In vitro antimalarial activity of Syzygium cumini fruit fraction

Abstract

Background:

Malaria is still one of the most severe public health problems worldwide. The development of treatment, prevention, and control of malaria is one of the substantial problems in the world.

Aims:

To investigate the in vitro antimalarial activity of Syzygium cumini methanol fruit fraction.

Methods:

Syzygium cumini L fruit powder was macerated with methanol (PA) and the extract obtained was fractionated using the liquid–liquid partition method with n-hexane, ethyl acetate, butanol, chloroform, methanol, and water solvents. In vitro antimalarial assay was conducted using the culture of Plasmodium falciparum 3D7 strain culture that had reached >5% growth and was examined for IC₅₀ values using a 24-well microplate in duplicate. Each treatment and control well contained 1,080 μl of complete media. Well, number 1 was added with 120 μl fraction, and then the solution was diluted until it reached 0.01, 0.1, 1, 10, and 100 μg/ml the final concentration in the microtiter well. The control only contained complete media and infected erythrocytes without the addition of anti-malarial drugs. The microplate was incubated for 48 hours. After 48 hours, a thin blood smear was made fixed with methanol and stained with 20% Giemsa for 20 minutes to determine the IC₅₀ value by plotting sample concentrations and percentage of parasitemia in Excel.

Results:

The IC₅₀ values of ethyl acetate fraction, n.hexane fraction, butanol fraction, and water fraction were 1.189, 76.996, 1,769, and 15.058 μg/ml, respectively. Whereases the IC₅₀ values of C1 fraction (mix fraction from chloroform: methanol 100:0 and 90:10) and C4 fraction (mix fraction from chloroform: methanol 20:80, 10:90, and 0:100) were 100.126 and 1.015 μg/ml, respectively. The results showed that the IC₅₀ value of ethyl acetate, butanol, and C4 fraction were lower than 10μg/ml and were considered as good activity (strong antimalarial activity).

Conclusion:

The ethyl acetate, butanol and C4 subfraction from S. cumini fruit have the potential to be developed as an antimalarial agent.

Introduction

Malaria is still one of the most severe public health problems worldwide. The development of treatment, prevention, and control of malaria is one of the substantial problems in the world. The use of common antimalarial drugs has been challenged variously due to the development of parasite resistance to some antimalarial drugs. There is also no effective vaccine to control malaria infection due to the complex life cycle of the Plasmodium parasite (Batista et al., 2009).

The problem of antimalarial drug resistance increases and there are difficulties in accessing effective antimalarial drugs, due to the spread of malaria-resistant multidrug parasites, so it is urgently needed to develop new antimalarial drugs from natural materials. One of the many medicinal plants found in Indonesia is juwet (Syzygium cumini). The results of a study showed that S. cumini has strong radical scavenging and antioxidant activity (Zhang and Lin, 2009). Phenolic compounds and flavonoids contained in S. cumini have antioxidant and anti-inflammatory activities (Borges et al., 2017). The anti-malarial activities of flavonoids inhibit the fatty acid biosynthesis (FASII) of the parasite as well as inhibiting the influx of L-glutamine and myoinositol into infected erythrocytes (Rudrapal and Chetia, 2016).

The use of some parts of the S. cumini plant as an antimalarial showed that S. cumini fruit as a therapeutic adjuvant has a better inhibitory effect on Plasmodium as compared to the leaves and bark (Maslachah and Sugihartuti, 2018; Maslachah et al., 2020). Determining the antimalarial activity of compounds in S. cumini L fruit can be made by in vitro. In vitro antimalarial testing is a test that does not involve the intervention of the host physiological factor, so the results obtained are the result of a parasite that is directly exposed to the test material and can be examined for inhibition of growth and maturation into schizont (Maji, 2018). The result of in vitro testing on malarial material can be determined based on the value of 50 concentration inhibitors (IC₅₀), so this research was conducted with the fraction of S. cumini L fruit as in vitro antimalarial assay against Plasmodium falciparum 3D7 strain to determine the IC₅₀ value.

