Antimalarial activity test and GC-MS analysis of ethanol and ethyl acetate extract of snake plant ( Sansevieria trifasciata Prain)

Snake plant or Mother-in-law's tongue ( Sansevieria trifasciata Prain) is a plant possessing of many ethnobotanical uses one of which is for medicinal. Previous pharmacological studies showed that this plant has several properties such as healing lesions, anthelmenthic, antimicrobial and cytotoxic. This research was conducted to investigate phytocompounds constituents of ethanol and ethyl acetate of S. trifasciata and their antimalarial activity potential. The phytochemical constituents were analyzed using Gas Cromatography and Mass Spectroscopy (GCMS). The antiplasmodial properties of the extract against malaria parasites were determined by calculating the percentage of parasitaemia suppression which was then used as a variable in probit analysis to determine the IC 50 value of the extract. There were 4 and 24 constituents of compounds detected using GC-MS in the ethanol and ethyl acetate extracts of the plant leaves respectively. Antiplasmodial assay of the extracts showed that ethyl acetate solvent gave stronger suppression against Plasmodium falciparum with an IC 50 value of 21,29 µg/mL compared to ethanol solvent with an IC 50 value of 21,29 µg/mL In conclusion plant leaf extract of Sansevieria trifasciata is potential to be developed as antimalarial ingredient.


Introduction
Sansevieria trifasciata Prain is a succulent, herbaceous, perennial plant in the Asparagaceae family that is native to Africa with several synonyms namely: Aletris hyacinthoides var. zeylanica L., Sansevieria jacquinii N.E.Br., Sansevieria laurentis De Wild., and Sansevieria zeylanica var. laurentii (De Wild.) L.H.Bailey [1]. The plant with the vernacular name snake plant or Mother-in-law's tongue has many ethnobotanical uses, such as for ritualistic purpose, medicinal use, horticultural use, food additives, and materials [2,3]. In addition, this plant is also known to have the potential to be used as raw material for the textile industry because of its high lignocellulosic fiber content [4].
Regarding the medicinal potential of Sanseviera, it is known that this plant has several properties such as healing lesions, anthelmenthic, antibacterial and anti-fungal. A study reported by Afrasiabian et al (2017) indicated that ointment formulated from this plant extract is efficacious in healing lesions on the skin of the toes [5]. An in-vitro study conducted by Karomo and Rwai (2016) showed that the extract of Mother-in-law's tongue leaves possess ananthelmintic activity against Fasciola hepatica [6]. Antimicobial test reported by Febriani et al (2019) revealed that plant extract of Sansevieria trifasciata significantly showed inhibition activities against Escherichia coli and Staphylococcus aureus [7]. Another important pharmacological property of this plant is its cytotoxic potential. By using brine shrimp bioassay test of the roots and leaves extracts, Berame at al (2017) suggested that Sansevieria trifasciata plant has cytotoxic properties [8].
Referring to snake plant extracts which are cytotoxic, anthelmintic, and contain main plant compounds such as alkaloids, flavonoids, tannins, phenols and triperterpenoids, we are compelled to test the antimalarial properties of this plant extract.

Plant samples and extraction
The plant samples of Sansevieria trifasciata were collected from suburb of Bandar Lampung, Lampung Province, Indonesia. The leaves were washed and rinsed under running water. Once clean, the samples were cut into small pieces and then air-dried by placing them in the sun covered with a black cloth until completely dry. Furthermore, the dry samples were blended into a fine powder (simplicia). The simplicia was then separated into two groups of 500 g each. The first group was macerated using ethanol solvent and the other one was extracted using ethyl acetate. Maceration was carried out for 3 x 24 hours with a ratio of 1: 10. After filtration the extracts were evaporated in a rotary evaporator until a thick extract was obtained. The thick extracts were then stored at 4°C as stock extract.

GC-MS analysis of the extract
For GC-MS analysis each stock extract was redissolved using ethanol and ethyl acetate solvents. The GC-MS analysis was performed on a GC-MS equipment (Shimadzu QP 2010 SE) equipped with Rtx-5MS (5% diphenyl/95% dimethyl polysiloxane) and Carbowax (Polyethylene glycol) standard capillary column. The identification of components was based on molecular structure, molecular mass, and calculated fragments. Interpretation of data was based on mass spectral matching with standard reference compounds in Wiley Spectral Library version 7.

