Antiplasmodial Activity of Stigmastane Steroids from Dryobalanops oblongifolia Stem Bark

Abstract Three stigmastane steroids: 6-hydroxystigmast-4-en-3-one (1), stigmast-4-en-3-one (2), and 3-hydroxystigmast-5-en-7-one (3) were successfully isolated from the acetone extract of Dryobalanps oblongifolia stem bark. The structural determination of isolated compounds was carried out on the basis of data analysis of NMR and MS spectra. In order to identify the antiplasmodial activity, the isolated compound was put to test against Plasmodium falciparum 3D7. Antiplasmodial activity of the isolated compounds showed that the IC50 values of 6-hydroxystigmast-4-en-3-one were 37.29 μg/mL while the IC50 values of stigmast-4-en-3-one were 43.54 μg/mL and the IC50 values of 3-hydroxystigmast-5-en-7-one were 13.34 μg/mL (chloroquine phosphate was used as a positive control, IC50 0.006 μg/mL). Judging from the results, the isolated compounds were proven to demonstrate mediocre antiplasmodial activity. Compound (3) indicated a better antimalarial activity than compound (1) and (2), even though there was no satisfactory activity that indicated its ability to combat chloroquine. Therefore, it might not be developed as an antimalarial drug.


Introduction
Malaria is one of the infectious diseases that has become a major problem of health. It is found in nearly most of all tropics, particularly developing and poor countries. Plasmodium, a parasitic protozoa genus, is what causes malaria in humans. The parasite that derived from the genus namely Plasmodium falciparum is the lethal part that causes acute infection worldwide with an annual death toll of 1-2 million people [1,2].
Quinine which isolated from cinchona tree has been widely used to cure malaria, yet it is still powerless to comprehensively break the life cycle of Plasmodium parasites [3]. Artemisinin, a sesquiterpene lactone, is reported as a potential antimalarial drug and have the ability to kill all phases of the parasites' life cycle through interaction with heme, yet animal experiment shows neurotoxic and cardiotoxic effect [4]. Development of synthesized drugs, such as chloroquin, pyrimethamine, cycloguanil, and sulfadoxine, have indicated the decline of effectivity caused by the resistance of Plasmodium [3,5,6,7]. Therefore, it is crucial to develop alternative medicines from plants by constituent exploration as potential antimalarial drugs.
Dryobalanops oblongifolia belongs to the family of Dipterocarpaceae and is widely found in Indonesia and Malaysia [8].The phytochemical screening of fruit of D. oblongifolia revaled the presence of steroids compounds in this species [9]. Dryobalanops is known to produce oligostilbene constituents with various interesting activity such as antibacterial, antioxidant, antimalarial and cytotoxic [10,11,12,13,14]. In continuation for searching bioactive compounds from Indonesia's plants, a study towards D. oblongifolia was conducted by isolating the agents and examining the antiplasmodial activity against Plasmodium falciparum 3D7. Based on our knowledge this three stigmastane steroids (1-3) were first report from family Dipterocarpaceae and these isolated metabolites expressed only moderate antiplasmodial activity.

General procedures
Firstly, CDCl 3 was used to dissolve 1 H and 13 C NMR and 2D NMR of stigmastane steroids spectra. While using TMS as the internal standard, JEOL J-500 spectrometer was used to record and it was utilized in CDCl 3 at 125 MHz ( 13 C) and 500 MHz ( 1 H). On a TSQ Quantum Access MAX Triple Quadrupole Mass Spectrometer, mass spectrometry was analyzed. Gravitation column chromatography (GCC) was conducted with Merck Si gel 60 (700-200 mesh). Vacuum liquid chromatography (VLC) and radial chromatography were conducted using Si gel 60 PF 254 and Si gel 60 GF 254 . The analysis of Thin Layer Chromatography (TLC) was done on Merck kieselgel 60 GF 254, precoated Si gel plates, with a thickness of 0.25 mm. This research used already distilled analytical and technical grade solvents.

Plant Material
Mount Mali was the place where D. oblongifolia Dyer stem bark was originally collected. The mount is located in Tempunak, Sintang, West Kalimantan of Indonesia. The researchers then proceeded to the identification step by sending the plant specimen to the Biological Research Center of LIPI in Bogor, Indonesia. A voucher specimen was put in safekeeping at the herbarium.

Extraction and Isolation
At room temperature, as much as 5 kg of D. oblongifolia stem bark powder was pulped twice in acetone. It is meant to afford the extraction of brownish gummy after the process of vacuum evaporation. The extract was subsequently separated into 2 fractions: 1 fraction is able to be dissolved in acetone -diethyl ether while the other fraction is insoluble. As much as 48 g of the soluble fraction was divided into fractions using vacuum liquid chromatography (VLC) (n-hexane -ethyl acetate, enhancing polarity) to give four main fractions which are fraction A-D. By using radial chromatography techniques and Gravitation Colum Chromatography (GCC), as much as 1.7 g of Fraction B was separated and purified. The process led to the production of compound 1 with a total of 6.5 mg and compound 2 with a total of 3 mg. In order to isolate both compounds and enhance the polarity, n-hexane and ethyl acetate mixtures were used. As much as 1.6 g of Fraction C was separated and refined by using the same chromatography techniques and solvent mixtures, resulting in the production of compound 3 with a total of 3.4 mg.