Materials and Methods

Materials

The solvents used to make S. cumini fractionation were n-hexane (Merck), chloroform (Merck), Methanol (Merck), Butanol (Merck), ethyl acetate (Merck), and aquadest. Materials used to make P. falciparum culture media and to make in vitro test solutions were aquadest, HEPES buffer (Sigma), RPMI 1640 (Gibco BRL, USA), sodium bicarbonate, gentamicin, plasma, and human erythrocytes, DMSO (Sigma-Aldrich), Giemsa dyes, methanol, and emersion oil.

Plasmodium isolate

Isolate of P. falciparum 3D7 (Chloroquine-sensitive) strain used in this study from the University Faculty of Pharmacy Airlangga Surabaya Indonesia.

Plant material

Syzygium cumini L fruit was obtained from Lumajang city of East Java, Indonesia, and was identified in the Herbal laboratory of Materia Medica, Batu with letter number 067/1873/102.20/2023.

Preparation of fruit S. cumini L extract

The cleaned fruit is dried by aerating it in the open air. After drying, it is ground to a fine powder. Syzygium cumini L fruit powder was macerated with 96% methanol (PA) for 3 × 24 hours then the filtrate was evaporated with rotavapor at 50°C and then dried using a freeze dryer to produce a thick extract.

Preparation of Syzygium fruit cumini L fraction

Extract fractionation uses the solid-liquid method with n-hexane, ethyl acetate, butanol, and water solvents. The filtrate results are evaporated. After drying, all fractions were weighed to find out each weight. Extract fractionation with chloroform and methanol was carried out by vacuum liquid chromatography, methanol extract of 5 g, was put in a porcelain cup. Silica gel 60 GF254 weighs as much as 20 g and takes a third part to be mixed into the extract and crushed until silica and extract form a homogeneous powder. The elution process uses eluents with a series of motion phases as follows: CHCl_3: MeOH (100:0), CHCl_3: MeOH (90:10), CHCl_3: MeOH (80:20), CHCl_3: MeOH (70:30) and CHCl_3: MeOH (60:40), CHCl_3: MeOH (50: 50), CHCl_3: MeOH (40:60), CHCl_3: MeOH (30:70), CHCl_3: MeOH (20:80), CHCl_3: MeOH (10:90), and CHCl_3: MeOH (0:100). The fractions obtained were evaporated and conducted for thin layer chromatography (TLC) investigation using mobile phase chloroform: methanol 7:3. Fractions that have the same Rf value and spot color are combined into one mixture.

In vitro testing procedure

In vitro activity antimalarial test was conducted using the culture of P. falciparum 3D7 strain. Plasmodium falciparum 3D7 strain which had been stored in liquid nitrogen was thawed using the Rowe method. In vitro culture using the Trager and Jensen method. O-type human red blood cells, hematocrit 5% in RPMI 1640 medium (GIBCO BRL, USA), supplemented with 22.3 mM HEPES (sigma), Hypoxanthine sodium bicarbonate with 10% plasma from human O-blood type. In vitro tests were carried out in good culture 24 with 1% parasitemia (1 ml suspension/well). For the analysis of sample concentrations starting from 0.01, 0.1, 1, 10, and 100 μg/ml. The culture was incubated at 37°C for 48 hours, a thin blood smear was made fixed with methanol and stained with Giemsa staining 20% for 20 minutes. Then washed with water and dried. After that, the percentage of parasitemia P. falciparum 3D7 strain was calculated by counting the number of infected erythrocyte cells per 1,000 erythrocyte cells under a light microscope at 1,000× magnification.

Plasmodium falciparum 3D7 strain culture that had reached >5% growth was examined for IC₅₀ values using a 24-well microplate in duplicate. The serial concentration of fractions that we tested for antimalarial activity were 0.01, 0.1, 1, 10, and 100 μg/ml. The final concentration in the microtiter well with two repetitions. The control only contained complete media and infected erythrocytes without the addition of anti-malarial drugs. The microplate was incubated for 48 hours. After 48 hours a thin blood smear was fixed with methanol, stained with 20% Giemsa for 20 minutes, washed with water, and dried.