Assay for antimalarial activity
Both ethanol and ethyl acetate extracts of Sansevieria trifasciata were evaluated for their antimalarial activity against Plasmodium falciparum strain 3D7 (chloroquine sensitive). The P. falciparum isolates were obtained from C-NPMRD (Center-Natural Product Medicine Research and Development), ITD (Institute of Tropical Disease), Universitas Airlangga, Surabaya, Indonesia.
Before being used in testing, plasmodium isolates were subjected to continuous cultivation and maintained using type O-positive human erythrocytes. The antiplasmodial assay was started by diluting 1 mg extract in 100 mL of dimethyl sulfoxide (DMSO). Serial concentrations of each seaweed extract were made in 5 levels namely: 100, 10, 1, 0.1 and 0.01µg/ml.
The extract (2 µl) was added to a microplate well which already contained 198 µl of parasite suspension with an initial parasitemia of 1%. The test plates were placed in a chamber containing mix gas (O2 5%, CO2 5%, N2 90%) under controlled condition and incubate at 37°C for 48 hours. After incubation, the cultures were then harvested for preparation of thin blood smears stained using 20% Giemsa dyes on a glass slide.

Parasitaemia measurement
To determine the number of parasites in the blood smear, the slide was then observed under a microscope. Ten fields on each slide were observed to calculate the percent of parasitaemia using formula below (Equation 1).

IC50 determination
To determine the seaweeds extract concentration that causes half-maximal inhibition against Plasmodium falciparum (IC50 values) a probit analysis in Minitab statistical software package was used. The IC50 values were determined by plotting concentration of extract on X-axis and percentage of suppression on Y-axis with dose-response curves The antiplasmodial activity of the samples extracts categorized into four groups based on the IC50 values i.e. very active (IC50< 5 μg/ml); active (5<IC50 <50 μg/ ml); weakly active (50<C50 <100 μg/ ml); and inactive (IC50 >100 μg/ ml) [11].

GC-MS metabolite profiles
The GC-MS analysis of the ethanol extract of Mother-in-Law's leaves resulted in metabolite profiles as depicted by a chromatogram presented in Figure 1. The metabolite constituents of the ethanol extract based on the GC-MS analysis mentioned above are listed in Table 1.    Table  2. Based on the data in Figures 1 and 2, as well as Tables 1 and 2, it can be emphasized that ethyl acetate solvent dissolves more phytochemical compounds in Sansevieria trifasciata leaves than ethanol. This finding seems confusing considering that ethyl acetate is less polar solvent than ethanol. According to common rules, polar solvents usually dissolve more plant compounds [12].
Even so, there is nothing wrong with the findings of this research because ethyl acetate actually dissolves more components than ethanol for certain compounds in certain plants. Shalmashi and Golmohammad (2010), for example, found that the solubility of caffeine in ethyl acetate was much higher than in ethanol [13].Next, da Nóbrega et al (2022) reported that ethyl acetate solvent produces more betulinic acid from the crude extract of the Eugenia florida DC plant leaves [14]. Chen et al (2022) also found that the active compound from the Portulaca oleracea plant extracted using ethyl acetate showed strong antioxidant activity compared to the ethanol extract [15]. Therefore it is not an exaggeration to say that ethyl acetate is the most preferred solvent for the extraction of active ingredients from certain plant species [16].

Antimalarial activity
The antiplasmodial properties of the ethanol extract and ethyl acetate extract of S. trifasciata against P. falcifarum described by percentage of parasitaemia growth ( Figure 3) and percentage of suppression ( Figure 4). While the IC50 values obtained through probit analysis are presented in Table 3.   [17]. Papaya (Carica papaya) leaf extracted using ethyl acetate also revealed to show antimalarial acivity against P. falciparum 3D7 and Dd2 [18]. Another study reported by Belay et al (2022) indicates that stem bark of Periploca linearifolia (Asclepiadaceae) extracted using ethyl acetate solvent significantly suppressed parasitemia of Plasmodium berghei [19].

Conclusion
GC-MS analysis and antiplasmodial assay of ethanol and ethyl acetate extracts of snake plant leaves presented a different result. Ethyl acetate solvent dissolved more constituent of phytocompounds and showed stronger parasitaemia suppression against Plasmodium falsiparum than that of ethanol. It suggests therefore that plant leaf extract Sansevieria trifasciata is potential to be developed as antimalarial ingredient.