Antiplasmodial Activity of the Low Molecular Weight Compounds from Streptomyces sp. GMR22

1. Study Program for Biotechnology, Graduate School, Universitas Gadjah Mada, Barek, Yogyakarta 55281, Indonesia 2. Department of Agricultural Microbiology, Faculty of Agriculture, Universitas Gadjah Mada, Bulaksumur, Yogyakarta 55281, Indonesia 3. Research Division for Natural Product Technology, Indonesian Institute of Sciences, Jl. Jogja – Wonosari Gunungkidul, Yogyakarta 55861, Indonesia 4. Department of Chemistry, Universitas Gadjah Mada, Sekip Utara PO Box: BLS21, Yogyakarta 55281, Indonesia 5. Department of Pharmacology and Therapy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia


INTRODUCTION
Molecular weight compound is one of the considerations for the successful discovery of drug compound candidates. This consideration emerged after High Throughput Screening (HTS) era, where many compounds have high molecular weights. The higher molecular weights cause compounds tend to be lead-like rather than drug-like. According to the Rule of 5 (Ro5), drug-like compounds have low molecular weights of less than 500g/mol. Low molecular weight is one of the keys of drug-likeness for the discovery of oral bioavailability candidates (Lipinski, 2004).
Low molecular weight (LMW) compound is one of the products of secondary metabolites produced by microbes, particularly soil-dwelling bacteria (Tyc et al., 2017). Streptomyces, the soildwelling bacteria, are a well-known source for the Volume 31 Issue 4 (2020) production of secondary metabolites, including LMW compounds. The LMW compounds of less than 500g/mol or 500 Da are commonly volatile (Schmidt et al., 2015), and known to possess biological activities (de Lima Procópio et al., 2012;Wu et al., 2015;Xing et al., 2018).
Streptomyces sp. GMR22 is one of the soildwelling bacteria isolated from Cajuput rhizospheric soil at Wanagama I Forest, Yogyakarta, Indonesia (Nurjasmi et al., 2009). This microbe is known to produce LMW compounds which possessed biological activity (Sukmawati et al., 2018), and predicted to encode biosynthetic pathways of terpenes (Herdini et al., 2017). The LMW compounds could be extracted and isolated using an organic solvent mixture. One of the LMW compounds was known as terpene (Jiang et al., 2016).
Previous studies revealed that monoterpenes and sesquiterpenes isolated from essential oils exhibited antiplasmodial activity in vitro against the W2 and K1 strain of P. falciparum (Boyom et al., 2011;Durant et al., 2014;Mota et al., 2012). Moreover, triterpenes also exhibited in vitro antiplasmodial activity (Isaka et al., 2010). The antiplasmodial compounds isolated from bacteria, particularly Streptomyces have not been widely reported. Therefore, Streptomyces sp. GMR22 is considered to produce LMW compounds, including terpenes as antiplasmodial.

Streptomyces sp. GMR22 isolate
Streptomyces sp. GMR22 was isolated from Cajuput rhizospheric soil at Wanagama I Forest, Yogyakarta, Indonesia, as obtained from the previous study (Nurjasmi et al., 2009). GMR22 isolate has been deposited at Indonesian Culture Collection (InaCC A148), Research Center for Biology, Indonesian Institute of Sciences, and NITE Biological Research Center (NBRC), Japan (NBRC 110112). GMR22 16S RNA sequence has been submitted in the National Center for Biotechnology Information (NCBI), accession code MN922646.

Fermentation, extraction, and isolation of LMW compounds
GMR22 isolate was inoculated on International Streptomyces Project-2 (ISP-2) medium (Alimuddin et al., 2010). The spore culture was inoculated into 50mL tryptic soy broth (TSB) and incubated at 28 °C for 3 days on a rotary shaker (150 rpm) as seed culture. Then, it was inoculated into 450 mL starch nitrate broth (SNB) for metabolites production (soluble starch 20.0 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4.3H2O 0.5 g, MgSO4.7H2O 0.5 g, FeSO4.7H2O 0.01 g, per liter) and incubated at 28 °C for 11 days on a rotary shaker (150 rpm). The supernatant was collected by centrifugation at 5000 rpm for 10 minutes then filtered by Whatman No.1 filtrate paper. The extraction method was carried out using an organic solvent of nhexane:EtOAc (85:15v/v) mixture, according to Jiang et al. (2016) obtained a solid brown extract.

