1

Centella asiatica extract modified growth development on intermittent hypoxia

embryos model and molecular prediction pathway on Insulin-like Growth Factor-1

Receptor Signaling

Ariani Ariani1,2, Husnul Khotimah3, Nurdiana3, Masruroh Rahayu4

1
Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya,
Malang, Indonesia
2
Department of Pediatric, Faculty of Medicine, Universitas Brawijaya, Malang,
Indonesia
3
Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang,
Indonesia
4
Department of Neurology, Faculty of Medicine, Universitas Brawijaya, Malang,
Indonesia

Corresponding author: Ariani,MD., Doctoral Program of Medical Science, Faculty of

Medicine, Universitas Brawijaya, Malang, Indonesia. Address: Jl. Veteran, Malang,

East Java, Indonesia. Phone: +62341343343, E-mail address: arianidr@ub.ac.id.

ORCID ID: https://orcid.org/0000-0002-3653-0834.

Abstract

Introduction: Hypoxia during pregnancy generates oxidative stress that alters the

growth and development of the human fetus. Centella asiatica (CA) has powerful

antioxidant properties that have the potential to be developed to prevent growth

impairment in the hypoxia condition.

Aim: The aim of this study was to investigate the effect of CA extract (CAE) on the

developments of intermittent hypoxia zebrafish embryos model and molecular docking

prediction in insulin-like growth factor 1 (IGF-1) receptor signaling.

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Methods: Embryo of zebrafish at 2-hour post- fertilization (hpf) were assigned to

control, intermittent hypoxia (IH), and IH with CAE 1.25, 2.5, and 5 µg/ml respectively.

Hypoxia treatment was conducted 4 hour/day for 3 days, while CAE was administered

for 3 days (2 - 72 hpf). The body and head length were evaluated at 3, 6, and 9 days post

fertilization (dpf). Molecular docking was performed to explore the binding affinity of

Asiatic acid to IGF-1 by Molegro Virtual Docker ver.5 software. The data was analyzed

by two-way ANOVA. 

Results: The body length and head length of larvae in IH and treatment groups were

shorter than the control group at 3 dpf (p<0,001). However, the lengths tended to be

longer than IH groups at 6 and 9 dpf (p>0,05). Molecular docking showed the agonist

effect of Asiatic acid on IGF-1 receptor signaling.

Conclusions: The administration of CAE provides benefits to zebrafish embryo

development through IGF-1 receptor activation.

 Keywords: Centella asiatica, zebrafish embryo, body length, IGF-1

1. BACKGROUND

Antenatal hypoxic stressor adversely impacts fetal programming and contributes to

growth restriction of the human fetus to cause growth retardation in later life (1,2,3).

Hypoxia during pregnancy can develop from several maternal conditions such as

maternal smoking, anemia, placental insufficiency, hypertension, asthma, and

respiratory tract infection (4). Low oxygen pressure induce stress oxidative thus the

administration of antioxidant provides benefit outcomes (5)

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Centella asiatica (CA) was previously reported to have antioxidant properties and

protects embryos from oxidative stress in rotenone-induced zebrafish embryos (6,7).

Asiatic acid is one of the potential antioxidants that may have activity on insulin-like

growth factor-1 (IGF-1). Zebrafish embryos are proposed as a commonsense model for

pediatric research according to their superiority aspects compared to other animals (8).

The effect of CA in the zebrafish embryo model of hypoxia is still limited as well as the

predicted pathway of CA in .  

2. AIM

This study aimed to investigate the effect of administration Centella asiatica extracts on

the body and head length of larva zebrafish model of intermittent hypoxia as well as the

molecular interaction mechanism prediction using two- and three-dimensional approach

on insulin-like growth factor 1 (IGF-1) receptor

3. MATERIAL AND METHODS

Materials

Adult wildtype of zebrafish was obtained from the Faculty of Fisheries and Marine

Science, Universitas Brawijaya. The simplicia of Centella asiatica was gained from

Materia Medica, Malang, East Java. All reagents and chemicals were analytical grades

from E-Merck.

Subject

Adult zebrafish from both sexes were kept in a 60- liter glass aquarium with a female

and male ratio of 2:1. The temperature was set at 28±1°C and 14:10 hours of light/dark

cycle period. Food was provided twice a day with TetraMin (TetraMin Tropical Fish
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Flakes, USA). The collection of embryos was carried out following the beginning of the

light period (9).

