In vitro Test for Inhibition of Plasmodium falciparum 3D7 Parasites using Streptomyces hygroscopicus subsp. hygroscopicus strain i18, isolated from a Pineapple Farm in lampung

Increasing natural ingredient awareness and utilization has created an increased demand for sources of natural medicinal ingredients, including sources of compound used to treat malaria. Streptomyces is a genus of prokaryote well recognized for its production of antibiotics and other pharmaceutically useful compound. This study aimed to assess the ability of unpurified fermentation metabolites to inhibit Plasmodium parasites. A strain of bacteria identified as Streptomyces hygroscopicus subsp. hygroscopicus strain i18 were isolated from pineapple fields in Lampung province, and was cultured and fermented on liquid synthetic Gause medium for 10 days. The supernatant was separated from the cells and extracted with ethyl acetate-methanol (1:1). Plasmodium falciparum 3D7 was used for antiplasmodial testing. Metabolites were tested qualitatively using a phytochemical approach. Saponins and triterpenoids were found to be present in the extract. Parasite inhibition as measured using probit analysis and yielded an iC50 value of 11.07 g.m/L. These findings suggest further examinations of this extract (e.g. assessment of off-target effects) are warranted.


iNtRODuCtiON
Malaria remains a significant medical challenge, particularly in tropical countries 1 . Plasmodium parasites are responsible for malaria, which is transmitted by the bite of female Anopheles mosquitoes 2 . Malaria is often found in tropical coastal and rainforest habitats. It is endemic to Lampung province with a high rate of incidence 3,4 . Therefore, better resources for malaria control are needed.
Malaria control using a variety of natural ingredients are now widely pursued 5 , due to increasing resistance to synthetic antimalarial drugs. Exploration for antimalarial natural products has not been limited to plants. Animals and microbes have also been assessed 6 . Streptomyces is one of many microbial genera that has long been known for its anti-parasitic and anti-pathogenic metabolites, including antimalarials 7 .
Streptomyces are Gram-positive bacteria found scattered across various habitats. These bacteria are capable of controlling pathogen populations and producing biosurfactants 8 . A previous study succeeded in isolating the bacteria Streptomyces hygroscopicus subsp. hygroscopicus i18 9 . These bacteria were shown to inhibit the growth of pathogens that caused root and stem rot, namely Dickeya zeae.
Plasmodium development is inhibited by the same bacterial strains, according to previous reports 10 . Determining the lethal doses of distilled extracts has been the subject of many antiparasitic studies. Distillation-based purifications are expensive and time-consuming 11 . It is important to know whether a crude extract obtained from Streptomyces hygroscopicus subsp. hygroscopicus can inhibit Plasmodium parasites without purification. This study thus aimed to learn more about Plasmodium inhibition by unpurified Streptomyces hygroscopicus subsp. hygroscopicus strain i18 fermentation extract.

Cell culture and fermentation
Bacteria were cultured in liquid Gause media (LGM) with an adjusted pH of 7.3 12 .
LGM media consists of soluble starch 20.0 g, KNO 3 1.0 g, NaCl 0.5 g, MgSO 4 x 7 H 2 O 0.5 g, K 2 HPO 4 0.5 g, FeSO 4 x 7 H 2 O 10.0 mg, dissolved in 1000 mL distilled water. Culture supernatant for extraction was prepared by scaling up cultures. Initially, a 10 mL test tube of LGM was inoculated with a single loop inoculum. After 24 h incubation, the 10 mL starter culture was used to inoculate 90 mL of LGM. This culture was incubated at room temperature for 10 days. Supernatant was obtained by centrifuging the culture for 30 min at 6000 rpm. Crude supernatant was filtered through filter paper and extracted with a 1:1 methanolethyl acetate solvent mixture 13 . The solvent was then evaporated using a rotary evaporator, yielding a paste that was lyophilized to form a powder.

Qualitative metabolites screening
Secondary metabolites content was determined by analyzing samples of the powdered extract. Testing was performed using a previously described qualitative approach 14 . Phenols, alkaloids, flavonoids, saponins, triterpenoids, anthraquinone glycosides, and tannins were prominent among identified compounds. infrared spectroscopy Infrared (IR) spectra of the crude extract were obtained using an Agilent Cary 630 spectrometer. Infrared spectra with wave numbers ranging from 400 to 4000 cm -1 were obtained. Resolution was reported in two sizes per wave number.

