Open Veterinary Journal, (2023), Vol. 13(5): 629–637

Original Research

10.5455/OVJ.2023.v13.i5.16

Asiatic acid in Centella asiatica extract towards morphological development in an intermittent hypoxia intrauterine embryo model and molecular prediction pathway of insulin-like growth factor-1 receptor signalling

Ariani Ariani^1,2*, Imelda Febriana Ghofar³, Husnul Khotimah⁴, Nurdiana Nurdiana⁴ and Masruroh Rahayu⁵

¹Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia

²Saiful Anwar General Hospital, Malang, Indonesia

³Esto Ebhu Hospital, Sumenep, Indonesia

⁴Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia

⁵Department of Neurology, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia

*Corresponding Author: Ariani Ariani. Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia. Email: arianidr [at] ub.ac.id.

Submitted: 06/02/2023 Accepted: 17/04/2023 Published: 17/05/2023

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Abstract

Background: Hypoxia during pregnancy generates oxidative stress that alters the growth and development of the human fetus. Insulin-like growth factor-1 (IGF-1) receptors are essential for normal fetal growth. Asiatic acid in Centella asiatica (CA) has antioxidant properties to prevent growth impairment in hypoxia.

Aims: This study aimed to investigate the effect of asiatic acid on the morphological development of an intermittent hypoxia (IH) zebrafish embryo model and analyze molecular docking prediction in IGF-1 receptor (IGF-1R) signaling.

Methods: Embryos of zebrafish at 2 hours postfertilization (hpf) were assigned to control negative (C), IH, and combination IH and CA extract groups consisting of 1.25 (IHCA1), 2.5 (IHCA2), and 5 (IHCA3) µg/ml. Hypoxia treatment (conducted 4 hours/day) and CA extract were administered for 3 days (2–72 hpf). The parameters of body length and head length were evaluated at 3, 6, and 9 days postfertilization (dpf). The data were analyzed by a two-way analysis of variance (p < 0.05). Molecular docking was performed to explore the binding affinity of asiatic acid to IGF-1R by Molegro Virtual Docker ver.5 software.

Results: The body length and head length of embryos in the IH and treatment groups (IHCA) were shorter than those in the control group at 3 dpf (p < 0.05). However, the body length was more prolonged in the IHCA1 group, but the head length was longer in the IHCA2 group than in the IH group at 6 and 9 dpf. Molecular docking showed the reliable interaction of asiatic acid with IGF-1R signaling in an IH animal model.

Conclusion: The administration of CA extract benefits IH through the development and growth of zebrafish embryos at a dose of 2.5–5 µg/ml. Asiatic acid has a binding affinity for IGF-1R signaling.

Keywords: Asiatic acid, Centella asiatica, Zebrafish embryo, Morphology, IGF-1R.

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Introduction

Oxidative stress may link altered placental function to fetal programming (Rodríguez-Rodríguez et al., 2018). Fetal programming occurs when the usual pattern of fetal growth in humans or animals is disturbed by a deviant stimulus, such as inadequate nutrition and hypoxia (low ambient oxygen), which can occur at a crucial period during intrauterine life (Kwon and Kim, 2017). The hypoxic state of pregnancy is highly implicated in fetal programming by interfering with the development of the placenta and fetus (Fajersztajn and Veras, 2017). During fetal development, hypoxia is involved in several embryonic processes, including placentation, angiogenesis, and haematopoiesis (Perrone et al., 2016). Hypoxia during pregnancy can develop from several maternal conditions, such as maternal smoking, anemia, placental insufficiency, hypertension, asthma, and respiratory tract infection (Jang et al., 2015).

Hypoxia is also involved in the pathogenesis of other human diseases, such as tumorigenesis (Kamei and Duan, 2018). Hypoxia is one of the most significant stressors affecting aquatic ecosystems globally, and its severity and incidence are expected to rise due to increased nutrient intake and climate change (Fitzgerald et al., 2016). In rats, prenatal hypoxia impairs endothelium-dependent relaxation in the mesenteric circulation of the newborn child (Fowden et al., 2006).