In Vitro Antiplasmodial Assays
The antiplasmodial activity of compound 1-3 was determined in the Tropical Disease Institute of Universitas Airlangga, which is located in Surabaya, Indonesia. In this part, the method used was equivalent with the former method described by Widyawaruyanti et al. [15]. The dissolution of these samples was conducted in DMSO and they were stored at −20°C until use. A culture plate with 24 wells was used to cultivate the P. falciparum clone. The concentration range of each compound was 100, 10, 1, 0.1, and 0.01 µg/mL. As a positive control, a drug with antimalarial characteristics namely Chloroquine phosphate was used. The antiplasmodial activity measurement of compound 1-3 and chloroquine phosphate was calculated in replica. The monitoring process of parasitaemie was conducted when 48 hours had passed by firstly making a blood test fixed with MeOH and spattered with Geimsa (Merck). With the aim to determine the parasitaemia average ratio and average inhibition, the researchers calculated the total number of infected erythrocytes from originally 1000 healthy erythrocytes. The researchers used IC 50 value to state the antiplasmodial activity of compound 1-3. IC 50 value is the concentration of compounds that causes 50% inhibition of the parasite growth. The IC 50 value was obtained by using probit analysis processed by the SPPS program.
Ethical approval: The conducted research is not related to either human or animal use.

Result and Discussion
The acetone extracted from D. oblongifolia stem bark was fractionated and purified using radial chromatography, gravitation column chromatography, and vacuum liquid chromatography. It was intended to produce three stigmastane steroid compounds, specifically 6-hydroxystigmast-4-en-3-one (1), stigmast-4-en-3-one (2), and 3-hydroxystigmast-5-en-7-one (3). The isolated compound structures were identified on the basis of 1 Hand 13 C-NMR spectral data, and 2D NMR experimentations and contrast with the reported data and MS spectral data.  (Table  1). Aside from the analysis of the HMBC spectrum, the determination of hydroxyl group location can also be achieved by doing a TOCSY test. Compound 1 structure was indicated as 6-hydroxystigmast-4-en-3-one (Figure 1) [16].
Compound 2 was isolated as an achromatic formless powder with a [M+H] + ion at m/z 413.244. It was isolated during ESI-MS analysis and it corresponded to C 29 H 48 O molecular formula. Compound 2 was discovered by the NMR data as a steroid with stigmastane skeleton. Although compound 2 has a high resemblance with compound 1 in terms of 1 H-NMR and 13 C-NMR spectrum chemical shifts, compound 2 does not possess any hydroxyl group (Table  1). Compound 2 structure was indicated as stigmast-4-en-3-one ( Figure 1) [17,18].
Compound 3 was isolated too as an achromatic formless powder that has C 29 H 48 O 2 molecular formula (ESI-MS, [M+H] + ion at m/z : 429.227). Compound 3 has high resemblance with compound 1 and 2 in terms of NMR spectrum chemical shifts, exposing that compound 3 was a stigmastane steroid. There were 29 carbon signals displayed by 13 C-NMR and DEPT spectra. These carbon signals consisted of 6 methyl carbons, 10 methylene carbons, 9 methine carbons, and 3 quaternary carbons, and carbonyl ketone. The HMBC spectra proved that 1 hydroxyl group and the α, β-unsaturated carbonyl system were present with links between H-4/C-2, H-4/C-3, H-4/C-5, H-4/C-6, H-4/C-10 ; H-6/C-4, H-6/C-8, and H-6/C10 ; H-8/ C-7, H-8/C-9. There are indications initiated by the HMBC analysis that the hydroxyl group and the α, β-unsaturated carbonyl system was located on different positions. The location of the former was at the position of C-3 in ring A, while the location of the latter was in ring B (Table 1). Compound 3 structure was identified as 3-hydroxystigmast-5-en-7-one. The confirmation can be seen through a contrast with stigmast 3-hydroxy-5-en-7-one chemical shifts, which is similar to the previously published research (Figure 1) [19,20].
The examination of antiplasmodial activity against P. falciparum 3D7 was carried out by in vitro to three stigmastane steroid compounds. The test results showed that the IC 50 value of 6-hydroxystigmast-4-en-3-one (1) was as much as 37.29 µg/mL. Meanwhile, for stigmast-4-en-3one (2), the IC 50 value was as much as 43.54 µg/mL, whereas the IC 50 value of 3-hydroxystigmast-5-en-7-one (3) was as much as 13.34 µg/mL. Chloroquine phosphate was used a positive control with as much as 0.006 µg/mL IC 50 (Table 1). Judging from the results, mediocre antiplasmodial activity was found in the three stigmastane steroid compounds [21]. Compound 3 showed better antiplasmodial activity than the others. The structure of stigmastane steroids chemical compound revealed that the presence and position of hydroxyl group can influence their antiplasmodial activity. The location of hydroxyl group in compound 3 is easier to interact with extracellular and intracellular fluids so that it can be easily carried to target molecule [22]. However, compound 3 is considered as lacking the ability to fight against the chloroquine so it may not be promoted as an antimalarial agent.

Conclusion
There were three stigmastane steroids that were successfully isolated from the acetone extract derived from the stem bark of Dryobalanops oblongifolia. The evaluation on antiplasmodial activity was performed to all of the isolated compounds, specifically 6-hydroxystigmast-4-en-3-one (1), stigmast-4-en-3-one (2), and 3-hydroxystigmast-  5-en-7-one (3). The antiplasmodial activity indicated that there was a total of 37.29 µg/mL of IC 50 value in 6-hydroxystigmast-4-en-3-one. Meanwhile, the IC 50 value of stigmast-4-en-3-one was as much as 43.54 µg/mL while as much as 13.34 µg/mL of IC 50 value was found in 3-hydroxystigmast-5-en-7-one (chloroquine phosphate was used as a positive control, IC 50 0.006 µg/mL). Compound 3 was the most active isolated compounds although there was not enough activity to fight against chloroquine. As the consequence, compound 3 did not meet the standard of an antimalarial drug and may not be developed as a proper medication of the disease.