In vitro antiplasmodial testing procedure
Antimalarial activity testing was performed in vitro 15 . This study used Plasmodium falciparum strain 3D7, which chloroquinesensitive. One mg of sample extract was diluted in 100 mL of DMSO (stock solution, concentration 1.000 mg/mL). Serial dilution were made from the stock solution. In this study, synchronous parasites (ring stage) approximating 1% parasitemia were used. Fresh type O-positive human erythrocytes were used to maintain cultures, which were suspended at 4 percent hematocrit in complete medium 16 . Parasites were added to 96-well plates using a high-throughput liquid handler. Two microliters of various dilutions of extract were then added. Final extract concentrations were 100, 10, 1, 0.1, and 0.01 μg/mL. Plates were incubated under controlled gas condition (O 2 5%, CO 2 5%, N 2 90%). Plates were incubated for 48 h at 37°C, then erythrocyte slides prepared using 20% Giemsa staining.
Data were analyzed by counting the number of infected erythrocytes per 1000 total erythrocytes microscopy. These data were used to calculate growth and inhibition percentages. The following formula was used to determine the growth percentage: Where D 0 represents initial 1% parasitemia prior to incubation (%) Inhibition is expressed as a percentage, and calculated using the formula below; Percent inhibition =100 % -((Xu/ Xk) x 100% .

..(2) Description
Xu = % growth in the presence of extract Xk = % growth in the negative absence of extract Extract concentration that inhibited parasite growth by up to 50% (EC 50 ), was calculated using the two formulae above. The inhibitory concentration (IC 50 ) was determined using probit analysis and inhibition percentage results.

Results
Streptomyces is genus of soil bacteria known to produce numerous secondary metabolites with useful human applications. Saponin and triterpenoid class compounds are produced by Streptomyces hygroscopicus subsp. hygroscopicus i18. Table 1 presents the findings of our secondary metabolite survey.
The bandwidth at 3212.96 cm -1 observed by FTIR analysis (Fig 3) is postulated to be linked to intermolecular H bonds. A =C-H bond, which can be found in any organic compounds was observed at 3060.61 cm -1 in the spectrum. Carboxylate

DisCussiONs
Streptomyces bacteria are considered to be probiotic bacteria that offer a variety of health benefits. Member of this genus have long been known to produce antibiotics 17 Antioxidants, pigment, and anticancer agents are   3. Infrared spectroscopy using FTIR at biosurfactant produced by Streptomyces hygroscopicus subsp. hygroscopicus strain i18 fermentation extract also produced by these bacteria 18 . The i18 strain produces saponins and triterpenoids, according to preliminary screening results for secondary metabolites (Table 1). Saponins have been described as having biosurfactant 19 . Biosurfactants can lower water's surface tension, disrupting the conformation of a target microorganism's cell membranes 20 . Saponins can produce emulsifying foam which can disrupt the stability of the hydrophobic core of phospholipid bilayers, causing micelles to form. Saponins have been shown to inhibit growth of pathogenic bacteria 21 .
Saponins and triterpenoids are closely related. Compounds in this group can be cyclic or non-cyclic, alcohol (OH), aldehyde, and carboxylic acid groups 22 . Spectroscopic analyses revealed the presence of a carboxylate group (Fig. 3). This carboxylate-containing isoform could be antidiabetic from a physiological standpoint. Some natural pigments are triterpenoids 23 . Triterpenoids dissolve readily in polar solvents, including water, owing to their composition. Geraniums' triterpene content has also been reported to be antibacterial 24 .
Since saponins in corporate glycosides in their structure 25 , the existence of anthraquinone glycosides in our Streptomyces exctract was not unexpected. This compound is structurally related to anthracene, but has a carbonyl group on each of the two atoms on opposite sides of the central ring. The color of this pigment ranges from yellow to orange. When ammonia is applied to anthraquinones, the majority develop a characteristic color reaction in Bontrager reagent tests 26 . In our extract, the solution became reddish, while the anthrone and dianthrone remained golden. Carboxyl groups are also present in anthraquinones, which can be extracted using alkaline media. Anthrones and anthranols (which reduce glycosides) are made from anthraquinones 27 .
The antimalarial compound gancidin was isolated from Streptomyces sp 7 . At doses of 6.25 and 3.125 μg/kg, it can inhibit nearly 80% of malarial parasites. In these experiment purified gancidin was used. Unpurified extract from strain i18, displayed an inhibitory concentration of 11.07 μg/mL. The toxicity of a candidate compound has no bearing on its antiplasmodial efficacy. We did, however, consider the toxicity of non-antimalarial compounds. Despite high toxicity toward Plasmodium, the extract could poison non-target cells 28 . Because this was a preliminary investigation, the compounds used in this analysis were not purified. Further testing would be needed in the future due to the demand for pure compound that could be used for mechanistic and in vivo testing. Effect on metabolism and cell damage could explain any toxicities in human usage.

CONClusiONs
Preliminary studies were carried out using crude extract fermented by Streptomyces hygroscopicus subsp. hygroscopicus strain i18, which contained of saponins, triterpenoids, and anthraquinone glycosides. This crude extract displayed the potential to inhibit the growth of Plasmodium parasites. Its half maximal inhibitory concentration (IC 50 ) towards Plasmodium parasites was 11.07 μg/mL. The results of this preliminary study need to be followed up with further research to establish physiological mechanisms and efficacy of pure compounds as antimalarial agents.