppb, Rotenone, and CA extract concentration 2.5, 5, and 10 μg/ml, respectively exposed from 2-hours post fertilization (hpf) until 3-days post-fertilization (dpf). The expression of IGF-1 and IRS were conducted at 9 dpf by immune-histochemistry whole-mount. The body length was measured at 3, 6, and 9 dpf using Image Raster software. The data were analyzed using one-way ANOVA. The results showed that the administration of CA extract could increase the expression of IGF-1 and IRS in rotenone-induced zebrafish larvae. The measurement of body length showed that rotenone reduced the body length of larvae after 6 dpf, and 5 μg/mL of CA extract significantly increased the body length at 6 and 9 dpf. It can be concluded that CA extract increased the body length of zebrafish larvae through the increasing of IGF-1 and IRS signaling.

INTRODUCTION

Stunting is linear growth retardation (LGR) in children under five due to long-term malnutrition and chronic infection. Stunting is according to the standard of WHO Child Growth Standard in 2010, based on body length index compared to age (length/age) or height compared to age (height/age) with a value (z-score) <-2 SD (Standard Deviation) [1-3]. Indonesian basic health research by 2013 noted that the prevalence of national stunting reached 37.2%, the highest one was in Southeast Asia [1,4].

Genetic and environmental factors influence growth and development. The environmental factors are toxic chemicals, such as pesticides or other xenobiotic ingredients, which disrupt synthesis, distribution, metabolism, and the action of hormones in the body, thus increasing abnormalities in growth and development [5]. Rotenone is a natural pesticide from the roots of tuba (Derris eelliptica (Wellich) Benth). The mechanism of action of rotenone inhibits mitochondrial I complex, which causes an increase in ROS, resulting in reduced ATP, oxidative stress, and cell death6.

International Conference on Life Sciences and Technology (ICoLiST)AIP Conf. Proc. 2231, 040036-1–040036-9; https://doi.org/10.1063/5.0002606

Published by AIP Publishing. 978-0-7354-1990-2/$30.00

040036-1

Stunting mechanism can be associated with malnutrition, disruption of growth hormones, and inflammation. Growth hormone has a role in stimulating the formation of IGF-1. IGF-1 receptor (IGF-1R) activates tyrosine kinases which will undergo a phosphorylation reaction, so that the IRS (insulin receptor substrate) as an effector, performs the main function of the receptor as signal transduction, by forming various complex pathways, through MAPKinase and PI3Kinase, which consequences for differentiation cell, proliferation, anti-apoptosis, pre, and postnatal growth, which are related to nutritional intake [7-10].

Gotu kola (Centella asiatica) is one of the medicinal plants that has low toxicity and complete nutritional content, namely macronutrients, such as carbohydrates, and proteins, micronutrients, such as various vitamins (A, B1, B2, B3, C, and βCarotene), and minerals (Na, K, Ca, Mg, P and Fe), as well as phytonutrients (flavonoids, triterpenes, carotenoids, alkaloids, and glycosides). Triterpenoids (asiaticoside, madecasosside), the major composition acts as antioxidants and anti-inflammatory that inhibit the damage due to rotenone exposure [11-13].

Zebrafish was used as an animal model due to some of its advantages. Some of them are the 70% genetic homologous and physiological similarity with human genes14, rapid embryogenesis, producing 300 eggs per week, transparent embryos and larvae that facilitate observation on microscopes directly, and zebrafish is cheaper than other vertebrates [15-17]. Comparison of zebrafish age analogy with humans is based on the ontogeny of the sleep-wake cycle. Zebrafish at 3, 6, and 10 dpf (day post-fertilization) is analogous to newborns baby, 2 and 8 years old babies18. This study aimed to determine the effect of CA extract on the expression of IGF-1 and IRS in the rotenone-induced zebrafish embryo.

EXPERIMENTAL DETAILS

Zebrafish Maintenance

The wild-type adult zebrafish was maintained under standard procedure [19]. Briefly, 40 zebrafishes (20 male and 20 female) were reared in the 60 L tank. The temperature range was 26-28 °C, conductivity 300-1500 μS, dark:light cycle 14:10 and dissolved oxygen > 6.0 mg/L. The zebrafish were fed three times a day. When the light was on, the female zebrafishes released eggs, and the males released sperm. The embryo was washed and placed in six wells plate in an embryonic medium. The embryos were divided into five groups: control, rotenone 12.5 pub exposed, rotenone, and CA extract concentration of 1.25, 2.5 and 5 μg/mL. All procedures were already approved by the ethical committee in the Faculty of Medicine, Universitas Brawijaya No.154/EC/KEPK/04/2017.

