Exploring the Bioactive Metabolites with Anti-Malarial Properties

Derived from Endophytic Microbial Resources Indigenous to

Indonesia

Elsera Br Tarigan

, Partomuan Simanjuntak

, Dwinna Rahmi

, Praptiwi

Erwahyuni Endang Prabandari

, Danang Waluyo

, Muhammad Ilyas

and Andria Agusta

Research Center for Agroindustry, National Research and Innovation Agency Indonesia, Indonesia

Research Center for Pharmaceutical Ingredient and Traditional Medicine,

National Research and Innovation Agency Indonesia, Indonesia

Research Center for Vaccine and Drug, National Research and Innovation Agency Indonesia, Indonesia

Research Center for Biosystematics and Evolution, National Research and Innovation Agency Indonesia, Indonesia

Keywords: Fusarium sp, Hypomycetes, High-Throughput Screening, PfDHODH, Medicinal Plants.

Abstract: Malaria, caused by parasite infection, is still difficult to cure due to partial treatment resistance. This

emphasizes the need for innovative molecules with different modes of action, particularly those derived from

unconventional sources such as endophytic microbes. This study investigates endophytes from medicinal

plants in order to find possible antimalarial drugs that target Plasmodium falciparum dihydroorotate

dehydrogenase (PfDHODH). Endophytes were associated to Mitragyna speciosa, Zingiberaceae, Uncaria

gambir, and others, yielding 586 species. Screening using a 96-well plate produced a z-factor of 0.92,

indicating trustworthy findings. Hypomycetes (from Physalis angulata L.) and Fusarium sp. (from Hornstedtia

scyphifera) showed considerable anti-malarial activity, suppressing PfDHODH by 51.53% and 66.37%,

respectively. Metabolomic profiling using LC-HRMS demonstrated that Hypomycetes included bioactive

substances such as uracil, kojic acid, and phloroglucinol, whereas Fusarium sp.

1 INTRODUCTION

Anopheles mosquitoes carrying the Plasmodium

infection bite humans, transmitting the infectious

disease malaria. The prevalence of malaria in the

world in 2019 was 227 million, and it increased to 241

million people in 2020. Indonesia itself experienced

an increase; in 2013, there were 0.4% of cases, and

this increased in 2018 to 1.4% (Kemenkes 2018).

There are five Plasmodium species that cause malaria

in humans, but Plasmodium falciparum is the most

potent and deadly.

The most effective treatment for malaria infection

is artemisinin-based combination therapy, which has

https://orcid.org/0009-0009-8361-1651

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https://orcid.org/0000-0003-1280-5984

https://orcid.org/0000-0002-9226-6265

no side effects. However, over time, Plasmodium

showed indications of partial resistance to artemisinin

derivatives. Antimalarial drugs can inhibit key

enzymes responsible for cell growth and parasite

development. A group of genes known as P.

falciparum lactate dehydrogenase (PfLDH), P.

falciparum dihydroorotate dehydrogenase

(PfDHODH), and P. falciparum dihydrofolate

reductase (PfDHFR) play a big role in the growth of

plasmodium parasites. PfDHODH is an important

component of parasite metabolism and plays a key

role in the de novo pathway of pyrimidine

biosynthesis. This enzyme facilitates the conversion

of dihydroorotate (DHO) into orotate. This compound

Tarigan, E. B., Simanjuntak, P., Rahmi, D., Agusta, A., Praptiwi, , Prabandari, E. E., Waluyo, D. and Ilyas, M.

Exploring the Bioactive Metabolites with Anti-Malarial Properties Derived from Endophytic Microbial Resources Indigenous to Indonesia.

DOI: 10.5220/0013554600004612

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of BRIN’s 2nd International Conference for Health Research (ICHR 2024), pages 47-53

ISBN: 978-989-758-755-9

is essential for generating the pyrimidine bases

required for DNA and RNA synthesis (Akinnusi et al.

2023). However, it is essential to emphasize that

targeted therapy must include safety and specificity,

as these treatments must suppress the parasite while

inflicting minimal harmful effects on the human

body.

Developing antimalarial drugs targeting

PfDHODH is a growing area of research to overcome

malaria drug resistance and improve malaria

treatment. The development of new malaria drugs

begins with the exploration of bioactive plant

metabolites and even plant microorganisms, one of

which is endophyte. Endophytic fungi are

microorganisms that grow in host tissue and produce

bioactive compounds similar to those of their host

without causing disease symptoms. Secondary

metabolites produced by endophytic fungi have many

activities, such as antioxidants and anti-bacterial.