Experimental design

This research was a true experimental study with a post-test-only control group design

approach. A total of 20 embryos at 2 hours post- fertilization (hpf) were involved in

each group of experiments. The experiments consisted of a control group, intermittent

hypoxia (IH), IH + extract of Centella asiatica (CA) at 1.25, 2.5, and 5 µg/ml,

respectively. All treatment was conducted from 2 hpf to 72 hpf (10). Afterward,

treatments ceased then subsequently embryo monitoring was performed in intermittent

schedules (Figure 1).

Figure 1. Timeline of the measurement

Intermittent hypoxia

Hypoxia was performed by exposing the embryos to a water chamber that supplied

nitrogen gas to reach oxygen pressure PO2~5 kPa, 2 mg O2/L. Hypoxia was applied as

an intermittent schedule at 2, 24, 48, and 72 hpf for 4 hours duration of exposure (at

08.00-12.00 am) followed by normoxia for the rest through medium substitution. The

level of oxygen concentration was tightly monitored by a dissolved oxygen meter (11).

Centella asiatica extraction, preparation, and treatment

Simplicia of 100 g Centella asiatica has macerated in 98% ethanol 900 ml for 24 hours

followed by filtration. This procedure was replicated 3 times then the filtrate was

collected for further rotary evaporation. The extraction was crude extract within pasta

then diluted in normal saline and stored at 0͐°C. The extract was dissolved in an embryo
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medium that contained a mixture of embryonic medium and CAE in 3 different

concentrations, 1.25, 2.5, and 5 µg/ml (10). The medium contained extract solution was

administered from 2 hpf until 72 hpf simultaneously with intermittent hypoxia in

treatment groups.

Embryo monitoring

Zebrafish embryos at age of 2 hpf were placed in a 28°C incubator. After hatching,

larvae were kept in at embryonic medium at room temperature. Embryos were divided

into 5 groups (control group, IH group, IH + extract of Centella asiatica (CA) at 1.25,

2.5, and 5 µg/ml) and monitored for body and head length measurements at 3, 6 and 9

days post fertilization (dpf).

Body and head length measurement

After hatching, larvae were kept an embryonic medium and monitored for body and

head length measurements at 3, 6, and 9 days post fertilization (dpf). Body length was

measured from the tip of the snout to the caudal fin. The head length was scaled from

snout to operculum. Measurement of body length and head length was carried out using

Image Raster software. Each larva in each group was observed under a stereo

microscope (Olympus SZ61) which was connected into an Optilab camera and Optilab

software 2.0 version.

Molecular docking analysis

The protein sequence of insulin-like growth factor 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 Pubchem NCBI (CID 119034). Software

of Molegro Virtual Docker ver.5 was used to predict the active site of the IGF-1
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receptor with 5 van der Waals parameters. Molecular docking was carried out Molegro

Virtual Docker ver.5 with Grid X96.16; Y107.61; Z157.85; radius 11. Docking

validation was set as RMSD < 2,0 and insulin as a native ligand (12).

4. RESULTS

Effect Centella asiatica extract on body and head length of zebrafish larvae with

intermittent hypoxia.

The body length of larvae among intermittent hypoxia (IH) and treatment groups was

significantly shorter than the control group at 3 days post fertilization (dpf). However, at

the same point of age, the body length in the group of IH group is similar to Centella

asiatica (CA) extracts treated groups (Figure 2). Further, larvae at 6 and 9 dpf

demonstrate longer body length than 3 dpf larvae. Despite the body length was not

significantly different between groups at 6 and 9 dpf, the body length of CA-treated

larvae tends to be longer than the IH group. The head length of larvae among groups is

displayed in Figure 3. Similarly, the head length in treatment groups at 3 dpf is shorter

than in control. The application of CA extracts tend to enhance the head length

particularly at 1.25 µg/ml and 5 µg/ml at the age of 3, 6, 9 dpf. The head length of the

IH group had a similar length to the 2.5 µg/ml CA group at the age of 3, 6, and 9 dpf.

Figure 2.  The body length of larvae in 3, 6, and 9 dpf among different treatment
applications. The larvae in the control group are shown longer than in other groups. At 3
dpf point time, the group of IH and combination of IH+CA were significantly lower
than the control (p<0,0001). The larvae in IH+CA groups tend to longer than the IH
group at 6 and 9 dpf (p>0.05). Data is shown as mean ± SD.
  
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Figure 3.  The head length of larvae in 3, 6, and 9 dpf among different treatment
applications. The larvae head in the control group are shown longer than in other
groups. At 3 dpf point time, the group of IH and combination of IH+CA were
significantly lower than the control (p=0,0005). The larvae in IH+CA groups tend to be
longer than the IH group at 6 and 9 dpf (p>0.05). Data is shown as mean ± SD.
 