Calculation percentage of parasitemia and percentage of growth inhibition

The percentage of parasitemia is calculated by the following (Ljungstrom et al., 2004; Garcia et al., 2008):

$$\%\mathrm{Parasitemia}=\frac{\mathrm{The}\mathrm{number}\mathrm{of}\mathrm{infected}\mathrm{erythrocytes}}{\mathrm{1}\mathrm{,}\mathrm{000}\mathrm{erythrocytes}}\times 100\mathrm{\%.}$$

The calculation of parasitemia percentage was made in 1,000 erythrocytes using a light microscope with 1,000× magnification, the growth and the percentage of inhibition were done with the following equation (Inbaneson et al., 2013):

$$\%\mathrm{Inhibition}=100\%-\frac{\mathrm{(\%}\mathrm{Parasitemia}\mathrm{treatment}\times 100\%)}{\%\mathrm{Control}\mathrm{parasitaemia}}.$$

Then, the SPSS 15 probit analysis program is used to determine the IC₅₀ value.

Results

Results of activity test of antimalarial in vitro with the culture of P. falciparum strain 3D7 using the Trager and Jensen (1976) methods. The results of the percentage of parasitemia, percentage of growth, percentage of inhibition, and IC₅₀ P. falciparum 3D7 are shown in Tables 1 and 2.

Table 1. The average percent of parasitemia of P. falciparum 3D7 gives a fraction of S. cumini L extract in vitro.

Fractions	Dosage (μg/ml)	% Parasitemia
		0 hours	48 hours
Ethyl acetate	-	0.70	2.58
	100	0.70	0.48
	10	0.70	1.29
	1	0.70	1.66
	0.1	0.70	2.05
	0.01	0.70	2.33
n-Hexane	-	0.70	2.23
	100	0.70	0.51
	10	0.70	1.82
	1	0.70	1.88
	0.1	0.70	2.12
	0.01	0.70	2.35
Butanol	-	0.70	2.80
	100	0.70	1.21
	10	0.70	1.49
	1	0.70	1.86
	0.1	0.70	2.22
	0.01	0.70	2.32
Water	-	0.48	1.62
	100	0.48	0.42
	10	0.48	1.12
	1	0.48	1.32
	0.1	0.48	1.40
	0.01	0.48	1.61
C1	-	0.92	4.13
	100	0.92	0.70
	10	0.92	3.22
	1	0.92	3.61
	0.1	0.92	3.81
	0.01	0.92	4.26
C4	-	0.70	2.26
	100	0.70	0.76
	10	0.70	1.34
	1	0.70	1.54
	0.1	0.70	1.74
	0.01	0.70	2.05

Table 2. Percent growth, percent inhibition, and IC₅₀ P. falciparum 3D7 administration of S. cumini L extract fraction in vitro.

Fractions	Dosage (μg/ml)	Average percent growth (%)	Average percent inhibition (%)	IC50 μg/ml
Ethyl acetate	-	1.88	-	1,189
	100	0.00	100.00
	10	0.59	68.62
	1	0.96	48.94
	0.1	1.35	28.19
	0.01	1.63	13.30
n-Hexane	-	1.53	-	76,996
	100	0.00	100.00
	10	1.12	26.80
	1	1.18	22.88
	0.1	1.42	7.19
	0.01	1.65	0.00
Butanol	-	2.10	-	1,769
	100	0.51	75.71
	10	0.79	62.38
	1	1.16	44.76
	0.1	1.52	27.62
	0.01	1.62	22.86
Water	-	1.14	-	15,058
	100	0.00	100.00
	10	0.64	43.86
	1	0.84	26.32
	0.1	0.92	19.30
	0.01	1.13	0.88
C1	-	3.21	-	100,126
	100	0.00	100.00
	10	2.30	28.35
	1	2.69	16.20
	0.1	2.89	9.97
	0.01	3.34	0.00
C4	-	1.56	-	1,015
	100	0.06	96.15
	10	0.64	58.97
	1	0.84	46.15
	0.1	1.04	33.33
	0.01	1.35	13.46

Results extract fractionated with technique partition liquid–liquid. The results of the fractionation process graded from the fraction test are shown in Table 3.

Table 3. Fractionation results graded extract methanol fruit S. cumini L.

Fraction test	Added solvent volume (ml)	Weight fraction (g)
n-Hexane	900	5
Ethyl acetate	800	11.5
Butanol	800	34
Water	300	137

Results TLC observations and Wavenumber (cm⁻¹) of several extract fractions of S. cumini L are shown in Tables 4 and 5.

Table 4. Results TLC observations.