Malnutrition, stress, and hypoxia can alter the concentrations of hormones that cross the placental barrier in the mother and fetus, including growth hormone (GH), insulin-like growth factor (IGF), insulin, glucocorticoids, catecholamines, leptin, thyroid hormone, placental hormone eicosanoids, sex steroids, and placental lactogen (Fowden et al., 2006). IGF-1 is synthesized in the liver via the pituitary/liver-GH/IGF-1 system axis, which also involves GHs. IGF-1 and its receptor (IGF-1R) are expressed in various extrahepatic organs (Acharjee et al., 2017). The placenta secretes IGF-1 during pregnancy. IGF-1 then stimulates the transfer of essential nutrients from the placenta’s mother to the fetus (Hellström et al., 2016). IGF-1 plays an important role in normal fetal growth and stimulates fetal cell proliferation, differentiation, and synthesis of protein and glycogen mediated via IGF receptors and binding proteins (Malhotra et al., 2019).

Hypoxia induces oxidative stress; thus, administering antioxidants provides beneficial outcomes (Merino et al., 2014). The health benefits of Centella asiatica (CA) are fighting free radicals, reducing inflammation, protecting nerve cells, and accelerating wound healing (He et al., 2023) by triggering blood microcirculation and collagen formation. Other benefits are diarrhea and stomach ulcers (Mishra et al., 2022). CA was previously reported to have antioxidant properties and protect rotenone-induced zebrafish embryos from oxidative stress (Cory’ah et al., 2020). A previous study demonstrated that CA extracts at concentrations of 1.25–5 μg/ml could enhance body length and locomotor activity in zebrafish larvae in the chronic constant hypoxia model (Ariani and Widiyastuti, 2022). The bioactive compounds from CA include triterpenoids, phenolic compounds, essential oils, pectins, free amino acids, vitamins, and minerals (Tripathy and Srivastav, 2023). The most dominant triterpenoids for health are asiatic acid, madecassic acid, asiatic acid-coside, and madecassoside (Gray et al., 2018). Asiatic acid is known as a strong anti-inflammatory agent. tory and wound healing abilities as well as regulating the effect of apoptosis in several chronic diseases, such as neurodegeneration, cancer, and hypertension (Kukula et al., 2022). Asiatic acid, an active compound in CA and the family of triterpenoids, is a potent cellular antioxidant (Wang et al., 2021) that may have activity on IGF-1. Zebrafish embryos are proposed as a commonsense model for pediatric research according to their superiority compared to other animals. This study investigates the effect of asiatic acid in intermittent hypoxia (IH) zebrafish embryos and analyses the predicted interaction pathway of asiatic acid with IGF-1R signaling.

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Material and Methods

Materials

Adult wild-type zebrafish were obtained from the Faculty of Fisheries and Marine Science, Universitas Brawijaya. The simplica of CA leaves was obtained from Materia Medica, Malang, East Java, Indonesia. All reagents and chemicals were analytical grade and obtained from E-Merck.

Experimental design

Adult zebrafish of both sexes were kept in a 60-l glass aquarium with a female and male ratio of 2:1. Adult fish were maintained at a density of 4–5 fish l^-1 (Mikloska et al., 2022). The temperature was set at 28°C ± 1°C, and the light/dark cycle was 14:10 hours. Food was provided twice a day with TetraMin (TetraMin Tropical Fish Flakes, USA) (Robertson et al., 2014). Embryo collection was carried out following the beginning of the light period. Egg water was replaced twice a day. Neither eggs nor embryos were reared for 12 hours:12 hours in light: dark radiation (Mikloska et al., 2022).

This research was a true experimental study with a posttest-only control group design approach. A total of 20 embryos at 2 hours postfertilization (hpf) were involved in each group of experiments. Every group contained three replications (Fitzgerald et al., 2016). The experiments consisted of five groups: a control group (normoxia), IH, and IH + extract of CA (µg/ml) at 1.25 (IHCA1), 2.5 (IHCA2), and 5 (IHCA3) (Darwitri et al., 2018).

Intermittent hypoxia

Hypoxia was performed by exposing the embryos to a water chamber that supplied nitrogen gas to reach oxygen pressure PO₂~5 kPa, dissolved oxygen (DO) 2 mg O₂/l as hypoxia status. Hypoxia was applied as an intermittent schedule at 2, 24, 48, and 72 hpf for 4 hours of exposure (at 08.00–12.00 am) followed by normoxia (PO₂~20 kPa, DO 8 mg O₂/l) for the rest through medium substitution. A DO meter tightly monitors the oxygen concentration (Kamei and Duan, 2018).

Preparation and treatment of CA extract

Simplicia of 100 g of CA was macerated in 900 ml of 96% ethanol for 24 hours, followed by filtration. This procedure was replicated three times, and then the filtrate was collected for further rotary evaporation. The crude extract was obtained from pasta, diluted in normal saline, and stored at 0°C. Preliminary research showed that based on the liquid chromatography mass spectrometry test, one of the components in the ethanol extract of CA is acetic acid. The extract was dissolved in an embryo medium that contained a mixture of embryonic medium and CA extract (Darwitri et al., 2018). The medium-containing extract solution was administered simultaneously from 2 until 72 hpf, with IH in the treatment groups. The medium was changed every day.