Plasmodium culture and in vitro antiplasmodial assay
The 3D7 strain of P. falciparum was obtained from the Eijkman Institute, Jakarta, Indonesia. In vitro antiplasmodial assay was carried out using donor blood type O + . The Plasmodium was cultured in vitro continuously, according to Trager and Jenson (1978), incubated in an incubator at 37°C with 5% CO2. The trophozoite-phase of Plasmodium was synchronized by the addition of 5% D-sorbitol every 48h, as reported by Lambros and Vanderberg (1979) and Mustofa et al. (2007).
One milligram n-hexane:EtOAc extract was dissolved in 10µL DMSO stock solution (100,000µg/mL). The extract was diluted using RPMI medium to obtain final tested concentration of 0.78, 6.25, 25, 50, 100, 200µg/mL. One hundred microlitre RPMI medium (negative control) and tested concentration of extract, respectively, were plated in 96-well microplate and followed by the addition of 100µL Plasmodium culture 275 (trophozoite-phase at 0.5-1% parasitemia, 1% hematocrit) resulting from the synchronization. The antiplasmodial assay was carried out with triplicate, incubated for 72h in an incubator at 37°C with 5% CO2. Parasitemia was observed by making thin blood films stained by 10% Giemsa stain. Microscopic observation of the parasitemia was done using light microscopy (Nikon) at 1000x magnification in 1000 observed erythrocytes. The percentage of Plasmodium growth inhibition was obtained by counting the parasitemia on the control group multiplied by 100% (Jenett-Siems et al., 1999).

Confirmatory antiplasmodial test by flow cytometry
Confirmatory antiplasmodial test of n-hexane:EtOAc extract was analyzed using flow cytometry. Plasmodium DNA was stained using SYBR Green I (Invitrogen, USA) (Dery et al., 2015), and CD235a (anti-human, eBioscience, USA) as a marker for erythrocyte. The confirmatory test was carried out independently in the same culture manner as the previous antiplasmodial assay. The final concentration of the tested extract was 50µg/mL, and DMSO (0.1% v/v) as the extracting solvent was used as a control group. The test was performed in duplicate with Plasmodium uninfected-erythrocyte was used as a comparison.
Two hundred microlitre of each treatment was centrifuged at 7000rpm for 10min. Then, each of 50µL erythrocyte pellet was transferred into a microtube. Afterward, 2µL of CD235a and 2µL of SYBR Green I (1000x final concentration) were added consecutively then it was incubated in the dark at room temperature for 30min. The pellet was washed once by adding 1mL of PBS and centrifuged at 2000rpm for 5min. Pellet was then collected and dissolved in 400µL flow cytometry buffer for subsequent analysis using a flow cytometer (BD FACSCanto™ II).

Ethical clearance
In vitro antiplasmodial assay used donor blood from an adult male with blood type O + , and approved by the Medical and Health Research Ethics Committee (MHREC) of Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia (KE/FK/0869/EC/2019). The ethics committee was recognized by The Forum for Ethical Review Committees in Asia and the Western Pacific (FERCAP).

Statistical analysis
The result of in vitro antiplasmodial assay was presented as mean ± standard error of the mean (SEM) of parasitemia percentage and growth inhibition percentage. One-way ANOVA followed by the LSD post hoc statistical test was done using SPSS 24 software (IBM Corp., USA). The antiplasmodial activity was defined by 50% inhibitory concentration (IC50) obtained through probit analysis (95% confidence interval) using SPSS 24 software (IBM Corp., USA).

Identification of LMW compounds and druglikeness screening
The detected compounds profile from n-hexane:EtOAc extract after analysed using GC-MS is presented on the chromatogram (Figure 1). Twenty one compounds were identified as alcohol, hydrocarbon, ester, aromatic/diester, diester, fatty acid, and triester classes, which have molecular weights of less than 500g/mol ( Figure 1). Meanwhile, 4 silicates have molecular weights of more than 500g/mol ( Table I). The result of druglikeness screening using SwissADME based on the Ro5 showed that 3 compounds were drug-likeness, 16 compounds were non-drug-likeness, and 2 compounds could not be predicted. The classified drug-likeness compounds are phenylethyl alcohol, ethyl citrate, and di-n-butyl phthalate (DBP).
Volume 31 Issue 4 (2020) It hypothesized that Streptomyces sp. GMR22 might secrete enzymes and could lead to the synthesis of silicate such as Actinobacter sp., which has been reported by Singh et al. (2008).
Extraction using an organic solvent mixture aimed to obtain the LMW compounds, including the class of terpenes. However, the terpenes were not identified. The GC-MS used in this study was RTX-5 capillary column, which was different from Jiang et al. (2016). Yamada et al. (2015) reported that 2methylisoborneol was only the trace that could be detected in liquid culture after analyzed using GC-MS. Therefore, the terpenes level in the extract might also be trace.
This study found that the classified druglikeness compounds were phenylethyl alcohol, ethyl citrate, and DBP from alcohol, ester, and aromatic/diester classes, respectively. This elucidates that the compounds have physicochemical properties, which are associated with acceptable aqueous solubility and intestinal permeability. Therefore, the compounds are predicted to be suitable for oral bioavailability candidates (Daina et al., 2017;Lipinski, 2004). Table I. Identification of the LMW compounds from n-hexane:EtOAc extract and drug-likeness screening for potential compound candidates. a: Retention time (minute); b: molecular weight; c: source: https://pubchem.ncbi.nlm.nih.gov; d: SwissADME prediction analysis based on Ro5 (Lipinski et al., 2001); v: eligible to Ro5; x: not eligible to Ro5; o: could not be predicted 277 The previous studies revealed that phenylethyl alcohol possessed antifungal activity (Xing et al., 2018), whereas DBP was reported to possess antimicrobial activity (Roy et al., 2006). Meanwhile, ethyl citrate was the first report in this study. This finding showed that the classified druglikeness compounds were considered to have other biological activities.
One of the classified non-drug-likeness compounds was DOP, which possessed biological activities. Fitriastuti et al. (2020) reported that ethanol extract fraction of temu mangga (Curcuma mangga Val.) contained DOP, which also identified using GC-MS. They reported that DOP possessed potency as an antimalaria compound. Moreover, DOP was reported to possess antimicrobial activity (Zothanpuia et al., 2018).

