INTRODUCTION:

The rapid growth of health services infrastructure and the advancement of technology have not reduced morbidity and mortality from vector-borne infectious illnesses. Malaria infection is one of the vector-borne diseases that fatalities approximately 400 thousand people worldwide. each year and produces morbidity that affects 200 million people worldwide¹. One of the malaria-endemic countries is Indonesia. In 2018, the Annual Parasite Incidence/API per 1000 Indonesian population was 0.84, with Papua, West Papua, East Nusa Tenggara, and Maluku having the highest API index².

Malaria is an arthropod-borne disease caused by Plasmodium sp, a protozoan that infects human red blood cells. Malaria infection is caused by five different species of Plasmodium sp. including P. vivax P.ovale, P. falciparum, P.malariae, and P.knowlesi³. Malaria infection causes a variety of clinical manifestations, including flu-like illness, and other symptoms caused by the rupture of human red blood cells (anemia, jaundice, hemoglobinuria, etc), and systemic inflammation, which causes kidney disorders, coagulation abnormality, decreased blood sugar, shock, cerebral malaria, and even death⁴^,⁵. In 2019, P. falciparum was the cause of 94 percent of malaria infection cases and the leading cause of mortality globally⁶.

Several factors impede the malaria eradication program, including increasing deletion of histidine-rich protein 2 (HRP2) gene, which jeopardizes the ability to diagnose and treat falciparum malaria; a lack of funds in the malaria eradication program, insecticide resistance, and the evolution of resistance to artemisinin-derived regimens⁷. The evolution of resistance to artemisinin-derived regimens, which are standard malaria therapy,challenges the eradication of the disease. As a result of this incidence, it is vital to investigate numerous natural compounds that are effective as antimalarials.

The ocean region accounts for over 70% of the total area on the planet, and it has a diverse aquatic biota that contains a variety of active compounds that may be effective in the treatment of a variety of disorders. Marine and freshwater biota have a substantially higher phylogenetic or diversity than land biota. Because marine biota is confronted with various environmental stimuli that necessitate different adaptations, they can synthesize unique secondary metabolites and engage in pharmacological actions⁸.

Curry fish or S. hermanni, is an aquatic biota with active compounds that have not been completely explored. Curry fish is a sea cucumber species that contains a variety of bioactive chemicals, including saponins, chondroitin sulfate, glycosaminoglycans, sulfated polysaccharides, sterols (glycosides and sulfates), phenolics, peptides, and cerebrosides⁹^,¹⁰^,. Several studies have demonstrated that curry fish exhibit various biological activities, including accelerate wound healing, relieve pain, inhibit bacterial and fungal growth, and have anti-tumor properties⁹^,¹⁰^,¹¹^,.

The solvents involved in the extraction process are categorized as polar (water, methanol, and ethanol), semipolar (ethyl acetate, chloroform, and acetone), and non-polar ((n-hexane and ether)¹². This type of polarity solvent can dissolve active substances with the same polarity qualities. The polarity of the solvent affects the active chemicals extracted and has a direct impact on biological activity, extract quality, quantity, extraction speed, inhibitory substances, toxicity, other biological activities, and biosafety ¹³^,¹⁴. The major goal of this study was to explore phytochemical assy and the antimalarial efficacy of curry fish extract and compare its phytochemical result and antimalarial activity with two different polarity solvents (semipolar and non-polar solvents). No researchers have explored the antimalarial effects of curry fish, so the findings of this study serve as the foundation for future research to determine the antimalarial biomolecular mechanism of curry fish using experimental animals.

MATERIAL AND METHODS:

Materials used in the research work include: P.falciparum 3D7 (Malaria Laboratory -Institut Tropical Disease Universitas Airlangga), curry fish, ethyl acetat (Merck), n-hexane (Merck), Meyer's reagent, Dragendroff's reagent, Wagner's reagent, distilled water (Smart-Lab), lead acetate (Merck), FeCl3 (Merck), chloroform (Merck) and H2SO4 (Merck), HCl (Merck), NaHCO₃(Merck), HEPES buffer (Merck), gentamycin (Merck), hypoxanthine (Merck), RPMI 1640 medium (Merck), Blood donor (Red Cross of Surabaya), DMSO (Merck) and Giemsa solution (Merck), methanol (Merck), immersion oil (Merck). The equipment used includes an analytical balance (Kern ABT), a Buchner funnel (Aldrich), a vacuum rotary evaporator, candle jar (sigma aldrich), light microscope (Olympus BX53), microwell plate (96 well Nunc), yellow tip steril (CLS4711), blue tip steril (eppendorf), micropipette (Eppendorf), falcon tube (eppendorf), Erlenmeyer flask (pyrex), Laminar Air Flow (LAF), and incubator (Nuve EN055).