Body length and head length measurement

After hatching, the larvae were kept in an embryonic medium and monitored for body and head length measurements at 3, 6, and 9 days postfertilization (dpf). Body length was measured from the tip of the head to the end of the trunk (before the caudal fin) (Collery et al., 2014). The head length was scaled from the snout to the operculum. The body length and head length were measured using Image Raster software. The observation was conducted under a stereomicroscope (Olympus SZ61) connected to an Optilab camera and Optilab software version 2.0.

Molecular docking analysis

The protein sequence of the IGF receptor (GenBank ID NP_694501.1) was retrieved from the GenBank database and then modeled by SwissProt with 6JK8 as a template. Asiatic acid was downloaded from the PubChem NCBI database (CID 119034). Molegro Virtual Docker ver.5 software was used to predict the active site of the IGF-1R with 5 van der Waals parameters. Molecular docking was carried out by Molegro Virtual Docker ver.5 with Grid X96.16; Y107.61; Z157.85; radius 11. Docking validation insulin as a native ligand and IGF-1R were set as root-mean-square deviation (RMSD) <2.0 Armstrong (Å) (Zhang et al., 2020).

Data analysis

The body length and head length data were analyzed by two-way analysis of variance (p < 0.05) followed by post hoc comparisons performed by Tukey’s test. Statistical analysis was performed using the IBM Statistical Program Service Solution version 18 program.

Ethical approval

This research was approved by the Health Research Ethics Committee of Universitas Brawijaya with reference number 92/EC/KEPK-PPDS/03/2019.

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Results

Zebrafish embryos are used in animal models of IH. Modifying ambient oxygen tension and observing phenotypic changes in real time is simply because of the zebrafish embryo’s rapid development and transparent performance. Additionally, critical components of the hypoxia factor system and their genes have been found in zebrafish that are highly comparable to humans and mammals (Kamei and Duan, 2018).

Effect of CA extract on the body length of zebrafish embryos under IH

The study results of the average body length of zebrafish embryos using the intermittent intrauterine hypoxia model and administration of CA ethanol extract at various concentrations at 3, 6, and 9 dpf (Fig. 1). The results showed that there was a significant difference in body length between the control group and the treatment group at 3, 6, and 9 dpf (p < 0.05). There was a significant difference in body length (p < 0.05) between the mean (mm) of the IH group (0.50 ± 0.04), IHCA1 (0.51 ± 0.01), IHCA2 (0.50 ± 0.02), IHCA3 (0.48 ± 0.03), and group C (0.65 ± 0.05) at 3 dpf. Group C showed a longer body length than the IH-induced group. The group given CA therapy showed a significant increase in embryo body length compared with the group not given CA. Zebrafish embryos aged 6 and 9 dpf showed an increase in body length compared with those aged 3 dpf in all treatment groups. The study also showed that the body length in the IHCA1 group (0.84 ± 0.05), IHCA2 (0.84 ± 0.07), IHCA3 (0.85 ± 0.07), and C (0.89 ± 0.08) differed significantly from that in the IH group (0.78 ± 0.07) at 6 dpf. Group IH (0.84 ± 0.05), IHCA1 (0.90 ± 0.07), IHCA2 (0.87 ± 0.05), and IHCA3 (0.88 ± 0.07) differed significantly from group C (0.90 ± 0.07) at 9 dpf. There was no significant difference in body length between the IHCA1, IHCA2, and IHCA3 groups with C at 6 and 9 dpf (p > 0.05), but IHCA1 tended to have higher body length than the other CA extract treatment groups. The group of zebrafish embryos given CA showed faster body length gain than those not given CA in the IH animal model.