Molecular interaction prediction between Asiatic acid from Centella asiatica and

Insulin-like Growth Factor-1 (IGF) receptor.

Three dimensional molecular docking prediction was shown interaction binding

between Asiatic acid with IGF-1 receptor. The interaction was facilitated by 5

conventional hydrogen bonds and 2 hydrophobic bonds at amino residues of ASN717,

ASP328, VAL326, ASN349, VAL326, and ALA710 (Figure 4). Those bonds interacted

with van der Waals energy thus resulting in energy binding of -191,2 kJ/mol (Table 1).

 Figure 4. Molecular interaction prediction between Asiatic acid from Centella

asiatica and IGF receptor. (A-B) Three-dimensional interaction showed ligand
interaction at 7 amino residues. (C) Two-dimensional interaction revealed the hydrogen
and hydrophobic bond as well as van der Waals energy.
 

Table 1. The binding interaction between Asiatic acid and IGF-1 receptor

5. DISCUSSION

This study found an increase in body length and head length of zebrafish larvae

following the combination of IH and CA at the age of 3, 6, and 9 dpf. The increase of

body length in the CA group compared with the IH group was close to the control group

at 6 and 9 dpf (Figure 2). Intermittent hypoxia has been shown to cause a reduction in

body length especially in the first year of life and administering CA with 3 different

concentrations can increase body length compared to the treatment group. The increase

in body length was demonstrated by a previous report using rotenone-induced zebrafish

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larvae by reducing MDA oxidant as well as elevation of SOD and catalase enzymes.

The reduction of oxidative stress was proposed as an underlying pathway following CA

treatment in the rotenone-induced maldevelopment zebrafish model (10). Abnormal

embryogenesis was reported on oxygen deprivation of zebrafish models by activation of

hypoxia-inducible factor proteins (13). Oxygen has a central role in fetal growth and

development as well as adult physiology mechanisms (14). Therefore, low oxygen

pressure (hypoxia) has consequences on growth, development, organogenesis, and

resulting growth restriction in human fetuses even in other vertebrate species such as

fish (14,15). Intermittent hypoxia as defined as recurrent hypoxia-reoxygenation cycles

induces inflammation cascades that impact multisystem morbidity for instance

retinopathy of prematurity, growth retardation, defect in neuronal development, and

respiratory-circulation system. Previous studies are reported many adverse effects of

intermittent hypoxia on behavior, academic achievement, and cognition in children.

These effects correspond to duration, severity, and onset age of hypoxia (16–18).   

The significantly lower head length at the age of 3 dpf in zebrafish which is equivalent

to 6 months of age in humans indicates that intermittent hypoxia conditions greatly

affect organ growth, especially early in human life, whereas in larvae aged 6 and 9 dpf

there was an insignificant difference in terms of head length. This indicates that very

rapid growth of the human brain occurs in the first months of life so that if there is a

disturbance in that period it will greatly affect brain growth, while at the age of 6 dpf

which is equivalent to the age of 2 years in humans, brain growth is not too fast,

indicated by a decrease in head length that was not significant compared to the control

with the administration of Centella asiatica (17,18).

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The benefits of Centella asiatica on the growth of the zebrafish model of growth

retardation were facilitated by the improvement of insulin-like growth factor-1 (IGF-1)

protein (7). Centella asiatica promotes vascular growth through the elevation of

vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2)

expression (6). The modulation of the IGF-1 pathway was proposed as a molecular

mechanism prediction pathway. Asiatic acid as a major metabolite active from CA has a

similar interaction with insulin on the active site of the IGF-1 receptor (12,19). This

indicates that CA activates the insulin signaling pathway as an agonist interaction.

Further studies on IGF-1 receptor signaling molecular parameters are necessary to

develop molecular therapy targets of CA in future drug developments.

CONCLUSION

The administration of CAE provides benefits effect on zebrafish embryo development

that is predicted through IGF-1 receptor activation. 

Acknowledgments:

Author’s contribution: AA, IFG, HK, and MR gave substantial contributions to the

concept and design of the study as well as data acquisition via the experimental

laboratory works, analysis, and interpretation. AA, HK, ND, and MR contributed to the

initial draft, revisions, and proofreading of the article. All authors have approved the

final version of the manuscript.

Conflicts of interest: There are no conflicts of interest.

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Conflicts of interest: There are no conflicts of interest.

Financial support and sponsorship: Nil.

Acknowledgement: This study was supported by the Department of Pharmacology,

Medical Faculty, Universitas Brawijaya

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