Research on endophytic fungi as a source of anti-

malarial bioactive metabolites is still limited, whereas

Indonesia is rich in germplasm sources.

Research related to the potential of endophyte

bioactive metabolites as antimalarials was carried out

by Ibrahim et al. (2017). The bioactive compound

fusaripeptide found in Fusarium sp., which was

isolated from the roots of the Mentha longifolia L.

plant, potentially kill malaria parasites (Ibrahim et al.,

2017). Fusarium endophytes from Cinchona calisaya

trees could also stop Plasmodium berghei from

growing by 62.18% to 78.41% (Hasbi, 2019).

The objective of this research was to: (1)

Screening of extracts was carried out on 587

endophytic fungi that had PfDHODH inhibitory

activity; (2) up-scaling cultivation and extraction of

Fusarium sp. endophytes. which is associated with

Phyisalus angulata and Hornstedtia scyphifera var.

fusiform, which has a percentage of >50% inhibition

of the PfDHODH enzyme; (3) identified the profile

compounds of extract by using HRMS.

2 METHODS

2.1 Materials

The sample population consisted of 586 endophytes.

Those endophytes was associated with the Mitragyna

speciosa (36); Soultalum album L (15); Phyllanthus

urinaria (4); Myristica fragrans (41); Andrographis

paniculata (18); Piper sarmentosum (36); Physalis

angulata L. (15) ; Chilorantus officinalis (5);

Staurogyne longata (5); Coleus amboinicus (19);

Nigelia sativa (22); Ziziphus mauritiana (50);

Artemisia annua (8); Artemisia vulgaris (13);

Uncaria Gambier (74); Zingiberaceae (95); Piper

nigrum (21); Kaempferia parviflora (26); Murraya

koenigii (4); medicinal plants from Mandalika

Lombok (35); and medicinal plants from Bali (44).

Enzyme inhibition testing was: DMSO, HEPES,

milli-Q water, KOH, NaCl, Triton-X 100, decyl

ubiquinone (d-UQ), absolute ethanol, aluminium foil,

parafilm, L-dihydro orotate (L-DHO),

dichloroindophenol (DCIP), recombinant enzyme

PfDHODH.

2.2 Endophytic Bioproduction and

Extraction

The endophytic fungi were cultivated in potatoes

dextrose broth (PDB) medium. Cultivation was

carried out in large-scale media, 7–10 L, at room

temperature for several weeks until bioactive

metabolites were formed. The process of cultivating

endophytic fungal isolates into PDB media was

carried out sterilely in laminar air flow (LAF). The

extraction was carried out by liquid-liquid extraction

technique using ethyl acetate solvent. The extracted

material was evaporated using a rotary evaporator,

followed by a subsequent drying process with

nitrogen gas. The dried extract was stored at a

freezing temperature for further processing.

2.3 PfDHODH Inhibition Screening

Assay

Measurement refers to the method developed by

Pramisandi et al. (2021), with some modifications.

Screening of extracts/isolates was carried out on 586

endophytic fungi that had PfDHODH inhibitory

activity. The inhibitory activity of key target cells was

assessed using the recombinant enzyme

dihydroorotate dehydrogenase (PfDHODH) derived

from P. falciparum. The inhibition experiments of

PfDHODH were conducted by quantifying the

decrease in the reduction of the electron acceptor 2,6-

dichloroindophenol (DCIP). The 96-well plate was

filled with 2 µL of extract and 190 µL of test solution.

Centrifugation was used to mix the the solutions for

30 seconds at 500 rpm. L-dihydroorotate (L-DHO) 5

mM were added to each well to start the enzymatic

reaction. Inhibitor activity was determined by

spectrophotometer at 600 nm. Inhibitory activity was

calculated based on equation (1).

% Inhibition = 100 −

[



.. 

. 



x 100%]

(1)

ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)

The endophyte fungi that had an inhibition percentage

above 50% were selected for bioproduction.

