ABSTRACT  A part of our continuing search for antiplasmodial compounds from Indonesian marine sponge, two steroidal alkaloids, epoxysarcovagenine-D (1) and epoxyepapakistamine-A (2), have been isolated from the ethyl acetate extract of the Indonesian marine ponge, Xestospongia sp. Their structures were identified on the basis of spectroscopic data analysis and by comparison with published spectroscopic and physicochemical properties data. Compounds 1 and 2 were isolated first time from marine sponge, Xestospongia sp., and showed strongest plasmodial activity against Plasmodium falciparum 3D7 strain with IC₅₀ values of  0.013 and 0.158 µM, respectively.

Keywords: Steroidal alkaloid, Xestospongia sp, Petrosiidae, Antiplasmodial activity

INTRODUCTION

          The genus Xestospongia is the marine sponge belong to Petrosiidae family, comprises approximately 40 species that are mainly distributed in north-western Australia, Papua New Guinea, Solomon Island, Thailand and Indo-Malaysia Peninsula (Fromont, 1991; Calcul, 2003; Laurent et al., 2006; Aguinaga et al., 2010). Xestospongia is known to settle and grow on a variety of substrates, such as sand, rock beds, dead coral rubble and coral heads (Kerr and Borges, 1994; Williams et al., 1998; Krisanapuntu et al., 2001; Bell and Smith, 2004; Armstrong et al., 2006). Chemical investigations on the species of this genus have led to the isolation of a large array of structurally diverse secondary metabolites with significant biological activities including antimalarial alkaloids (Girard et al., 2004; Darumas et al., 2007; Ashok et al., 2014), antifungal alkaloids (Moon et al., 2012), cytotoxic and inhibition of the aspartic protease of the quinones and hydroquinones (Aguinaga et al., 2010; Dai et al., 2010), antimalarial quinones (Laurent et al., 2006), antiplasmodial benzaldehyde (Murtihapsari et al., 2019) and antiplasmodial sterol (Renga et al., 2012).

          During the course of our continuing search for antiplasmodial compounds from Indonesian marine sponge, the methanolic extract of Xestospongia sp exhibited a significant antiplasmodial activity against Plasmodium falciparum 3D7 strain. Xestospongia sp is distributed in the eastern part of Indonesia, and previous investigation have led to the isolation of several steroid with antiplasmodial activity (Muptihapsari et al., 2019). Owing to our interest in antiplasmodial compounds from this species, we investigated the ethyl acetate extract of the Xestospongia sp and obtained two antiplasmodial steroidal alkaloid. Here, we describe the structural identification of the isolates and their antiplasmodial activity.

MATERIALS AND METHODS

General

         Melting points were obtained on an electrothermal melting point apparatus. Optical rotations were measured on an ATAGO AP-300 automatic polarimeter. IR spectra were obtained with a Perkin Elmer spectrum-100 spectrophotometer using KBr pellets. Mass spectra were recorded on Water Qtof HR-MS XEV^otm mass spectrometers. 1D and 2D-NMR spectra were run on a JEOL ECZ A-600 spectrometer with tetramethyl silane as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Column chromatography was performed on silica gel (70-230 and 200-400 mesh, Merck, Germany) and RP-18 gel (20−45 μm, Fuji Silysia Chemical Ltd., Japan).  Fractions and spots were monitored by TLC (GF₂₅₄, Merck, Germany), and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH followed by heating.

Sponge materials

         Material of porifera Xestospongia sp, was obtained by scuba in about 10 m depth in the south west of Kaimana, West Papua, Indonesia (GPS: 4°20.341’ S-133°30.265’E). It has been identified taxonomically as Xestospongia sp. (Darumas et al., 2007), the species belongs to the Xestospongia. Identification taxonomic and nomenclature were provided by the Laboratory Biology and Conservation, Jakarta Fisheries University, Ministry of Marine Affairs and Fisheries (LIN BIOVASI, No: 041/STP-V/2016, Catalogue: MS041.1-5).

Parasite cultivation

        Plasmodium falciparum strain 3D7, which  is resistant to chloroquine (Singh and Rosenthal, 2001), was cultured in sealed flask at 37 ^oC, in a 3% O₂, 5% CO₂ and 91% N₂  atmosphere in RPMI 1640, 25 µM HEPES, pH 7.4, supplemented with heat  inactivated 10% human serum and human erythrocytes to achieve a 2% haematocrit.  Parasites were synchronized in the ring stage by serial treatment with 5% sorbitol and studied at 1% parasitemia (Lambros et al., 1979).

