In Vitro Effect of 96% Ethanol Extract of Bitter Herbs (Andrographis

paniculata Nees) on Heme Detoxification Process of Plasmodium

falciparum Parasites

Safarianti Safarianti

, Aty Widyawaruyanti

, Hilkatul Ilmi

, Achmad Fuad

, Indah Tantular

Maryatun

Faculty of Medicine, University of Syiah Kuala, Darussalam, Banda Aceh, Indonesia

Institute of Tropical Disease, University of Airlangga, Surabaya, Indonesia

Keywords: The 96% Ethanol Extract Of Bitter Herbs, Andrographis Paniculata Nees, Heme Detoxification Process.

Abstract: Malaria still becomes a global health problem especially in tropical countries and developing countries. The

number of malaria infections in Indonesia is still high and it becomes endemic disease in some areas. The

high rate incidence of malaria can be caused by the emergence of malaria parasite strains that are resistant to

anti malarial drugs. Various efforts have been made to reduce the incidence of malaria, one of which was to

develop a new medicinal compounds from natural ingredients such as 96% ethanol extract of bitter herbs

(Andrographis paniculata Nees). This herb has been proven to have antimalarial activity both in-vitro and

in-vivo from previous studies. This study aims to determine the effect of 96% ethanol extract of bitter herbs

on heme detoxification process of Plasmodum falciparum through the inhibition of β-hematin formation

(synthetic heme). This research is a laboratory experimental research that was conducted in Malaria

Laboratory of Institute of Tropical Disease (ITD) University of Airlangga Surabaya. The results showed that

96% ethanol extract of bitter herbs inhibits the formation of β-hematin equal to 61,07 ± 4,69%. It can be

concluded that 96% ethanol extract of bitter herbs (Andrographis paniculate Nees) are able to inhibit heme

detoxification process of Plasmodium falciparum parasites.

1 INTRODUCTION

96% ethanol extract is a natural herb obtained

from bitter plants (Andrographis paniculataNees /

Sambiloto). This traditional medicinal plant that has

long been used empirically as an anti-malarial drug

(Widyawaruyanti, 2009). The compound substances

are contained with Sambiloto herbal plants include

diterpenoid lactone, flavonoid, xanton and

Andrographolide which are the main active

substances of this plant. The level of

Andrographolide in the form of ethanol extract 96%

is ± 14,91% (Okhuarobo et all, 2014; Safitri EY,

2007).

96% ethanol extract of Sambiloto herb are

obtained from extraction process of Sambiloto

powder through maceration process with its solution

is 96% ethanol to enable it to draw a range of

compounds contained in Sambiloto plant

(Widyawaruyanti, 2009). Earlier research has

indicated that 96% ethanol extracts of Sambiloto

herbs are active as antimalarial drug against

Plasmodium falciparum in vitro, and are also active

Against Plasmodium berghei in-vivo

(Widyawaruyanti et al, 2014; Rahman et al, 1999).

Other studies show that 96% ethanol extracts of

Sambiloto herbs were found to have antimalarial

activity at medium-to-moderate categories with

inhibitor concentration value (IC) 2,287 μg/ml that

inhibits the development of schizont stage

(Schizontocide) (Resi, 2013; Widyawaruyanti et al,

2015).

One of the important work targets of antimalarial

drugs in killing malaria parasites is by inhibiting the

process of heme detoxification from developing into

hemozoin crystals. This is a vital process for the

parasite and has been validated as one of the

antimalarial work targets. Therefore it is necessary

to study the effect of 96% ethanol extract of

Sambiloto herbs in inhibiting the process of heme

detoxification of Plasmodium parasite to figure out

In Vitro Effect of 96 .

DOI: 10.5220/0008791100720075

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 72-75

ISBN: 978-989-758-438-1

the mechanism of 96% ethanol extract of Sambiloto

herbs in killing parasites.

2 RESEARCH METHODOLOGY

This is a laboratory experimental research. The

antimalarial herb 96% ethanol extract Sambiloto

herbs at dose 100 μg / ml is incubated with hemin

chloride and then its effect is observed on heme

detoxification process by using spectrophotometer.