2.4 Compounds Characteristic Using

LC-HRMS

Dried extract (1 mg) was soluble using 1 mL of MS

grade MeOH. Sample analysis was performed

according to Windarsih et al. (2022) with some

modifications. The analysis was conducted using

liquid chromatography with the Thermo Scientific™

Vanquish™ UHPLC Binary Pump, along with

Orbitrap high-resolution mass spectrometry using the

ThermoScientific™ Q Exactive™ Hybrid

Quadrupole-Orbitrap™ High Resolution Mass

Spectrometer. Analytical column of Thermo

Scientific™ Accucore™ Phenyl-Hexyl 100 mm × 2.1

mm ID × 2.6 µm was used for liquid chromatography.

Using a gradient method and a flow rate of 0.3

mL/min, the mobile phases were MS-grade water

containing 0.1% formic acid (A) and MS-grade

methanol containing 0.1% formic acid (B). Initially,

the mobile phase B was set at 5% and then gradually

increased to 90% over a period of 16 minutes. After

that, it remained at 90% for 4 minutes and then

returned to the initial condition (5% B) until 25

minutes. The temperature of the column was adjusted

to 40 ◦C, while the injection volume was set at 3 µL.

An untargeted screening was conducted using full

MS/dd-MS2 acquisition mode in either positive or

negative ionization polarities/states. The spray

voltage was set to 3.30 kV, while the capillary

temperature and the auxiliary gas heater temperature

were set at 320 ◦C and 30 ◦C, respectively. The scan

range was conducted from 66.7 to 1000 m/z, with a

resolution of 70,000 for full MS and 17,500 for dd-

MS2, in both positive and negative ionisation modes.

3 RESULT AND DISCUSSION

3.1 Enzymatic Screening of Endophyte

Extract as an Anti-Malarial

Endophytic fungi are a type of fungus that live in

plant tissue without causing visible damage or disease

in their hosts (Ababutain et al. 2021). The metabolites

produced are generally similar as those of the host

plant. Metabolites generated by endophytes exhibit

numerous functions, including antimalarial activity.

Malarial assay can be carried out enzymatically,

parasitically and computationally. PfDHODH, P.

falciparum Dihydroorotate Dehydrogenase, plays a

vital role as an enzyme in the pyrimidine biosynthesis

pathway of the malaria parasite P. falciparum.

Primary screening was conducted on 586

endophyte extracts derived from a variety of plant

categories and parts, such as leaves, stems, roots, and

seeds. Those extracts were plated on a 96-well plate

for the PfDHODH assay, Fig.1. There are numerous

critical stages that comprise its mechanism. The

enzyme attaches to its substrate, dihydroorotate, at its

active site and facilitates the chemical reaction that

converts dihydroorotate into orotate. This procedure

entails the extraction of two electrons and two protons

from dihydroorotate. PfDHODH utilizes the

coenzyme FMN (Flavin Mononucleotide) as an

electron acceptor, facilitating the conversion of FMN

from its oxidized state (blue form) to its reduced state

(red color), Fig.2. FMN transports electrons to the

electron transport chain located in the mitochondrial

membrane of P. falciparum. This process produces

vital energy for the parasite's survival and growth.

The end product of the reaction, orotate, is liberated

from the active site of the enzyme, so concluding the

conversion process. PfDHODH plays a crucial role in

the formation of pyrimidine, making it an important

target for the development of antimalarial drugs.

Inhibiting this enzyme can effectively hinder the

growth and reproduction of P. falciparum (A. Phillips

dan K. Rathod 2012). PfDHODH facilitates an

enzymatic process that is linked to the reduction of

DCIP. PfDHODH transfers electrons to DCIP,

converting it from its oxidized blue form (DCIPox) to

its reduced colorless form (DCIPred), as it converts

dihydroorotate to orotate and reduces

decylubiquinone to decylubiquinol, Fig. 2.

During this study, extracts that demonstrated

inhibitory efficacy exceeding 50% were designated as

primary findings. There were two extracts identified

as the main hits (blue color), particulary endophytes

associated with Physalis angulata L (51,53%),

Hornstedtia scyphifera (66,37%). The two

endophytes were fungi belonging to the Hypomycetes

type, which was associated with Physalis angulata L,

and the Fusarium sp., which was associated with

Hornstedtia scyphifera. The batch codes for these

fungi were BgPa1 and HSFP3, respectively.

Exploring the Bioactive Metabolites with Anti-Malarial Properties Derived from Endophytic Microbial Resources Indigenous to Indonesia

Figure 1: Inhibitory activity of 586 microbial extracts

against PfDHODH; purple-dashed line represents threshold

line.