Code	Color	Rf
A	No colored	0.70
B	No colored	0.81
C	No colored	0.88
D	Green	0.96
E	Orange	0.96

Table 5. Wavenumber (cm−1) of several extract fractions of S. cumini L.

Fraction
Etyl acetat	n-Hexane	Butanol	Air	C1	C2	C3	C4	Bond
3,373	3,419	3,388	3,390	3,774	3,401	3,393	3,388	NH, CH OH
	3,010	2,938	2,937	3,390	2,929	2,938	2,935
	2,925			2,927	2,856
	2,854			2,855
			2,103			2,098		Triple bond C=C ( Nitril)
1,667	1,732	1,728	1,731	1,738	1,732	1,726	1,728	C=O, C=N, and C=C
1,615	1,698	1,634	1,633	1,652	1,667	1,638	1,634
1,523	1,633				1,523
	1,517
1,448	1,464	1,403	1,415	1,455	1,399	1,415	1,416	NO2, C=O C–N C–H
1,397	1,377	1,346	1,249	1,378	1,384	1,384	1,244
1,340	1,189	1,231	1,057	1,258	1,280	1,347	1,190
1,195	1,059	1,058	919	1,078	1,194	1,243	1,059
1,110	922	919	867	922	1,074	1,188	920
1,025	869	867	818	868	1,027	1,077	868
966	814	817	778	817	968	1,059	818
923	774	778	631	780	816	918	779
876	758	631	596	702	780	866	703
812	721	522		626	596	818	630
776	577				521	778
698						630
633						593
577						520
18 peaks	19 peaks	14 peaks	14 peaks	16 peaks	18 peaks	19 peaks	14 peaks

Results profile TLC chromatogram with eluent ethyl acetate: chloroform (7:3). Results identification to spots that appear were done with sighting stain citric borate, and then checked with UV lamp on Lambda 254 and 366 nm. The results show (A). Rf 0.70, colorless, (B) Rf 0.81, colorless, (C) Rf 0.88, colorless, (D) Rf 0.96, green, and E) Rf 0.96 orange color (Fig. 1). The results of Fourier transform infra red (FTIR) ethyl acetate were 18 peaks, N-hexane 19 peaks, butanol 14 peaks, water 14 peaks, C1 16 peaks, C4 14 peaks. FTIR spectra of several extract fractions of S. cumini L (Fig. 2).

Fig. 1. Chromatogram of TLC results with UV lamp on Lambda 254 and 366 nm. (1) Methanol extract; (2) ethyl fraction acetate; (3) fraction n-hexane; (4) water fraction; (5) fraction butanol; (6) fraction C1; (7) C2 fraction; (8) C3 faction; and (9) C4 fraction.

Fig. 2. FTIR spectra of several extract fractions of S. cumini L.

Discussion

The results of the antimalarial activity test showed that the C4 fraction (chloroform: methanol with a ratio of 20:80, 10:90, and 0:1,000 had the highest antimalarial activity with IC₅₀ value of 1.015 μg/ml, then ethyl acetate with IC₅₀ value 1.189 μg/ml, Butanol with IC₅₀ value 1.769 μg/ml, water with IC₅₀ value 15.058 μg/ml, n-hexane with IC₅₀ value of 76.996, fraction C1 (chloroform: methanol with a ratio of 100:0 and 90:10) with IC₅₀ value 100.126 μg/ml and C2 (chloroform: methanol; 80:20,70:30) and C3 (chloroform: methanol; 60:40, 50:5, 40:60, and 30:70) fraction not detected. A testing sample is considered to be a strong antimalarial inhibition if it has an IC₅₀ value less than 10 μg/ml, while moderate if the IC₅₀ value is 10–50 μg /ml and low activity if the IC₅₀ ranges from 50 to 100 μg/ml and if the IC₅₀ is above 100 μg/ml, is declared less active (inactive). From these data, it is shown that the antimalarial activity of the C4, ethyl acetate, and butanol fractions has strong antimalarial activity while the water fraction was moderate and the n-hexane fraction was low activity and the C1 fraction was less active (inactive) (Dolabela et al., 2008).