In vitro antiplasmodial assay
The result of antiplasmodial assay of n-hexane:EtOAc extract (Figure 2). It showed that the parasitemia percentage was inversely correlated to the growth inhibition percentage. One-way ANOVA statistical test showed that the extract reduced the parasitemia percentage significantly as compared to the control group (p < 0.00). Likewise, the LSD post hoc test showed there was a significant difference between each extract treatment compared to the control group (p<0.05). The extract activity as antiplasmodial obtained an IC50 value of 38.61±19.06µg/mL. This shows that the extract exhibited moderate antiplasmodial activity with IC50 below 50µg/mL as reported by Kaharudin et al. (2020).
The compound's content of the extract is believed to be active antiplasmodial compounds. DOP, one of the main compounds, was reported being potential as antimalaria (Fitriastuti et al., 2020). They reported that the antimalaria test was carried out by a heme polymerization inhibition method, and the result obtained an IC50 value of 1.479µg/mL. Although phthalates are commonly encountered as plasticizers or pollutants from industrial wastes (Xu et al., 2020), other studies revealed that phthalate derivatives could be biologically produced by either Streptomyces (Mangamuri et al., 2016;Roy et al., 2006;Zothanpuia et al., 2018) or filamentous fungi through shikimic acid pathway (Tian et al., 2016).
In this study, the use of donor blood type O + aimed to successfully facilitate the in vitro cultivation of Plasmodium parasites. Theron et al. (2018) reported that P. falciparum preferred to invade donor blood type O + rather type A + , B + , and AB + for an in vitro culture. Thereby, the donor blood type O + became an advantage for the in vitro cultivation of P. falciparum.

Confirmatory antiplasmodial test by flow cytometry
The confirmatory antiplasmodial test results and fluorescence intensity of Plasmodium DNA and detected erythrocyte are also presented ( Figure 3).
Area of SYBR Green I and CD235a, which stained the Plasmodium DNA and erythrocyte, respectively ( Figure 3). Q1 indicates the area of SYBR Green I negative and CD235a positive. Q2 indicates the area of P. falciparum infectederythrocyte and SYBR Green I positive. Q3 indicates the area of SYBR Green I negative, but CD235a positive for erythrocyte debris. Q4 indicates the area of P. falciparum uninfectederythrocyte and CD235a positive. The fluorescence intensity of SYBR Green I represents the DNA of Plasmodium infected-erythrocyte, and the fluorescence intensity of CD235a represents the detected erythrocyte. The % total erythrocyte represents the detected erythrocyte count. The % erythrocyte (uninfected) represents the P. falciparum uninfected-erythrocyte count, whereas the % parasitemia (infected) represents P. falciparum infected-erythrocyte count. The % inhibition is the P. falciparum growth inhibition percentage after the treatment compared to the control.

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Volume 31 Issue 4 (2020) The detected erythrocyte intensity in both groups was equal. Thus, the total erythrocyte count was proportional. The control group showed uninfected-erythrocyte intensity lower than the treatment. This is in line with Kotepui et al. (2015). They reported that P. falciparum infection with high parasitemia showed a decreased total erythrocyte count as compared to P. falciparum infection with low parasitemia. The parasitemia percentage was found inversely correlated to the inhibition percentage on the flow cytometry. This is in line with the result of previous microscopic analysis. Hence, it shows that the extract exhibited potency as antiplasmodial.

CONCLUSION
Further research was needed to reinvestigate and to identify other antiplasmodial active compounds besides DOP resulted/generated from Streptomyces sp. GMR22. Figure 3. The flow cytometry analysis results of Plasmodium DNA and erythrocyte on the group of (a) P. falciparum uninfected-erythrocyte and P. falciparum culture after treatment of (b) DMSO and (c) extract 50 µg/mL, respectively, which showed a distinction percentage among groups based on (d) the fluorescence intensity of the detected erythrocyte (both total and uninfected), the percentage of parasitemia (the DNA of P. falciparum infected-erythrocyte), and the percentage of inhibition of P. falciparum growth