Figure 2: Endophytes extracts are subjected to enzyme-

based screening assays to determine their inhibitory

activities against PfDHODH (Waluyo et al. 2021)

Figure 3: The z-factor value of each assay batch.

In this HTS study the z score value was also

measured. The Z'-factor is a statistical metric that

quantifies the reliability of data acquired from high-

throughput screening (HTS) experiments. A high Z'-

factor indicates that the assay is capable of accurately

differentiating between active and inactive

substances. The mean value of the z'factor in this

research was 0.92 (Figure 3), indicating excellent

results.

3.2 Untargeted Compounds Assay

Liquid chromatography-high resolution mass

spectrometry (LC-HRMS) was employed for

untargeted compounds, allowing for a comprehensive

screening of small compounds in the extract. The

most often used approach for analysis is reversed-

phase liquid chromatography (RPLC), specifically

the C18-based method. LCHRMS enables the precise

identification of a comprehensive metabolomic

profile, encompassing both major and minor

components in natural extracts. The novel

information obtained from LC/HRMS analysis of the

metabolomic profile of endophytes Hypomycetes

(associated with Physalis angulata) and Fusarium sp

(associated with Hornstedtia scyphifera) can be

useful for advancing study. Results of the

chromatogram analysis for each extract using

LC/HRMS are displayed in Figure 1.

The LCHRMS results for the ethyl acetate extracts of

endophytes associated with Physalis angulate and

Hornstedtia scyphifera are displayed in Table 1. The

three compounds with the largest areas for

Hyphomycetes endophyte extracts were uracil,

hexadecane, and 3-[(4-hydroxyphenyl)methyl]-

octahydropyrrole[1,2-a]pyrazine-1,4-dione. The

molecular structure of this chemical is complex, and

it exhibits antifungal, anticancer, and anti-

inflammatory properties, respectively (Zhang et al.

2013; Bao et al. 2023; Husnawati et al. 2023). A

fungus belonging to the Hyphomycetes class was

found in the flower of PA. This fungus showed both

antioxidant and antibacterial properties (Palupi et al.

2021). Regarding Fusarium sp. fungus isolates

related with the Hornstedtia scyphifera plant, the

three predominant chemicals were amide compounds,

specifically docosanamide, stearamide, and

hexadecanamide. According to research conducted

by (Gurning 2024) amide compounds exhibit

potential as antidiabetic. Research related to the

potential of endophyte bioactive metabolites as

antimalarials was carried out by Ibrahim et al. (2018).

The bioactive compound fusaripeptide found in

Fusarium sp., which was isolated from the roots of

the Mentha longifolia L, can kill malaria parasites

(Ibrahim et al., 2017). Fusarium endophytes from

Cinchona calisaya trees could also stop Plasmodium

berghei from growing by 62.18% to 78.41% (Hasbi

2019) Considering that Indonesia is known as a

source of germplasm with high biodiversity, it is

certainly a great opportunity to explore endophytic

bioactive metabolites as anti-malarials.

-20

Inhibition (%)

Number of Extract

0,3

0,5

0,7

0,9

1,1

1,3

0510

Z' Factor Value

Number of screening

Average Z' Factor: 0,92

ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)

Figure 4: Profile LC/HRMS of endophyte extract: (a) Hypomycetes of endophytes associated with Physalis angulate (b)

Fusarium sp. endophytes associated with Hornstedtia scyphifera.

4 CONCLUSIONS

There were two extracts had an inhibitory PfDHODH

of more than 50%, from endophytes associated to

Physalis angulata L (51.53%) and Hornstedtia

scyphifera (66.37%). These endophytes were

identified as Hypomycetes and Fusarium sp.

Analysis of untargeted compounds using LC-

HRMS on ethyl acetate extracts from endophytes

related to Physalis angulata and Hornstedtia

scyphifera revealed various metabolites with

potential biological activity. This research shows that

endophytes have significant potential as a source of

bioactive metabolites for the development of

antimalarial drugs. Especially considering

Indonesia's high biodiversity, further exploration of

endophyte bioactive metabolites as antimalarials is

very promising.

Exploring the Bioactive Metabolites with Anti-Malarial Properties Derived from Endophytic Microbial Resources Indigenous to Indonesia

Table 1: Metabolomics components with the highest peak area in the etyl acetate extract.

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Exploring the Bioactive Metabolites with Anti-Malarial Properties Derived from Endophytic Microbial Resources Indigenous to Indonesia