Effect of CA extract on the head length of zebrafish embryos with IH

The results of the mean head length of zebrafish larvae using the intermittent intrauterine hypoxia model and administration of CA ethanol extract at various concentrations at 3, 6, and 9 dpf (Fig. 2). The study results showed a significant difference in head length between group C and the IH-induced treatment group at 3, 6, and 9 dpf. There was a significant difference in head length (mm) between group C (0.09 ± 0.01) and group IH (0.07 ± 0.01), IHCA1 (0.07 ± 0.00), IHCA2 (0.07 ± 0.00), and IHCA3 (0.07 ± 0.00) at 3 dpf. In the IH-induced group, there was no significant difference between those given CA extract (IHCA) and those not given CA therapy at 3 dpf. Significant differences in CA concentration began to appear at 6 and 9 dpf. The two treatment groups, group C (0.12 ± 0.01) and IHCA1 (0.11 ± 0.01), did not show a significant difference in head length, but those groups were significantly different from the IH group (0.10 ± 0.01), IHCA2 (0.10 ± 0.02), and IHCA3 (0.11 ± 0.01) at 6 dpf. The C group (0.13 ± 0.01), IHCA1 (0.12 ± 0.01), and IHCA3 (0.12 ± 0.01) had significantly different head lengths than the IH (0.11 ± 0.01) and IHCA2 (0.11 ± 0.01) groups at 9 dpf. The IHCA1 group had a higher average head length than the IHCA2 and IHCA3 groups and was almost the same as the C group, while the IHCA2 group had a shorter head length trend than the other two treatment groups and was the same as the HI group at 6 and 9 dpf.

Fig. 1. The body length of embryonic zebrafish at 3, 6, and 9 dpf in the treatment groups. (a–g) Different notations show significant differences. Data are shown as the mean ± SD.

Molecular interaction prediction of asiatic acid with IGF-1R

Three-dimensional molecular docking prediction showed interaction binding between asiatic acid and the IGF-1R. The results showed interacting residues of the ligand and protein, mediated by five conventional hydrogen bonds and two hydrophobic bonds between amino residues Asn717, Asp328, Val326, Asn349, Val326, and Ala710, but interacting residues of the insulin and IGF-1R, mediated by all eight conventional hydrogen bonds at amino residues Arg351, Asp328, Asn349, and Val326 (Fig. 3). Those bonds interacted with van der Waals energy, thus resulting in an energy binding of asiatic acid to IGF-1R (191.2 kJ/mol) lower than insulin to IGF-1R (-7.2 kJ/mol) (Table 1).

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Discussion

Effect of IH in zebrafish embryos

Hypoxia in this animal model was applied as an intermittent schedule at 2, 24, 48, and 72 hpf. At 18 hpf, zebrafish embryos are very tolerant of anoxia, and the cardiovascular and nervous systems are just starting to form. At 24 hpf, the heart tube expands, and the heartbeat starts. At this age, anoxia tolerance decreases, and the embryo upregulates 148 genes in response to severe hypoxia. At 36 hpf, the cardiovascular system is well developed with circulating red blood cells, reduced anoxia tolerance, and embryos hatch into larvae up to 48 hpf (Robertson et al., 2014). In this study, the effect of IH was growth retardation in the body and head of zebrafish, but no inflammation was detected. IH, defined as recurrent hypoxia-reoxygenation cycles, induces inflammation cascades that impact multisystem morbidity, such as retinopathy of prematurity, growth retardation, defects in neuronal development, and the respiratory-circulation system). Many adverse effects of IH on children’s behavior, academic achievement, and cognition correlate with hypoxia duration, severity, and onset age. Cycles of hypoxia/reoxygenation frequently lead to a proinflammatory cascade with resulting multisystem morbidity, such as retinopathy prematurity, growth retardation, cardiorespiratory instability, and neurological defects (Serebrovskaya and Xi, 2015). In this study, zebrafish embryos were exposed to IH for 4 hours daily, although it could occur nocturnally. According to Fitzgerald et al. (2016), in a seasonal environment, during the warm season, hypoxia is related to the formation of a thermocline in surface waters, which results in excess oxygen consumption as organic matter decomposes in the lower water layers. Continuous exposure to hypoxia in the process of embryogenesis causes hypoxic events to persist in the long term.

Fig. 2. The head length of embryonic zebrafish at 3, 6, and 9 dpf in the treatment groups. The embryos in the control group were shown to have a significantly more extended head length than those in the other groups (p < 0.05). (a–e) Different notations show significant differences. Data are shown as the mean ± SD.

Fig. 3. (A–C) The molecular interaction prediction between asiatic acid from CA and the IGF-1R compared to the IGF-1-insulin complex (D–F).

Table 1. The binding interaction between asiatic acid and the IGF-1R.