Fractionation was carried out using the different polarity of solvents such as n-hexane, ethyl acetate, butanol, and water as well as chloroform and methanol to extract the active ingredients and to separate the active compounds from S. cumini fruit extract. Syzygium cumini contains a variety of compounds that have different polarities, such as triterpenoids, polyphenols, flavonoids, and anthocyanins. A previous study reported that in vitro antimalarial activity test on clove extract showed an association of antioxidant effects of phenol compounds and their derivatives such as flavonoids and tannins produced from clove plants with the ability to inhibit P. falciparum growth) (Brahmam et al., 2018; Dharmawati, 2019). Methanol extract of S. cumini contains alkaloids, amino acids, flavonoids, glycosides, phytosterols, saponins, steroids, tannins, and triterpenoids. The results of chemical studies reported that there is a relationship between the form of chemical conformation the antimalarial activity against Plasmodium vivax and P. falciparum (Kumar et al., 2009; Oliveira et al., 2009; Onguéné et al., 2013). The alkaloid component of Strychnos malacoclados bark also has antimalarial activity (Tchinda et al. 2011). Elfita et al. (2011) stated that 7-hydroxypiranopiridin-4-one alkaloid compound (C₈H₇NO₂) as antimalarial potential, isolated from the microbial endophytic microbial (Andographis paniculata Nees) IC₅₀ value of 0.201 μM. Several nonalkaloid compounds that act as antimalarials have been reported. Batista et al. (2009) suggested that antimalarial compounds are found in terpene, limonoid, flavonoid, chromon, xanton, antraquinone, and miscellaneous groups. Sutthivaiyakit et al. (2009) suggested that the compound components of diterpene, sesquiterpenes, and sesquiterpenicumarin from Jatropha integerrima plants have antimalarial activity in vitro. Uchôa et al. (2010) group of triterpenoid compounds from the Cecropia pachystachya plant are antimalarial. Antimalarial activity of lactone sesquiterpenes in vitro against P. falciparum was reported by Sulsen et al. (2011) on Ambrosia tenuifolia plant and Toyang et al. (2013) on Vernonia guineensis (Asteraceae). Artemicia annua plant has an antimalarial active compound namely artemisinin which belongs to the group of sesquiterpenes compounds (Guo, 2016). Some other compounds are proven to be antimalarial β-karyofilen (Kamaraj et al., 2017). Gallic acid derivatives (octyl gallate) with hydroxyl groups are found in the aromatic ring (Arsianti et al., 2017). Methyl galat (Arsianti et al., 2018), coumarin (Pingaew et al., 2014; Tanjung et al., 2016; Beaufay et al., 2017).

The antimalarial activity of these antioxidant potential compounds which have low molecular weight, and can interfere with the redox metabolism of malaria parasites, acts as an inhibitor of the formation of antioxidant enzymes and can interfere with the formation of hemozoin. Also, the nature of a compound determines its activity. The active compound contained in the test fraction (supposed as flavonoid) has semipolar properties so that it is easier to penetrate the semipolar layer than the nonpolar lipid layer which makes it more difficult for the compound to enter the cell membrane (Becker et al., 2004). The activity of flavonoids as an antimalarial with the mechanism of action by inhibiting the biosynthesis of fatty acids (FAS II) pathway, which is in the parasite’s apicoplast such as luteolin-7-O-β-d- glucopyranoside, reported as the first natural product to target the plasmodial FAB I enzyme which regulates the FAS II pathway (Waller et al., 2003). Some flavonoids also show the ability to inhibit l-glutamine and myoinositol influx in infected erythrocytes or work with interference with hemin degradation (Elford, 1986).

Conclusion

The ethyl acetate, butanol and C4 fraction from S. cumini fruit has the potential to be developed as an antimalarial agent. Further identification of the active compounds of these fractions is to be investigated.

Conflict of interest

The authors declare that there is no conflict of interest.

Authors contributions

Research project leader and coordinating research, designed study, analyzed data, drafted the paper and corresponding author by Lilik Maslachah. Processing, examination, and analysis of in vitro data and reading the final manuscript by Neny Purwitasari. All authors contributed to the manuscript’s reading, reviewing, revising, and approving the final version.

Funding

The funding support of the DRPM research grant the PTUPT research 2019 from the Ministry of Higher Education on Research and Technology.

Data availability

All data supporting the findings of this study are available within the manuscript.