The researcher employs an experimental method that includes the measurement of all variables at the ending of the treatment (Post Test Only Control Group Design), all parameters evaluated were tested at the end of the trial, except parasitemia levels, measured twice (pre and post of treatment). Malaria Laboratory - Tropical Disease Institute, Airlangga University, provided the research implementation and P.falciparum 3D7 culture medium. There are three primary groups:

1. P1: control group (P.falciparum culture medium without any extract or antimalarial drug)

2. P2: P.falciparum culture medium with n-hexane extract of curry fish

3. P3: P.falciparum culture media with an ethyl acetate extract of curry fish

Each treatment group will be further subdivided based on the amount of extract administered: 100; 10;1; 0.1; 0.01µg/ml, and each dose was duplicated twice, so there were ten mediums in each group study (5 types of doses x 2 replication = 10). The Hang Tuah Medical Faculty Ethics Committee has given its official approval for the study's implementation (no I/2021).

Curry fish material:

The curry fish used in this study were obtained during the rainy season from the sea area around Sapeken Island, Sumenep, Madura-East Java, Indonesia. Plant Bioscience and Technology Laboratory, Institut Teknologi Sepuluh November, Surabaya, Indonesia (No. 002/IT.1/Bioscience and Plant Technology/2021) conducted taxonomic tests on fresh samples of curry fish. Curry fish samples were evaluated for qualitative phytochemicals test to identify the types of bioactive components.

Extraction Process:

There are several advantages to using the maceration or cold extraction method in this research. The cost is low, the number of active ingredients that dissolve more, the volume of extract produced is large, and the method does not require heating, which reduces the risk of the active ingredients being damaged or decomposed¹⁵.

Extract preparation started by removing the internal organs from the meat. The meat was cut into small pieces and dried in a freeze dryer set to -50°C. The curry fish is pulverized into a powder after drying. As solvents, n-hexane (non-polar) and ethyl acetate (semipolar) were used to extract the powder. The extract is filtered and evaporated, affording a thick/dry extract of n-hexane and ethyl acetate. Extracts are kept refrigerated in an air-tight container until they are used¹⁵.

Phytochemical Analysis:

A phytochemical test is used to determine the active substance content of a specific material. There are two types of phytochemical tests: qualitative (only knowing the presence or absence of an active ingredient) and quantitative (knowing the presence or absence of multiple active ingredients) (knowing the amount of an active ingredient). A qualitative phytochemical test was used in this study¹⁶. The following method is used to identify components in curry fish:

1. Identification of Alkaloids:

The extract was dissolved in hydrochloric acid before being filtered. The filtrate will be treated with specific reagents, and the production of precipitates of varied colors indicated the presence of alkaloids. There are three types of reagents used:

a. Meyer's reagent, the filtrate was mixed with a solution of potassium mercuric iodide to produce a white precipitate¹⁶^,17^,¹⁸.

b. Dragendroff's reagent, the filtrate was mixed with a potassium, bismuth, and iodide solution to produce a reddish-orange precipitate¹⁶^,17^,¹⁸.

c. Wagner's reagent, the filtrate was mixed with iodine in potassium iodide to produce a brownish precipitate¹⁶^,17^,¹⁸.

2. Identification of Saponin Using the Froth Technique:

Saponins were identified by adding 20ml of distilled liquid to the extract and stirring it for 15 minutes until it dissolved in a barker glass, then observing for froth, indicating the presence of saponins¹⁷.

3. Identification of Flavonoid Using Lead Acetate Test:

Flavonoids were recognized by adding a few hatches of lead acetate to the extract, and the formation of a yellow precipitate showed the presence of flavonoid content¹⁶.