Effect of CA extract on body length and head length of zebrafish embryos with IH

This study found an increase in zebrafish embryo body length and head length following the combination of IH and CA at 3, 6, and 9 dpf. IH causes a reduction in body length and head length. Abnormal embryogenesis was reported on oxygen deprivation of zebrafish models by activation of hypoxia-inducible factor proteins. Oxygen is central to fetal growth and development and adult physiological mechanisms. Therefore, low PO₂ concentration (hypoxia) affects growth, development, organogenesis, and resulting in growth restriction in human fetuses, even in other vertebrate species such as fish (Fathollahipour et al., 2018).

In this study, measurements of body length and head length after administration of CA extract began when zebrafish embryos were at 3 dpf. Zebrafish aged 3–48 hpf elicited greater morphological effects because of the high variability of hormone receptor expression that appeared earlier, which has the potential to bind maternally inherited hormones and allow them to have responsive abilities early in development. Differentiation of the first endocrine gland occurs at 3 dpf (Kinch et al., 2016). According to Lantz-McPeak et al. (2015), the drug can spread through the skin and gills of the embryo and enter the body orally when the zebrafish begins to ingest at approximately 72 hpf.

However, this study showed that the mean body length was longer in the IHCA1 group, but the head length was longer in the IHCA2 group than in the other IHCA groups at 6 and 9 dpf. Giving CA extract at 2.5–5 µg/ml is an effective dose to increase the growth of zebrafish embryos under conditions of IH. The results of this study are the same as those conducted by Wardani et al. (2018), which showed that CA extracts at a dose of 2.5 could inhibit stunting and improve body length by increasing the expression of vascular endothelial growth factor (VEGF) and VEGF receptor-2 in the stunting model of zebrafish larvae. The increase in body length due to CA treatment was demonstrated by a previous report using rotenone-induced zebrafish larvae by reducing malondialdehyde and elevating superoxide dismutase and catalase as endogenous enzymes. The reduction in oxidative stress was proposed as an underlying pathway following CA treatment in the rotenone-induced maldevelopment zebrafish model (Darwitri et al., 2018). Asiatic acid is an aglycone of asiaticoside, which is a biomarker component and is an indicator of the quality of CA plant extracts because of its high content compared to other components (Gray et al., 2018; Mioc et al., 2022). Treatment with an adequate dose of CA could also reduce reactive oxidative stress (ROS) generation via the enhanced activity of mitochondrial complex I, including mitochondrial DNA, to electron transport of adenosine triphosphate production, which can eventually lead to apoptosis (Lu et al., 2018).

Molecular interaction prediction of asiatic acid with IGF-1R

The results of validation of insulin as a native ligand with the IGF-1R show an RMSD value of ≤2 Å. RMSD shows how much the protein-ligand interaction changes during docking to determine the deviation value. The docking method is valid if the RMSD value ≤2 Å, so the docking method can be used for docking compounds (Utami et al., 2022). In this study, an in silico test was also carried out to predict the bond between asiatic acid and IGF-1R. The hydrogen bonding of acetic acid and insulin is maintained to bind with Asn349.

It was observed at a distance of over 2 A. The distance from hydrogen bonds and the free energy of both of them exceeded the simulation time, which is very important to explain the potential compound affinity (Frimayanti et al., 2021). The most important bioactive compounds of CA are triterpene glycosides (saponins), such as asiaticoside, madecassosisi, asiatic acid, and madecassic acid. Asiatic acid exhibits antioxidant, anti-inflammatory, and antiapoptotic effects by reducing ROS production and caspase activity. CA promotes vascular growth by elevating VEGF expression. Signaling via IGF-1R in normal cells leads to its activation of several intracellular pathways mediated by receptor domain-associated tyrosine kinase, phosphatidylinositol 3-kinase, and serine/threonine kinase (Akt), which plays a role in increasing growth and survival (Soni et al., 2015). In this study, hydrophobic and hydrogen bonds played important roles in the interaction between asiatic acid and IGF-1. This study is in line with that of Susanto et al. (2018), who found that theaflavin 3,3’-Togliatti has strong binding activity with IGF-1R signaling via hydrogen bonds as an anti-obesity agent by inhibiting cell proliferation (apoptosis agonist). The modulation of the IGF-1 pathway was proposed as a molecular mechanism for predicting pathways. Over five conventional hydrogen bonds, fulfilling one of the criteria of the rule-of-five in drug-likeliness scores of ligands, namely, having five or fewer hydrogen bond donor sites. This study showed different residues from research conducted by Soni et al. (2015) in that the Glu1080, Glu1027, Glu1025, Glu1145, Met1028, Arg1003, Arg1064, Arg1029, Lys1150, Leu1081, and Thr1028 residues were found to be significant binding site residues in the IGF-1R protein in various human malignancies.