Embryonic Medium

The composition of embryonic medium was based on Darwitri [20]. The embryonic medium was made with a concentration of 10x by CaCl 0.25 g, KCl 0.15 g, NaCl 5 g, MgSO4 0.815 g, and 500 mL of distilled water.

CA Extract

The CA was gained and identified from Materia Medica, Batu, Malang East Java. The extraction used maceration in 96% of ethanol and was evaporated at 70 °C.

Administration of Rotenone and CA

Rotenone (sigma, R8875) was diluted for concentration of 12.5 ppb in embryonic medium (previously diluted in Dimethyl sulphoxide (DMSO) for stock solution. Administration of Rotenone and CA extract was conducted from 2 hpf to 72 hpf and refreshed for every 24 h.

Body Length and Ratio of Head over the Body Length Measurement

The body length and ratio of head over the body of larvae (after hatching) were measured at 3, 6, and 9 dpf. The standard length (SL) was measured from the tip of the snout to the caudal fin (snout-fin) [21] of the body. The larvae were observed and measured using Image Raster software from Optilab V.2 after the calibration procedure.

040036-2

FIGURE 1. Measurements of the head length and body length by using Image Raster

Whole-mount Immunohistokimia

Zebrafish larvae at 9 def were anesthetized based on NIH protocols [22]. Briefly, zebrafish larvae were placed in the iced water for five seconds until the larvae of zebrafish did not move. Then, larvae were fixed by paraformaldehyde (PFA) 4% at 4 °C overnight, and continued by soaking in 3% MeOH for five minutes and 3% H2O2 for 24 h for bleaching the pigment. The chromogen used in this staining was DAB (3,3’-Diaminobenzidine), while primer antibody anti IGF-1 (AbCam® lot 9J18V1) and anti IRS (SantaCruz® Tyr 989 lot 62611). The larva were observed under microscope Olympus SZ61 40x magnification and connected to the Panasonic DMC G6 Lumix for taking pictures.

Quantification of IGF-1 and IRS Expression

The photographs of larvae were analyzed for the intensity of expression each protein using Image J v.4.1. The brown color (positive expression) was analyzed for the integrated density in a pixel unit.

FIGURE 2. Left panel showed the photograph of zebrafish larvae by Olympus SZ41 in 40x magnification. Right panel

showed the process for quantification of protein expression using Image J v4.1 software.

040036-3

Data Analysis

The Data were analyzed using ANOVA from SPSS v.21.

RESULTS AND DISCUSSION

The Body Length and ratio of the length head and body length can be seen at Table 1. Table 1 showed that at 3 dpf there no significant different of body length. However, at 6 and 9 dpf, there were significant different. As it in stunting children, they born in normal condition but a after 2 years there are shorten of the body length which the differentiation is more than 2 SD of normal population. The ratio of all group were constant from 3 to 9 dpt like the stunting children that has normal proportion of the body. The administration of CA extract increased the body length the higher concentration the longer of the body, and the optimum results in this research was five μg/mL.

TABLE 1. The body length and ratio of head over the body of zebrafishlarvae.

Abbreviation :C=Control ; R=rotenone 12.5 ppt ; RCA 1.25=Exposure of rotenone 12.5 ppt and CA extract concentration 1.25 μg/mL ; RCA 2.5=Exposure of rotenone 12.5 ppt and CA extract concentration 2.5 μg/mL ; RCA 5 = Exposure of rotenone 12.5 ppt and CA extract concentration 5 μg/mL. Rotenone and CA extract were co-incubated from 2 hpf to 72 hpf.

FIGURE 3. The body length of zebrafish larvae on 3, 6 and 9 dpf

040036-4

Zebrafish Larvae Body Length

One of the parameters of this study is the length of the body, because the length of the body is one of the main indicators to determine the condition of stunting growth. Stunting is a linear growth retardation (RPL) in children under five, because of long-term malnutrition [23], so that there is an obstacle in the growth of height or body length compared to age, with a value (z-score) <-2 SD based on the standard standards of WHO Child Growth Standard in 2010 [1-3].

Based on table 1 and Figure 3 there is no significant difference (p = 0.113) body length among all groups at 3 dpf (analogue to newborn baby). At 6 and 9 dpf, analogues to children of 2 and 8 years old, there were significantly different in the body length (p = 0.000) between the control group and the rotenon group [9]. At 6 dpf the difference of body length in rotenone group to the control group was 0.23, while a standard deviation of 2 SD was 0.16. Therefore 0.23 > 2SD, so it can be concluded that rotenone can induce stunting. The results of this study were in accordance with the theory that stunting is the initial state of birth without the presence of congenital abnormalities, and it only will appear at 2 years old [23-25].