4. Identification of Tannin Using Braymer’s test:

The Braymer test was performed by combining three milliliters of distilled water with a few drops of 3 percent ferric chloride into the extract filtrate; the presence of a blue-green color change in the filtrate demonstrated the presence of tannins in the extract¹⁶.

5. Identification of Steroid:

The presence of steroids is indicated by a red layer under the chloroform layer after the addition of a mixture of 2 milliliters of chloroform and a concentrated H2SO4 solution into the extract sample (5 milliliters)¹⁷.

Cultivation of Plasmodium falciparum In vtro:

The culture technique necessitates using three basic ingredients: (1) A chloroquine-sensitive strain of P. falciparum (strain 3D7); (2) human plasma and packed red cells donated by the Red Cross; (3) RPMI 1640 medium (contains various components including human blood serum, sodium bicarbonat, HEPES buffer solution, gentamicin, and hypoxanthine). An In vtro study was implemented in this study using a modified Trager and Jensen approach. Strain 3D7 of P.falciparum was cultured in RPMI 1640 culture medium with a 5% hematocrit. The culture was incubated at 37°C in a modified candle jar. Determination of the density of P. falciparum parasites was carried out by microscopic examination of thin blood smears with Giemsa staining. The infected erythrocyte was examined under a light microscope at a magnification of 1000 times. The parasite included in this research was homogenized P.falciparum ring-stage and parasitemia density around 1%¹⁹^,²⁰.

Antimalarial Activity Analysis:

The first step is to prepare a stock solution by combining DMSO with a curry fish extract solution to create a stock solution 10,000µg/ml. The following step is continual dilution until the concentration of the extract solution is obtained based on the dose used in this study (100; 10; 1; 0.1; and 0.01 micrograms per milliliter). The micro well culture medium in groups P2 and P3 contained 198µl of parasite cell suspension and 2µl of curry fish extract solution, which was replicated twice at each concentration of the extract solution. All micro well culture medium is placed in a chamber with a gas mixture (oxygen: carbon dioxide: nitrogen ₌5%: 5%: 90%) and then incubated at 37°C for 2 days or 48 hours. Parasite cell suspensions were checked for thin blood smear before being implanted into the microwell to measure the level of parasitemia (at - 0 hours). After two days or 48 hours of incubation, a blood smear plate was generated utilizing a ten percent giemsa stain²⁰^,²¹. The density of parasite was monitored by counting total amount of infected erythrocytes in a thousand erythrocytes using light microscope (data on parasitemia level after curry fish extract administration). The parasitemia growth and inhibition percentages were calculated using this dataset. The growth percentage was calculated by subtracting the level of parasitemia after extract administration from the level of parasitemia before extract administration (% level parasitemia post treatment - % level parasitemia before treatment). The following equation was used to calculate the percentage of parasite growth inhibition/ inhibitory rate²⁰:

100- (% Plasmodium growth in curry fish administration group (P2/P3)/% parasite growth in control group (P1) x 100)

The half-maximal inhibitory concentration, or IC₅₀, is one method of determining a drug's or active ingredient's potency. In pharmacological research, the IC₅₀ value is used to assess the quantity of drug/active component minimum inhibitory biological processes by 50 percent²²^,²³. The IC₅₀ of curry fish extract was estimated as the last step in confirming its antimalarial activity using regression probit analysis.

Criteria for In vtro Antimalarial Activity:

There were four different groups of extract materials based on their IC₅₀ values in this research, compared to WHO recommendations and standard requirements for antiparasitic drug research activities: (1). If the IC50 is lower than or equivalent to 5 µg/ml, it is considered high efﬁcacy; if the IC50 is higher than 5 µg/ml or smaller and equivalent to 15µg/ml, it is considered encouraging efﬁcacy; if the IC50 is higher than 15 µg/ml or smaller and equivalent to 50µg/ml , it is considered moderate efﬁcacy; and anything above 50µg/ml is considered low efﬁcacy. Pure substances with IC₅₀ lower than or equivalent to 1µg/ml are considered very high efficacy ²⁰.

Analyses of data:

There are two stages in data analysis: (1) Entering all datasets, including parasite density, growth percentage, and parasite inhibition percentage, into Excel; (2) Calculating IC50 by entering all data, including parasite density, growth percentage, and parasite inhibition percentage, into SPSS for probit regression analysis. This study's probit analysis aims to determine the effective dose of curry fish extract to inhibit 50% of parasites^22,23.