In fish, the appearance of morpho anatomical deformities is not only influenced by oxygen tension conditions but also related to temperature (Mugwanya et al., 2022). In this study, IH zebrafish embryos that were given CA therapy also exhibited an increase in locomotor activity (zebrafish velocity) at the age of 6.9 dpf (unpublished data). The results of this study follow research conducted by Oliva et al. (2021), and there is a correlation between morphological changes and locomotor defects. Thus, acetic acid in CA may serve as an efficient support therapy of IH on IGF-1R targets. Asiatic acid-induced improvement in growth retardation in the concentration-dependent dose. Asiatic acid has a similar interaction with insulin on the active site of the IGF-1R against intermediate hypoxia mediated by the same conventional hydrogen bonds at amino residues Asp328, Asn349, and Val326, although the energy binding of asiatic acid to IGF-1R is lower than that of insulin. A more negative binding free energy means stronger binding (Frimayanti et al., 2021). This indicates that CA activates the insulin signaling pathway as an agonist interaction.

This study shows that changes in the head and body embryo length can go through alternative pathways, such as IGF-1R. This research is in line with Kinch et al. (2016), who stated that changes in head morphology, embryo length, pericardial morphology, or egg yolk morphology in zebrafish do not occur through activation of a single hormone receptor pathway but can occur through alternative pathways, such as estrogen-related receptors or IGF-1R hormone receptors. Growth, and glucocorticoid receptors, as components of the growth axis and endocrine stress expressed in the embryonic phase. Further studies need to research other roles of asiatic acid compounds in other receptor signaling molecules as therapeutic targets of IH and observe the criteria of the rule-of-five in the drug-likeliness scores of ligands so that they can be developed as natural drugs.

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Conclusion

The hypoxic state of pregnancy is highly implicated in fetal programming. Hypoxia induces oxidative stress and, thus, requires the administration of antioxidants. The administration of CA extract benefits IH through the development and growth of zebrafish embryos at a dose of 2.5–5 µg/ml. There is a binding affinity of asiatic acid to the IGF-1R that plays a role in the IH animal model. Further studies need to research other roles of asiatic acid compounds in other receptor signaling molecules as therapeutic targets of IH and observe the criteria of the rule-of-five in the drug-likeliness scores of ligands so that they can be developed as natural drugs.

Acknowledgments

The Department of Pharmacology, Medical Faculty, Universitas Brawijaya supported this study.

Conflicts of interest

There are no conflicts of interest among the authors in this research and publication.

Funding

This research was supported by the Ministry of Research and Technology of the Republic of Indonesia with grant Number 033/E5/PG.02.00/2022. The sponsor had no such involvement.

Author contributions

AR, HK, NU, MA: concept and design of the research; AR, IF: acquisition of data, analysis, and interpretation; critical revision and final approval: AR, HK, NU, MA.

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References

Acharjee, S., Upadhyay, A., Roy, A. and Ghosh, R. 2017. Analysis of insulin-like growth factor I and its receptor of an Indian Major Carp Labeo rohita: an in silico approach. MOJ Proteomics Bioinf. 6(3), 287–293.

Ariani, A. and Widiyastuti, T. 2022, The effect of Centella asiatica extract in zebrafish (Danio rerio) larvae as a chronic constant hypoxia (CCH) model. Pediatr. Sci. J. 1, 20–26.

Collery, R., Veth, K., Dubis, A., Carroll, J. and Link, B. 2014. Rapid, accurate, and noninvasive measurement of zebrafish axial length and other eye dimensions using SD-OCT allows longitudinal analysis of myopia and emmetropization. PLoS One 9(10), e0119779.

Cory’ah, F.A.N., Primaditya, V., Ariati, L.I.P., Zakiah, Wardani, D.W.K.K., Primihastuti, D., Yuningsih, Khotimah, H., Ali, M.M. and Nurdiana, 2020. Ethanolic extract of Centella asiatica increased IGF-1 and IRS expression in zebrafish (Danio rerio) rotenone-induced. AIP Conf. Proc. 2231(1), 040036.

Darwitri, Y.T., Nuraenah E., Zahara E., Khotimah H., Kalsum U.N. and Ali, M.M. 2018. Centella asiatica increased the body length through the modulation of antioxidant in rotenone-induced zebrafish larvae. Biomed. Pharmacol. J. 11, 827–833.

Fajersztajn, L. and Veras, M.M. 2017. Hypoxia: from placental development to fetal programming. Birth Defects Res. 109, 1377–1385.