The mechanism of stunting is closely related to the condition of malnutrition, impairment of growth hormone and inflammatory process. Stunting growth can be return to normal conditions estimated at only 3% [26]. Environmental factors and genetic factors can affect children's growth and development. One of the related environmental factors is the use of pesticides. Pesticides are toxic substances which include endocrine disrupting chemicals (EDCs) which are chemicals that can affect metabolic processes, synthesis, transport, secretion and action of hormones in the body [5,27,28]. Rotenon is a natural pesticide which mechanism in the cell by inhibiting mitochondrial complex I. It will be increased production of ROS, oxidative stress, failure of ATP formation and cell death [29,30]. Depletion of ATP production can cause hormonal disorders and growth factors (IGF-1) that play an important role in the process of neurogenesis, muscle and bone formation that impact to the growth and development including stunting growth [30]. Children who experience mitochondrial disorders show stunted stature and associated with a low level of IGF-1 in the body [29,31].Zebrafish was sensitive to the particular chemicals such as rotenone during early development so that it affect to the its growth [16]. Ridlayanti [32] and Wijayanti [33] proved that rotenone 10 ppb impaired the zebrafish body length.

IGF-1 and IRS Expression

Based on the Figure 4, rotenone decreases IGF-1 expression. Addition of CA extract can increase IGF-1 expression as concentration increases with significant effect was obtained on 5μg/ml. CA extract contain antioxidant properties because it can decreased MDA production and increased SOD and Catalase level [20,34]. It may inhibit ROS production due to rotenone-induced. It has strong correlation to the body length of zebrafish larvae. This is because IGF-1 directly has a role in mediating somatic cell growth and development, including muscle and bone, both in prenatal and postnatal period [10]. Signalling of IGF-1 also plays a role in determining skeletal patterns including bone shape and size [35,36]. Wood et al. [10] reported that IGF-1 concentration in zebrafish showed a significant increase in the larval phase compared to the embryo phase, because the larval stage had anterior growth of the head. In this study, whole-mount immunohistochemistry also found that IGF-1 expressed stronger in the head part of larvae.

040036-5

FIGURE 4. The expression of IGF-1 (upper section) and after quantified using Image J software (lower section)

The Figure 5 showed that the administration of CA extract tend to increase the expression of IRS. IRS is a major

effector for insulin receptors and is a substrate receptor that can act properly by carrying out phosphorylation of tyrosine kinase in the intracellular substrate, and binding to other signalling molecules [37]. IRS as a transduction signal pathway helps in metabolic processes of the body, including the process of glycogen synthesis, protein, pleats, growth, cell proliferation and gene expression and also contributes to the process of Glut-4 translocation to cell membranes [36,37]. The IRS as a signal of transformation forms various complex paths. If there are disturbances such as exposure to toxic substances in this case rotenone will be able to inhibit other signalling pathways, disability from signals, receptors and gene mutations, it can interfere with the expression of IRS [38] Rotenone works in inhibiting mitochondrial complex I causing oxidative stress marked by ROS production exceeds antioxidants, causing ATP levels to decrease, resulting in cell damage, mitochondrial damage and DNA damage [39-41]. Lack of ATP levels, causes energy requirements in metabolic and mitogenic processes to be inhibited. These conditions can cause the IRS-1 concentration to decrease and the presence of disruption in growth and weight loss [37].

With the fulfilment of good nutrition will be able to provide a good effect on the system in the body, including hormones and growth factors (IGF-1) [7] and IRS, and can stabilized the pattern of linear growth and body mass of children [9].

040036-6

FIGURE 5. The expression of IRS (upper section) and after quantified using Image J software (lower section).

Centella asiatica is one of the herbs that has low toxicity and has complete nutritional content, namely

macronutrients such as carbohydrates, and proteins, micronutrients such as vitamins and minerals, and phytonutrients (flavonoids, triterpenes, carotenoids, alkaloids and glycosides). CA has high triterpenoids, one of them is asiaticoside bioactive compounds as antioxidants and anti-inflammatory that inhibit the occurrence of oxidation reactions due to rotenone exposure [12,13,42]. Ethanolic extract of CA had antioxidant activity for inhibiting the oxidation reaction, reduce free radicals due to induction of rotenone, so that the addition of these antioxidants can stabilize the IGF-1 as a ligand on its receptor and produce other transduction signalling pathways including increasing IGF-1 concentration in body [38]. Decreased IGF-1 and IRS expression can be caused by induction of rotenone as a toxic material capable of causing cell damage and growth disturbances. Protein restriction has been shown to reduce IGF concentrations.