RESULT AND DISCUSSION:

Phytochemical Analysis:

The active components in curry fish extract were determined utilizing a qualitative method to a phytochemical investigation. According to the phytochemical results, the solvent of ethyl acetate and n-hexane in the curry fish extract generated the following results:

Table I. Active Compounds of Curry fish Extract Using Two Different Solvents

Active Compounds	Result	Type of Solvent
Active Compounds	Result	Ethyl Acetate	N-hexane
Flavonoid	Formation of a yellow precipitate	+	+
Alkaloid (using 3 methodes)
a. Meyer's reagent	a white precipitate formed	+	+
b. Dragendroff'sreagent	a reddish-orange precipitate formed	+	+
c. Wagner's reagent	a brownish precipitate formed	+	+
Tanin	a greenish-black solution formed	-	-
Saponin	Froth formed after shaking	+	-
Steroid	a layer of red color formed in the lower chloroform	+	+

The phytochemical findings showed that the active component content of curry fish extract with ethyl acetate solvent appears to include a variety of active compounds (flavonoids, saponins, steroids, and alkaloids) more than n-hexane solvent. However, n-hexane solvent has only small bioactive components (flavonoids, alkaloids, and steroids). This study found that using non-polar solvents (n-hexane) in curry fish extract produced fewer bioactive components than semipolar solvents (ethyl acetate). This study's result is parallel with the outcomes of another study, which found that the higher the polarity of a solvent, the more extract is formed and the more bioactive components are present²⁴. Because this study's phytochemical assessment is qualitative, the nominal amount of bioactive component content is unknown, and comparisons are only made based on the found components and those that are not.

The lack of saponins in the n-hexane extract of S. hermanni is due to saponins' high polarity, which makes it more soluble in high polarity solvents such as water, methanol, and ethanol²⁵. Saponins, on the other hand, are not soluble in nonpolar n-hexane¹². Because the solvent ethyl acetate is semipolar, it can dissolve components such as saponins. According to the outcomes of this research, no tannin component was identified when ethyl acetate and n-hexane were used as solvents. This study's result is connected with the OH group's chemical structure, which modifies its polarity, allowing the tannin component to dissolve in polar substances²⁶.

Pringgenies et al. (2018) conducted a phytochemical test on an ethanol extract of curry fish (S.hermanni) and identified bioactive materials in addition to tannin, saponin, flavonoid, steroid, and terpenoid but no alkaloid²⁷. This disparity's possible cause is a difference in sampling sites, variances in solvents, extraction methods, extraction temperature, and extraction duration. All of these variables impact the bioactive composition, number of bioactive components, biological activity, toxicity, and biosafety¹³^,²⁴.

Antimalarial Activity Analysis:

The results of parasitemia development (%) obtained by subtracting parasitemia levels after cuury fish extract administration and parasitemia levels before the administration curry fish extract are shown in the table below:

Table II: Parasitemia Growth Result Before And After Treatment at Several Doses of Extract

Study Groups	Parasitemia Growth (%)
	0 hours	After Incubation For 48 hours
	0 hours	0.01 µg/ml	0.1 µg/ml	1 µg/ ml	10 µg/ ml	100 µg/ml
Control (-)	1.03	5.7	5.7	5.7	5.7	5.7
N-Hexane	1.03	5.11	4.08	2.915	1.615	0
Ethyl acetate	1.03	5.065	4.225	3.535	1.935	0

According to parasite growth assessments, parasites developed more slowly when provided curry fish extract in larger doses. The administration of a dose of 100 µg/ml extract suppressed parasite growth to the point where the growth rate was zero. At a lowest dose of curry fish extract (0.01µg/ml), the n-hexane solvent extract group developed more parasites than the ethyl acetate extract group. However, the administration of extract doses ranging from 0.1 to 10µg/ml revealed that parasite growth was lesser in the group applying n-hexane extract of curry fish than in the group applying with ethyl acetate extract.

