Evaluation of Selectivity Index and Phytoconstituents Profile of Various Extracts from the Stem of Strychnos lucida R. Br. as Anti-malarial

Introduction: Strychnos lucida R. Br. or Songga was empirically used as an anti-malarial and immunostimulant in the Tetun tribe, Indonesia. However, almost all plants from the genus Strychnos contain the alkaloid toxic compound strychnine. Objectives: The aims of this study are to determine in vitro/in vivo anti-malarial activity, to analyze the selectivity index, and to measure the phytoconstituents of various extracts (water, ethanol, ethyl acetate, n-hexane) from S. lucida stem. Materials and Methods: In vitro anti-malarial study was conducted against Plasmodium falciparum 3D7-chloroquine-sensitive, and in vitro cytotoxic was performed against Monkey kidney Vero cell. Plasmodium berghei ANKA-chloroquine sensitive infected malaria mice were used as a model for evaluation of in vivo anti-plasmodial. Phytoconstituents profile was determined using Thermo scientific LC-HRMS, and the m/z data was analyzed using Compound Discoverer software with mzCloud MS/MS Library. Results: Water (W), Ethanol (E), and Ethyl Acetate (EA) extract exhibited more potential in vitro/in vivo anti-malarial activity than n-hexane (H) extract (IC 50 2.48±0.09; 2.45±0.02; 2.90±0.07; 7.64±0.30 μg/mL, respectively). The selectivity index of water, ethanol, ethyl acetate, and n-hexane extract are 211.47, 78.46, 249.62, and 32.39, respectively). According to the LC-HRMS profile, water and ethanol extract mainly contain alkaloid and phenolic compounds. Ethyl acetate and n-hexane mostly contain terpenoids and fatty acids. Conclusion: According to the result, we conclude that ethanol and water extract from S. lucida R. Br. display potential anti-malarial. Alkaloids and phenolic compounds probably have the most contribution to their anti-malarial activity.


INTRODUCTION
Malaria is an infectious disease that has become a major project in the Sustainable Development Goals (SDGs) program.In 2024, the Ministry of Health in Indonesia targets to increase the elimination of malaria cases in 405 cities, especially in the provinces of Maluku, West Papua, East Nusa Tenggara North Maluku, and Papua. [1]The use of medicinal plants by locals in malaria-endemic areas is still common.Some of the plants used empirically to treat malaria symptoms in Indonesia are stem barks of Alstonia spectabilis, Strychnos lucida, Fatuoa pilosa, roots of Calotropis gigantea, Neoalsomitra podagrica, the whole plant of Cleome rutidosperma, Physalis angulata, Alstonia scholaris, leaves of Melia azedarach, and Jatropha curcas. [2,3]From the results of ethnobotanical research, Strychnos lucida or Songga and Jatropha curcas are most often mentioned and used by the Tetun people on the island of Timor. [2]The stem of Strychnos lucida R. Br. also shows antioxidant, anti-bacterial, and anti-cancer activity. [4]owever, genus Strychnos contains strychnine which is a toxic compound.[7][8] Based on these data, it is required to study the anti-malarial activity and cytotoxic of various extracts of Strychnos lucida R. Br. stem both in vitro and in vivo.Secondary metabolites screening is also required to identify the phytoconstituents that contribute to its anti-malarial activity.
cm.The sample was identified by botanist and a plant voucher specimen was saved with code 787/IPH.06/HM/VII/2020 in Purwodadi Botanical Garden, East java, Indonesia.The stems are dried, powdered, and then stored in an airtight container.Strychnos lucida R. Br. stem powder was macerated using organic solvents of 80% ethanol, ethyl acetate, and n-hexane for 72 hr.Some stem powder is boiled with distilled water for 15 min (starting when the water starts to boil).The ratio of S. lucida stem powder and solvent is 1:10.The organic solvent from liquid extract was removed using a rotary evaporator and oven at temperature 400C, in addition the water extract was dried using freeze dryer.The dried extract was stored at 2-6°C for further procedure.