Symptoms of chronic malnutrition are cognitive disability, decreased motor ability and impaired body function and other have long-term effects [25]. Based on the research by Ridlayanti [32] it proved that CA extractincreased BDNF (brain-derived neurotrophic factor) that responsible for neuroprotection, neurodevelopment and cognitive function. Administration of CA extract increased osteoprotegerin (OPG) and decreased RANKL with the consequence is growth and development of the bone [43]. OPG expression is used for bone formation, RANKL as an indicator of osteoclastogenesis (absorption process bone) [40].It was supported by Primihastuti et al.44 that administration CA extract can improve ossification in the overall bone formation process.

SUMMARY

Rotenone 12.5 ppb significantly decreased the body length at 6 and 9 dpf through the decreasing of IGF-1 and IRS expression. Administration of 5 μg/mL CA extract significantly increased the body length by increasing IGF-1 and IRS expression at 9 dpf.

ACKNOWLEDGMENTS

We would like to thanks to Mr. Wibi Riawan, M.Biomed for the antibodies and immunostaining assistance and Mrs. Ferrida, ST for helping preparation of reagent. Faculty of Medicine Brawijaya University for the funding by 2016.

040036-7

REFERENCES

1 Kemenkes RI, Jakarta. (2016) 2 H. Sudirman, Media Penelitian dan Pengembangan Kesehatan, 18, (2008) 3 WHO, interpretation guide. (2012) 4 Trihono et al, Pendek (Stunting) di Indonesia masalah dan solusinya, (Lembaga Penerbit Balitbangkes, Jakarta.

2015) 5 Utami R.P., Suhartono S., Nurjazuli N., Kartini A. dan Rasipin R., Jurnal Kesehatan Lingkungan Indonesia, 12,

127 (2013) 6 Sherer T.B., Betarbet R., Testa C.M., Seo B.B., Richardson J.R., Kim J.H., et al., Journal of Neuroscience, 23,

10756 (2003) 7 Chitnis M.M., Yuen J.S., Protheroe A.S., Pollak M. dan Macaulay V.M., Clinical Cancer Research, 14, 6364

(2008) 8 Palacio A.C., Pérez-Bravo F., Santos J.L., Schlesinger L. danMonckeberg F, Nutrition, 18, 17 (2002) 9 Prendergast, J A., Humphrey dan H J., Paediatrics and international child health, 34, 250 (2014) 10 Wood A.W., Duan C. dan Bern H.A., International review of cytology, 243, 215 (2005) 11 Khotimah H., Ali M., Sumitro S.B. danWidodo M.A., Asian Pacific Journal of Tropical Biomedicine, 5 (11): pp.

948-954 (2015) 12 Chippada S.C., Volluri S.S., Bammidi S.R. dan Vangalapati M., Rasayan J Chem, 4, 457 (2011) 13 Hashim P., International Food Research Journal, 18(4): pp. 1215-1222 (2011) 14 Howe K., Clark M.D., Torroja C.F., Torrance J., Berthelot C., Muffato M., et al., Nature, 496, 498 (2013) 15 Belyaeva N., Kashirtseva V., Medvedeva N., Khudoklinova Y.Y., Ipatova O. danArchakov A., Biochemistry

(Moscow) Supplement Series B: Biomedical Chemistry, 3, 343 (2009) 16 Dai Y.J., Jia Y.F., Chen N., Bian W.P., Li Q.K., Ma Y.B., et al., Environmental toxicology and chemistry, 33,11

(2014) 17 Eimon, M P., Ashkenazi & Avi, Apoptosis, 15, 331 (2010) 18 Sorribes A., Þorsteinsson H., Arnardóttir H., Jóhannesdóttir I.H., Sigurgeirsson B., De Polavieja G.G., et al.,

Frontiers in neural circuits, 7, 178 (2013) 19 Avdesh, A., Chen, M., Martin-Iverson, M., Mondal, A., Ong, D., Rainey-Smith, S. Taddei, K., Lardelli, M.,

Groth, DM., Verdile, G., Martins, RN., Journal of Visualized Experintal. 69, 4196 (2012) 20 Khotimah, H., Darwitri, Yuliyani, T., Nuraenah, E., Zahara, E.,., Kalsum, Nurdiana, Ali., Biomedical and