The sample used in this study was hemin chloride

which is a synthetic pigment obtained from Sigma®.

The inhibition of the P. faciparum heme parasite

detoxification process is carried out by synthetically

testing the inhibition of β-hematin formation. In this

test, the active antimalarial compound of 96%

ethanol extract of Sambiloto herbs with the 100 μg /

ml concentration obtained from the ITD Science and

Technology Research Partnership for Sustainable

Development (SATREPS) Laboratory was incubated

in acetate buffer with α-chlorohemin (Sigma)

solution for 24 hours, with trial of duplication.

2.1 Preparing the Test Substance of

96% Ethanol Extract of Sambiloto

Herbs

The 96% ethanol extract of Sambilato herbs as the

test substance in this study was firstly prepared

through the following steps:

 The extract is to be weighed 10 mg of test

substance, dissolved in 100 μl DMSO to

obtain solution concentration of 100.000 μg /

ml as stock solution

 Measure as much as 10 μl of stock solution

with pipette then add a complete medium until

the volume reaches 500 μl which results in the

solution concentration of 2000 μg / ml

(Solution A)

 Measure as much as 10 μl of solution A with

pipette then add a complete medium until the

volume reaches 500 μl which results in the

solution concentration of 100 μg / ml

(Solution B)

2.2 Procedure of synthetic heme

detoxification inhibion Test (ß-

Hematin Inhibition Assay) (Tekwani

BL and Walker LA, 2005)

The inhibition test of heme detoxification process is

undertaken through the following steps:

 Turn solid natrium acetate into 8M natrium in

warm water. Do the mixing continously at

370C in waterbath

 Add 8 M acetic acid until it reaches pH 5.0

value. The addition of acetic acid is to be

done at 370C, then incorporate a total of 100

μl of the solution in the falcon tube.

 Add the 96% ethanol extract test substance of

50 μl with 100 μg / ml concentration in

DMSO in a falcon tube containing the acetate

buffer solution.

 Add a total of 50 μl of hemin chloride 8mM

(Sigma) in DMSO to the falcon containing the

test substance and acetate buffer solution.

 Undertake incubation for 24 hours at 35

with two replications.

 Centrifugalize β-hematin and insoluble

hematin (in the from of pellet) at 3000 rpm for

20 minutes at a temperature of 22

C, and

discard the supernatant.

 Add 200 μl DMSO to the obtained pellet to

dissolve unreacted hematin, then re-

centrifugalize it and discard the supernatant.

 Add 200 μl NaOH 0.2M to the pellet to

convert ß-hematin into a soluble hematin

alkaline, then make each sample of alkaline

hematin in dilution series with Aqua bidest.

 Measure absorbance using a

spectrophotometer at a wavelength of 414 nm.

 Calculate the effect of the test substance on

the production of ß-hematin and compare it

with the negative control. The negative control

used in this study was 50 µl of DMSO

solution.

In this study, the data analysis was undertaken

descriptively by comparing the group that has been

given 96% ethanol extract of sambiloto herbs with

the negative control group.The sentence must end

with a period.

3 RESULTS AND DISCUSSION

The Plasmodium parasite will consume

hemoglobin from erythrocyte cells as one of its

nutrients for its survival. The stage at which

hemoglobin is mostly consumed is the tropozoit

stage. Being in erythrocytes, the parasite

endocytoses a number of nutrients that are mostly

hemoglobin coming from erythrocyte cytosol.

In Vitro Effect of 96

Endocytosis occurs through the process of

invagination of parasitic plasma membranes known

as sitostoma. Once the endocytosis process is

complete, the neck of sitostoma is removed to

release the double membrane of the endocytic

vesicle that contains the cytoplasm of red blood

cells.

The digestion process of erythrocyte cytoplasm

is initiated by proteolytic gastrointestinal enzymes in

double membranes vesicular transport. The double

membrane vesicular transport is thought to originate

from the invagination of the parasitic plasma

membrane as the outer layer and the parasitophoruos

vacuoles membrane as the inner layer.