Fathollahipour, S., Patil, P.S. and Leipzig, N.D. 2018. Oxygen regulation in development: lessons from embryogenesis towards tissue engineering. Cells Tissues Organs 205(5–6), 350–371.

Fitzgerald, J., Jameson, H., Dewar, Fowler, V., Bond, G., Bickley, L., Webster, T.M., Bury, N., Wilson, R. and Santos, E. 2016. Hypoxia suppressed copper toxicity during early development in zebrafish embryos in a process mediated by the activation of the HIF signaling pathway. Environ. Sci. Technol. 50, 4502–4512.

Frimayanti, N., Aryani, F., Rishanti, N. and Yaeghoobi, M. 2021. In silico analysis towards exploring potential β secretase 1 (BACE1) inhibitors; the cause of Alzhemier disease. J. Phys. Conf. Ser. 2049, 012011.

Fowden, A., Giussani, D. and Forhead, A. 2006. Intrauterine programming of physiological systems: causes and consequences. Physiology 21, 29–37.

Gray, N.E., Magana, A.A., Lak, P., Wright, K.M., Quinn, J., Stevens, J.F., Maier, C.S. and Soumyanath, A. 2018. Centella asiatica—phytochemistry and mechanisms of neuroprotection and cognitive enhancement. Physiol. Behav. 17(80), 678–687.

He, Z., Hu, Y., Niu, Z., Zhong, K., Liu, T., Yang, M., Ji, L. and Hu, W. 2023. A review of pharmacokinetic and pharmacological properties of asiaticoside, a major active constituent of Centella asiatica (L.) Urb. J. Ethnopharmacol. 302, 115865.

Hellström, A., Ley, D., Hansen-Pupp I., Hallberg, B., Ramenghi, L., Löfqvist, C., Smith, L.E. and Hård, A. 2016. Role of insulinlike growth factor 1 in fetal development and in the early postnatal life of premature infants. Am. J. Perinatol. 33, 1067–1071.

Jang, E., Longo, L. and Goyal, R. 2015. Antenatal maternal hypoxia: criterion for fetal growth restriction in rodents. Front. Physiol. 6, 1–6.

Kamei, H. and Duan, C. 2018. Hypoxic treatment of zebrafish embryos and larvae. Methods Mol. Biol. 1742, 195–203.

Kinch, C., Kurrasch, D. and Habibi, H. 2016. Adverse morphological development in embryonic zebrafish exposed to environmental concentrations of contaminants individually and in mixture. Aquat. Toxicol. 175, 286–298.

Kukula, O., Kırmızıkan, S., Tiryaki, E.S., Cicekli, M.N. and Günaydın, C. 2022. Asiatic acid exerts an anti-psoriatic effect in the imiquimod-induced psoriasis model in mice. Immunopharmacol. Immunotoxicol. 44(3), 367–372.

Kwon, E. and Kim, Y.J. 2017. What is fetal programming?: a lifetime health is under the control of in utero health. Obstet. Gynecol. Sci. 60, 506–519.

Lantz-Mcpeak, S., Guo, X., Cuevas, E., Dumas, M., Newport, G., Ali, S., Paule, M. and Kanungo, J. 2015. Developmental toxicity assay using high content screening of zebrafish embryos. J. Appl. Toxicol. 35, 261–272.

Lu, Y., Kan, H., Wang, Y., Wang, D., Wang, X., Gao, J. and Zhu, L. 2018, Asiatic acid ameliorates hepatic ischemia/reperfusion injury in rats via mitochondria-targeted protective mechanism. Toxicol. Appl. Pharmacol. 338, 214–223.

Malhotra, A., Allison B., Castillo-Melendez, M., Jenkin, G., Polglase, G. and Miller S.L. 2019. Neonatal morbidities of fetal growth restriction: pathophysiology and impact. Front. Endocrinol. (Lausanne). 10, 1–18.

Merino, J., Roncero, C., Oset-Gasque, M., Naddaf, A. and González, M. 2014. Antioxidant and protective mechanisms against hypoxia and hypoglycaemia in cortical neurons in vitro. Int. J. Mol. Sci. 15, 2475–2493.

Mikloska, K., Zrini, Z. and Bernier, N. 2022, Severe hypoxia exposure inhibits larval brain development but does not affect the capacity to mount a cortisol stress response in zebrafish. J. Exp. Biol. 225(2), 243335.

Mioc, M., Milan, A., Malit, D., Mioc, A., Prodea, A., Racoviceanu, R., Ghiulai, R., Cristea, A., Căruntu, F. and Soica, C. 2022. Recent advances regarding the molecular mechanisms of triterpenic acids: a review (Part I). Int. J. Mol. Sci. 23, 7740.