Culture of Plasmodium falciparum 3D7-chloroquine sensitive
Plasmodium falciparum 3D7-chloroquine sensitive was obtained from the Institute of Tropical Disease (ITD), Airlangga University, Surabaya, Indonesia.The Plasmodium falciparum culture method refers to Trager and Jensen with some modifications. [9]The parasite was cultured on human blood group O+ in complete media.Complete media consisted of RPMI 1640 supplemented with 0.05 g hypoxanthine, 5.96 g HEPES, 50 g/mL gentamicin, 2.1 g NaHCO 3 , and 10% human O+ serum.Parasite cultures were stored in a petri dish and incubated at 37°C.Parasite growth monitoring was conducted by completing thin blood smears and Giemsa staining.Percent parasitaemia was calculated under the microscope at 1000x magnification.Subculture was performed if the parasitaemia percentage had reached 6%.

In vitro anti-malarial assay
The culture stock was diluted with complete media and O+ human blood to reach the parasitaemia percent of 1%, and most of the parasites were in the ring stage.The anti-malarial activity procedure refers to Khasanah with modifications. [10]Each sample was weighed 10 mg and dissolved in 100 μL DMSO.Serial dilutions were completed for each sample to obtain five concentrations (100; 10; 1; 0.1; 0.01 μg/mL).The anti-malarial assay was conducted using 24 well microplates.Each well contained 500 μL sample solutions and 500 μL parasite cultures.Chloroquine Diphosphate (CQ) was used as a standard anti-malarial drug.The microplates were incubated for 48 hr at 37°C.The slides from the anti-malarial assay were observed under a microscope with 1000x magnification and counted the number of parasite-infected erythrocytes per 1000 erythrocytes.Percent parasitaemia was calculated using the following formula: Growth percentage (% growth) = % Parasitemia -D 0 Percent of inhibition (% inhibition) = 100% − {Xe/Xc) × 100%}

Note
• D 0 = Parasitemia percentage of infected red blood cell on day 0.
• Xe = Growth percentage of experimental groups.
• Xc = Growth percentage of negative control.
If values were calculated using Probit analysis using SPSS 26.

In vitro toxicity assay
Monkey kidney Vero cells was obtained from the Institute of Tropical Disease (ITD), Airlangga University, Surabaya, Indonesia.The culture procedure of monkey kidney Vero cell lines refers to Dwivedi with modification. [11]Monkey kidney Vero cells were grown in a flash containing complete media (RPMI, Penicillin-streptomycin 1%, Fungizone 0.5%, FBS 10%).Subculture was performed when the cells became 90% confluent by disaggregating the cells using trypsin phosphate into suspension.The culture stock was diluted using complete media to obtain a 1 x 10 5 cells/mL density.The cell suspension was seeded into 96-well microtiter plates and incubated for 24-48 hr until the cells were 90% confluent.After the cell was attached, the complete media was removed.One hundred microliters extract solutions were added into each well and incubated for 24 hr at 37°C.
Cytotoxic activity was determined using the MTT assay.One hundred microliters of MTT solution (5 mg/mL) were added to each well and incubated for 4 hr.After incubation, the MTT solution was removed, and DMSO was added to each well.The absorbance was recorded using an ELISA reader at a wavelength of 540 nm, and the percent viability was used to calculate the cytotoxic concentration CC 50 value of each sample.

Plasmodium berghei ANKA-chloroquine sensitive
In vivo anti-malarial procedure had been reviewed and approved by the ethical commission faculty of medicine, Brawijaya university, with a number 311/EC/KEPK/10/2021. Plasmodium berghei ANKA-chloroquine sensitive was obtained from the parasitology laboratory, Faculty of Medicine, University of Brawijaya, Malang, Indonesia.Mus musculus Balb/C were obtained from the pharmacology laboratory of the Faculty of Medicine, Brawijaya University.The male mice were 2 months old and weighed 20-30 g.Mice were acclimatized for seven days and fed ad libitum before treatment.Experimental animals were divided into 14 groups: negative control (infectious mice and received CMC-Na solution), positive group (chloroquine 10 mg/ kg BW), ethanol extract group (dose of 100; 10; 1 mg/kg BW), water extract group (dose of 100; 10; 1 mg/kg BW), ethyl acetate extract group (dose of 100; 10; 1 mg/kg BW), n-hexane extract group (dose of 100; 10; 1 mg/kg BW).Each group consisted of 5 mice.
Donor mice were inoculated with 0.2 mL frozen stock of Plasmodium berghei intraperitoneally.Observation of percent parasitaemia was performed every day through a tail blood smear.Donor mice were sacrificed when the parasitemia percentage reached 10%.The number of erythrocytes was calculated using a hematocytometer, and each animal obtained 10 7 erythrocytes intraperitoneally.
The in vivo anti-malarial activity assay refers to the 4-suppressive daily test. [12]Each extract was dissolved in CMC-Na and administered orally for four days (day 0 -day 3).Thin blood smears from the tails were made for seven days (day 0 -day 6), then calculated percent parasitemia, parasitaemia growth percentage, and percent growth inhibition using the following formula:

Extraction, in vitro anti-malarial and cytotoxic assay
Strycnos lucida R. Br. extraction process produces the following percent yield: water extract (2.40% w/w); ethanol extract 80% (3.67% w/w); ethyl acetate extract (0.69% w/w); n-hexane extract (0.59% w/w).In anti-malarial activity showed that water and ethanol extracts could inhibit the growth of parasites more potentially than ethyl acetate and n-hexane extracts (Tables 1 and  2).Ethanol and n-hexana extract exhibited potential cytotoxic against Vero cell monkey kidney.In addition, selectivity index from the highest was ethyl acetate extract, water extract, ethanol extract, and n-hexane extract, respectively (Table 1).

In vivo anti-malarial activity
Paracitemia percentage of each sample followed the dose dependent manner, higher concentration possed lower paracitemia percentage (Figure 1).Each extract inhibited the paracitemia growth, and attenuated the replication of Plasmodium falciparum, which is shown by inhibition percentage.Therefore, in this study, water extract exhibited lower paracitemia percentage than other extracts (Figure 1).There was no significant difference between the growth percentage of water extract group and chloroquine group (Table 2).The inhibition percentage of each extract ranged from 26.74% -72.72%.Therefore, higher inhibition was shown by water extract at dose 100 mg/kgBW (Table 2).