The result of host cytoplasm endocytosis (80%

hemoglobin) will be transported by vesicle transport

into the food vacuole and degraded proteolitically

into heme and globin in the food vacuole namely an

oxygen-rich lysosome-like organelle at the pH

between 5-5,5 (Hong-Chang Z et al, 2009; Hoppe

HC, 2004).

The Plasmodium parasite consumes only globin

as its nutritional source, while the heme is released.

Free heme is toxic to the parasite and has the ability

to lyse the parasitic membrane of Plasmodium.

Therefore the parasite must detoxify the heme

through certain mechanisms, one of which is by

converting free heme into insoluble crystal

hemozoin (Egan TJ, 2007).

The effects of Plasmodium parasite's heme

inhibition can be known through β-Hematin-

formation inhibition test (ß-Hematin Inhibition

Assay). β-hematin has a structure and spectrum

identical to hemozoin, in which case the β-hematin

formation inhibition test may be used to determine

the effect of antimalarial drugs on the detoxification

process of heme parasite P. falciparum.

ß-hematin can be formed in vitro in acidic

atmosphere (acetate buffer solution pH 5.0) of

hematin or hemin chloride. Hemin chloride will

react to acetate buffer and form an insoluble β-

hematin after 24-hour incubation at 35

C. The

administration of NaOH solution in the β-hematin

pellet will convert it into alkaline hematine whose

absorbance can be measured using UV.8

spectrophotometer (Tekwani BL and Walker LA,

2005).

The result of calculation on percentage of β-

hematin formation inhibition in table 1 shows that in

the group which was given 96% ethanol extract of

Sambiloto herbs, β-hematin formation activity was

inhibited with the average percentage of β-hematin

formation inhibition of 61,07 ± 4,69%.

Frolich et al (2005) suggests that compounds

with β-hematin formation inhibition greater than

60% are stated to have good potential as β-hematin

inhibitors.

Compounds with β-hematin formation

inhibitions smaller than 40% are claimed to be weak

inhibitors on the formation of ß-hematin.12

Therefore, it can be stated that 96% ethanol extract

of the Sambiloto herbs has good potential as an β-

hematin formation inhibitor.

This result is supported by a research conducted

by Widyawaruyanti et al (2015) which states that

96% ethanol extract of Sambiloto herbs is known to

have activity of inhibiting the process of

detoxification of heme into crystalline polymer

hemozoin through observation of food vacuoles

parasit P.falciparum with TEM (Transmission

Electron Microscope) microscope. It was mentioned

that, in the parasite which was given 96% ethanol

extract Sambiloto herbs, it appears that the crystal

hemozoin was undergoing fragmentation.

On the contrary, in the control group, it appears

that the woven crystals are still clear and transparent

(Widyawaruyanti, 2015).

The aformentioned description shows that

ethanol extract of Sambiloto herbs has an inhibitory

effect on the detoxification process of heme parasite

Plasmodium falciparum and is one of ethanol extract

work target of 96% Sambiloto herbs. This

inhibition process can minimize the pathogenesis of

malaria and also inhibit the formation of hemozoin

in the liver and spleen of malaria patients.

Table 1: The percentage of β-hematin formation inhibition by 96% ethanol extract of Sambiloto herbs

Substance

Absorbance

(414 nm)

% β-hematin

formation

% β-hematin

inhibition

% mean

inhibition of β-

hematin

Control

0,73

100

0,71

100

0,26

35,62

64,38

96% ethanol

extract

61,07±4,69

0,30

42,25

57,75

4 CONCLUSIONS

In conclusion, 96% ethanol extract of Sambiloto

herbs have the activity of inhibiting the process of

heme parasite Plasmodium detoxification

ACKNOWLEDGEMENTS

We thank the staff in Institute of Tropical Disease

(ITD) Surabaya for their assistance in preparing a

test substance and for help with processing the

spectrophotometer sample

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(submiited)

In Vitro Effect of 96