Pharmacological Journal. 11, 827 (2018) 21 Spence R., Gerlach G., Lawrence C. dan Smith C, Biological Reviews, 83, 13 (2008) 22 Strykowski J.L. danSchech J.M., Journal of the American Association for Laboratory Animal Science, 54,

81(2015) 23 Shrimpton R. dan Kachondham Y., Analysing the causes of child stunting in DPRK, (New York: UNICEF. 2003) 24 Badham, Jane Sweet danLara, Stunting: an overview, Sight Life, 3, 40 (2010) 25 Reinhardt, Kristina & Fanzo J., Frontiers in nutrition, 1 (2013) 26 Caulfield, L. E., Richard, S. A., Rivera, J. A., Musgrove, P., & Black, R. E. Stunting, wasting, and micronutrient

deficiency disorders. In Disease Control Priorities in Developing Countries. 2nd edition. (The International Bank for Reconstruction and Development/The World Bank 2006)

27 Costa, L. G. Toxic effects of pesticides. Casarett and Doull’s toxicology: the basic science of poisons, 8, 883 (2008)

28 Grandjean P., Bellinger D., Bergman Å., Cordier S., Davey‐Smith G., Eskenazi B., et al., Basic & clinical pharmacology & toxicology, 102, 73 (2008)

29 Sherer T.B., Betarbet R., Testa C.M., Seo B.B., Richardson J.R., Kim J.H., et al., Journal of Neuroscience, 23, 10756 (2003)

30 Murphy M.P., Biochemical Journal, 417,1 (2009) 31 Wolny S., McFarland R., Chinnery P. & Cheetham T., Acta Paediatrica, 98, 553 (2009) 32 Ridlayanti, A. Proteksi Ekstrak Centella Asiatica Pada Model Stunting Larva Zebrafish (Danio Rerio) Yang

Diinduksi Rotenon Melalui Peningkatan Ekspresi Bdnf (Magister thesis, Universitas Brawijaya, 2016) 33 Wijayanti, A. Proteksi Ekstrak Pegagan (Centella Asiatica) Melalui Ekspresi Hsp60 Dan Bax Terhadap Model

Stunting Larva Zebrafish (Danio Rerio) Yang Diinduksi Rotenon (Magister thesis, Universitas Brawijaya, 2016).

040036-8

34 Yanusaga T., S.k., Noriyuki, Horikawa R.t., Toshiaki, Tanae A. dan Hibi I., Endocrine journal, 45, 221(1998) 35 Garrone S., Radetti G., Sidoti M., Bozzola M., Minuto F. dan Barreca A., The Journal of Clinical Endocrinology

& Metabolism, 87, 5460 (2002). 36 Bikle, D. D., Tahimic, C., Chang, W., Wang, Y., Philippou, A., & Barton, E. R. Bone, 80, 79 (2015) 37 Ozoe A., Sone M., Fukushima T., Kataoka N., Arai T., Chida K., et al., FEBS letters, 587, 2319(2013) 38 Kofoed E.M., Hwa V., Little B., Woods K.A., Buckway C.K., Tsubaki J., et al., New England Journal of

Medicine, 349, 1139 (2003) 39 Haruta T., Uno T., Kawahara J., Takano A., Egawa K., Sharma P.M., et al., Molecular endocrinology, 14, 783

(2000) 40 Li N., Ragheb K., Lawler G., Sturgis J., Rajwa B., Melendez J.A., et al., Journal of Biological Chemistry, 278,

8516 (2003) 41 Zima T., aacute, scaron, Fialová L., Mestek O., Janebová M., et al., Journal of biomedical science, 8, 59 (2001) 42 Xu X.-L., Shang Y. & Jiang J.-G., Food & function, 7,643 (2016) 43 Ariati, L. I. P. Pengaruh Ekstrak Etanol Pegagan (Centella Asiatica) Terhadap Ekspresi Osteoprotegrin (Opg)

Dan Receptor Activator Nuclear Kappa-Β Ligan (Rankl) Pada Stunting Larva Zebrafish (Danio Rerio) Yang Diinduksi Rotenon (Magister thesis, Universitas Brawijaya, 2017).

44 Primihastuti D. Pengaruh Ekstrak Etanol Pegagan (Centella asiatica) pada Osifikasi Tulang dan Osteoklastogenesis pada Model Stinting Larva Zebrafish (Danio rerio) yang diinduksi rotenon. (Magister thesis, Universitas Brawijaya, 2017).

040036-9