Mishra, A., Singh, D.Y., Singh, R., Kumar, R., Shukla, S., Prajapati, R.K., Aditi, Rawat, N., Kumar, A. and Pol, S.L. 2022. Ethano-pharmacology activity and Antioxidant activity of Centella asiatica plant parts. Neuroquantology 20, 1.

Mugwanya, M., Dawood, M.A.O., Kimera, F. and Sewilam, H. 2022. Anthropogenic temperature fluctuations and their effect on aquaculture: a comprehensive review. Fish. Aquac. J. 7, 223–243.

Oliva, C., Hinz, N.K., Robinson, W., Thompson, A.M.B., Booth, J., Crisostomo, L.M., Zanineli, S., Maureen, T., Lloyd, E., O’Gorman, M., McDole, B., Paz, A., Kozo, R., Brown, E.B., Kowalko, J.E., Fily, Y., Duboue, E.R. and Keene, A.C. 2021. Characterizing the genetic basis of trait evolution in the Mexican cavefish. Evol. Dev. 24, 131–144.

Perrone, S., Santacroce, A., Picardi, A. and Buonocore, G. 2016. Fetal programming and early identification of newborns at high risk of free radical-mediated diseases. World J. Clin. Pediatr. 5, 172.

Robertson, C., Wright, P., Köblitz, L. and Bernier, N. 2014. Hypoxia-inducible factor-1 mediates adaptive developmental plasticity of hypoxia tolerance in zebrafish, Danio rerio. Proc. R. Soc. B Biol. Sci. 281, 20140637.

Rodríguez-Rodríguez, P., Ramiro-Cortijo, D., Reyes-Hernández, C., López de Pablo, A., Carmen González, M. and Arribas, S. 2018, Implication of oxidative stress in fetal programming of cardiovascular disease. Front. Physiol. 9, 1–13.

Serebrovskaya, T. and Xi, L. 2015. Intermittent hypoxia in childhood: the harmful consequences versus potential benefits of therapeutic uses. Front. Pediatr. 3, 1–10.

Soni, N., Pardasani, K. and Mujwar, S. 2015, Insilico analysis of dietary agents as anticancer inhibitors of insulin like growth factor 1 receptor (IGF1R). Int. J. Pharm. Pharm. Sci. 7, 191–196.

Susanto, H., Kharisma, V.D., Listyorini, D., Taufiq, A. and Sunaryono, A. 2018. Effectivity of black tea polyphenol in adipogenesis related IGF-1 and its receptor pathway through in silico based study. IOP Conf. Series: J. Phys. Conf. Series. 1093, 012037.

Tripathy, S. and Srivastav, P.P. 2023. Encapsulation of Centella asiatica leaf extract in liposome: study on structural stability, degradation kinetics and fate of bioactive compounds during storage. Food Chem. Adv. 2, 100202.

Utami, J.P., Kurnianingsih, N. and Faisal, M.R. 2022. An in silico study of the Cathepsin L inhibitory activity of bioactive compounds in Stachytarpheta jamaicensis as a COVID-19 drug therapy. Makara J. Sci. 26(1), 25–36.

Wang, Y., Wang, H., Zhao, P., Cheng, J., Gong, W. and Zhang, J. 2021, Asiatic acid exerts neuroprotective effect against hypoxicischemic brain injury in neonatal rats via inhibition of oxidative damage. Trop. J. Pharm. Res. 20, 1903–1908.

Wardani. D.W.K.K., Ali, M., Khotimah, H., Nurdiana, Riawan, W., Primihastuti, D., Cory’ah, F.A.N., Puspita, L.I., Primaditya, V. and Yuningsih, Z. 2018. The effect of Centella asiatica to the vascular endothelial growth factor and vascular endothelial growth factor receptor-2 on the rotenone induced zebrafish larvae (Danio rerio) stunting model. GSC Biol. Pharm. Sci. 5, 88–95.

Zhang, X., Yu, D., Sun, J., Wu, Y., Gong, J., Li, X., Liu, L., Liu, S., Liu, J., Wu, Y., Li, D., Ma. Y., Han, X., Zhu, Y., Wu, Z., Wang, Y., Ouyang, Q. and Wang, T. 2020. Visualization of ligand-bound ectodomain assembly in the full-length human IGF-1 receptor by Cryo-EM single-particle analysis. Structure 28, 555–561.e4.