DISCUSSION
The in vivo anti-malarial activity showed that the water extract could inhibit the parasite's growth in mice infected with P. berghei more potently than the ethyl acetate, ethanol, and n-hexane extracts.The water and ethanol extract exhibited the same secondary metabolite profile; mostly contained alkaloids, and phenolic derivates.Some alkaloid compounds in water and ethanol extracts were choline, betaine, pyrazoline alkaloid derivatives: nicotinamide, and nicotinic acid.
Choline-derived compounds show anti-malarial activity by inhibiting membrane phospholipid biosynthesis in P. falciparum and interacting with metabolites resulted from hemoglobin degradation in food vacuoles. [15,16]Betaine lipid derivative compound, monoacylglyceryl trimethyl homoserine, isolated from Heterospora chenopodii, inhibited the growth of Plasmodium falciparum in vitro with an IC 50 7 μM. [17]In vitro anti-malarial assay showed that nicotinamide and nicotinic acid performed as competitive inhibitors of PfSir2. [18]Plasmodium falciparum, "silent information regulator 2" (PfSir2), is a class III member of histone deacetylases (HDACs) that play a role in the epigenetic regulation of virulent genes in the pathogenesis of malaria.Inhibition of PfSir2 activates the silence var gene through chromatin modification. [19]HDAC and Histone Acetyltransferases (HATs) play a role in modifying histones' covalent bonds.Histones affect chromatin-based events such as transcription, replication, and DNA repair.Inhibition of HDAC causes histone hyperacetylation, thereby changing the lysine composition and transcription of Plasmodium falciparum DNA. [20]In the culture of Plasmodium falciparum strains CS2 and 3G8, nicotinamide inhibited parasite growth with IC 50 6.9 mM and 2.2 mM, respectively.The combination of nicotinamide, artemisinin, chloroquine, and pyrimethamine could show a synergistic effect. [21]nzylamine was identified as an alkaloid from ethyl acetate extract.Previous study showed that naphthaquinone, dibenzylamine derivative compounds, inhibited the growth of Plasmodium falciparum KI (multidrug-resistant) with IC 50 between 0.77 -4.05μg/mL. [22]In addition, the ethyl acetate extract also contained indole strychnine alkaloids.Bisindol compounds from the genus Strychnos; isosungucine, hydroxyisosungucine, and strychnogucine B, showed anti-malarial activity against Plasmodium falciparum W2-chloroquine resistance with IC 50 168; 85; 85 nM, respectively. [23]Strychnogucine B inhibited the growth of Plasmodium berghei in murine models by 36% on day 5 and 60% on day 7. [24] The indole alkaloid reserpine, isolated from Corynanthe pachyceras, inhibited the growth of Plasmodium falciparum-chloroquine resistance with IC 50 8.1 μM. [25]e phenolic compounds in the water and ethanol extracts that showed anti-malarial activity included: caffeic acid, cinnamic acid, pyrogallol, coumarin, and hydroxybenzoic acid.Caffeic acid has an IC 50 of 80.5 μM in inhibiting the growth of Plasmodium falciparum 3D7-chloroquine sensitive. [26]Several compounds derived from cinnamic acid (myristicine) showed in vitro anti-malarial activity. [13]Gallic acid derivative compounds with phenol functional groups exhibited inhibition of Plasmodium growth with IC 50 between 20 micromolar and selectivity index >5. [27]One of the gallic acid derivatives, methyl gallate, performed in vitro anti-malarial activity against Plasmodium falciparum 3D7 with IC 50 0.0128 μM. [28]Coumarin derivatives exhibited anti-malarial activity through inhibition of hemozoin formation, resulting in toxicity to vacuole cells. [29]Methyl 4-benzoxy-3,5-dihydroxybenzoate is a water-soluble derivative of hydroxybenzoic which inhibited Plasmodium falciparum growth with an IC 50 3.72 mM. [27]rpenoid compounds that showed anti-malarial activity are Caryophyllene and limonin.Limonin inhibited the growth of parasites in the ring phase with an IC 50 2.7 μM. [30] The nanoparticle delivery system of Caryophyllene inhibited the growth of Plasmodium falciparum 3D7 chloroquine sensitive with IC 50 2.34 μg/mL and showed cytotoxic activity in lung cancer cells. [31]veral compounds belonging to the fatty acid derivates could also inhibit the growth of Plasmodium parasites, including oleamide, palmitic acid, and olealinic acid.Some studied showed that palmitic acid was ineffective in inhibiting the ring phase maturation process into scizont on Plasmodium (MIC > 50 μg/ mL). [32]One of the plants that contain oleamide was Blumea balsamifera.Blumea balsamifera inhibited plasmodium's growth in vitro with IC 50 9.66 g/mL and SI >20.70. [33] is required to measure the selectivity index to decide whether the sample has the potential for further development.An extract can be observed further if the selectivity index is > 10. [34] According to the result, all extracts were selective as anti-malarial with SI value > 10.However, based on the in vitro and in vivo anti-malarial assays, water, ethanol, and ethyl extracts acetate exhibited more effective activity.Water and ethanol extracts were categorized as highly active as anti-malarials (IC 50 < 5 μg/mL), meanwhile, ethyl acetate and n-hexane extracts were categorized as active (IC 50 > 5 to 50 μg/mL). [35]The phytoconstituents of the ethyl acetate extract displayed more abundant fatty acids than the ethanol and water extracts.38] According to the LC-HRMS profile and prior anti-malarial investigations, the alkaloids and phenols in the ethanol and water extracts contributed more to the anti-malarial activity of Strychnos lucida R. Br.

CONCLUSION
Ethanol and water extract of the stem from Strycnos lucida R. Br. provide selective anti-malarial activity according to selectivity index.The alkaloid and phenol groups contribute to the anti-malarial activity of water and ethanol extract based on LC-HRMS profiles, in vitro and in vivo assay.This research proves the effectiveness of Strychnos lucida decoct as anti-malarial that has been used empirically by local people in Indonesia.However, further studies are required to isolate and identify active compounds from Strcyhnos lucida that exhibit anti-malarial activity.

ACKNOWLEDGEMENT
This research was supported by Institute of Research and Community Service Faculty of Medicine, Brawijaya University, Malang, Indonesia (Contract number 234/2021).

Figure 2 :
Figure 2: Percentage of secondary metabolites profile from each extract.