Table III. Inhibitory Rate And IC₅₀ Assay Result

Type of solvents	IC₅₀	Inhibitory Rate (%)
Type of solvents	IC₅₀	0.01	0.1	1	10	100
N-Hexane	1.07	10.35	28.42	48.86	71.67	100
Ethyl acetate	2.20	11.4	25.88	37.98	66.5	100

The parasite inhibitory rate assessment data revealed that the stronger the inhibitory rate, the higher the dose of curry fish extract administered. The control group did not calculate the inhibitory rate because it just presented P.falciparum cells and no other remedies. The group that got n-hexane and ethyl acetate solvent at a 100 µg/ml concentration demonstrated a 100 percent inhibition rate. Based on the results of parasite growth and inhibitory rate measurements in this experiment, it is reasonable to conclude that administration of curry fish extract with n-hexane and ethyl acetate solvents showed antimalarial action. In both solvents, IC₅₀ measurements revealed high antimalarial activity in compliance with WHO recommendations, specifically IC₅₀ ≤ 5µg/ml ²⁰. The IC₅₀ value of n-hexane is lower than that of ethyl acetate, which implies that the use of n-hexane solvent in S. hermanni extract has greater antimalarial activity than ethyl acetate solvent.

The antimalarial activity of S. hermanni is not yet known because earlier investigations focused on its antibacterial and antifungal properties. The discussion focuses on the comparability of the results in this study, which was conducted based on the antimalarial activity of the bioactive components found in curry fish, including flavonoids, saponins, steroids, and alkaloids, using a phytochemical assay (Table 1). Flavonoid compounds have been shown to have antiplasmodial activity In vtro²⁸. Although the biomolecular mechanism of flavonoids' antimalarial activity is not entirely understood, it is believed that flavonoids decrease fatty acid biosynthesis. Flavonoids work indirectly in the parasite's feeding vacuole because they are acidic (a phenolic–OH group). Under cellular oxidative stress (in vivo), these polyphenolic flavonoids' phenolic–OH groups are readily converted to stable phenoxy radical anions, which lead extensive oxidative stress injury to parasite biological components or direct tissue injury via irreversible breakdown covalent bond (destruction of proteins, RNA, and DNA)²⁹. And other study showed that flavonoids exert antimalarial effects by lowering hemoglobin breakdown by inhibiting the cysteine protease enzyme falcipain-2²⁰. A vast number of saponin's biological effects have been linked to its action on cell membrane permeability. They have a unique capacity to create pores in membranes. Saponins have a lytic effect on the membranes of erythrocytes³⁰. There is evidence that alkaloids can diminish parasitemia levels by decreasing parasite DNA and RNA synthesis(1). Steroids and their derivatives have been discovered to have antimalarial effects in several studies, although the mechanism remains unknown²²^,³¹. For example, Krieg et al. (2017) revelaed that lipophilic steroid carriers to this antiparasitic have been shown to aid in modulating cellular absorption and intracellular transport pathways, thereby increasing the antiplasmodial's efficacy., and also increases cellular oxidation³². Research results have proven the presence of active compounds in curry fish and high antimalarial effects. However, further quantitative investigation of the complete content of bioactive components is required. The next step should be an in-depth investigation of the mechanism of action of curry fish extract against parasite growth inhibition and its anti-inflammatory activity to regulate cytokine storm in severe malaria, its effective dose, and toxicity.

CONCLUSION:

Data from phytochemical tests show that the semipolar ethyl acetate solvent has more bioactive components than nonpolar solvent n-hexane. However, the antimalarial activity test revealed that both solvents demonstrated strong antimalarial performance, even though the IC₅₀ value of n-hexane was lower than that of ethyl acetate. The lower the IC₅₀ of a bioactive component, the greater its biological activity. The lack of data on curry fish or S. hermanni's biological effects on malaria parasites has encouraged a more in-depth research of its components and mechanism of action as an antimalarial²⁶.

ACKNOWLEDGMENTS:

There is no conflict of interest in this study and article's research. We are grateful to Hang Tuah University's Faculty of Medicine for providing funding for research and publication. We also thank the Malaria laboratory - Institute for Tropical Diseases, Universitas Airlangga, Plant Bioscience and Technology Laboratory, Institut Teknologi Sepuluh November and other parties participating in and assisting with the research's implementation.

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflicts

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Received on 18.11.2021 Modified on 16.04.2022

Research J. Pharm. and Tech 2023; 16(2):645-651.

DOI: 10.52711/0974-360X.2023.00110