In vitro antiplasmodial

          Extracts and isolated compounds were prepared as 20 µg/mL stock solutions in DMSO (dimethyl sulfoxide), diluted as needed for individual experiment and tested in triplicate. The stock solutions were diluted in supplemented RPMI (Rosewell Parla Memorial Institute medium product) 1640 medium so as to have, at most, 0.2% DMSO in the final reaction medium. An equal volume of 1% parasitemia, 4% haematocrit culture was there after added and gently mixed thoroughly. Negative controls contained equal concentration of DMSO. Positive control contained an artemisinin. Cultures were incubated at 37 ^oC for 48 hours (one parasite erythrocytic life cycle). Parasites at ring stage were thereafter fixed by replacing the serum medium by an equal volume of 1% formaldehyde in PBS. (Phosphate Buffered Saline) aliquots (50 µL) of each culture were then added to 5 mL round-bottom polystyrenes tubes containing 0.5 mL 0.1% Triton X-100 and 1 nM YOYO nuclear dye (barand YOYO^®-1) in PBS. Parasitemias of treated and control cultures were compared using a Becton-Dickinson FAC (brand BD-FAC™)  Sort flow cytometer to count nucleated (parasitized) erythrocytes. An antiplasmodial examination was used 96 wells, each well filled by parasitemia culture 1%. About 50 µL of the compounds filled into the well with the following concentration 10^-9 to 10^-2 µg/mL. These data were normalized to percent control activity and 50% inhibitory concentration (IC₅₀) calculated using Table probit.

         Different concentration of the extract and isolated compounds were incubated at 37 ^oC with cultured 3D7 strain of P. falciparum parasites for 48 hours. Parasites were thereafter fixed and strained, and parasitemias of treated and control cultures were determined. Results are means, compared to untreated controls from 3 experiments. Error bars represent standard deviations of results (Bagavan et al., 2011).

RESULTS 

Extraction and isolation

          The milled fresh of marine sponge, Xestospongia sp (38 kg), were extracted with ethanol (30 L) at room temperature for 3 days. The ethanol extract was evaporated in vacuum to give a semisolid residue (376 g). After being suspended in water (2.0 L), the solution was successively fractionated with n-hexane, EtOAc and n-BuOH. Evaporation in vacuum resulted in the crude extracts of n-hexane (10.3 g), EtOAc (11.4 g), and n-BuOH (19.6 g), respectively. All extracts were evaluated for their antiplasmodial activity against P. falciparum 3D7 and showed antiplasmodial activity with IC₅₀ values of 1.95, 0.41, and 1.68 µg/mL, respectively.

         The EtOAc extract (11.4 g) was applied to silica gel column chromatography, eluted with n-hexane-EtOAc (100:1 to 1:100, v/v) to produce eight fractions (F.1-8). Fraction F.4 (420 mg) was subjected to silica gel column chromatography eluted with n-hexane-acetone (100:1 to 1:100, v/v) to produce six subfractions, 4A-4F. Subfraction 4C (130 mg) was applied to silica gel column chromatography using CHCl₃-MeOH (9.5:0.5, v/v), then octa desyl silane (ODS) eluting with MeOH-H₂O (2:3, v/v) to afford 1 (12.3 mg). F.6 (370 mg) was subjected to silica gel column chromatography eluted with n-hexane-acetone (100:1 to 1:100, v/v) to produce seven subfractions, 6A-4G. Subfraction 6E (130 mg) was applied to silica gel column chromatography using n-hexane-acetone (8:2, v/v) to afford 2 (14.6 mg). 

Epoxysarcovagenine-D (1). Yellowish solid, m.p. 113-115 ^oC; [α]²⁰_D +16.5^o (c = 0.1, CHCl₃); UV λ_max (MeOH) nm (log ɛ) 280 (5.2); IR (KBr) n_max  cm^-1:  3440, 3240, 2920, 2880, 1670, 1645, 1146, 1056; ¹H-NMR (CDCl₃, 600 MHz): Table 1;  ¹³C-NMR (CDCl₃, 125 MHz): Table 1; HR-TOFMS m/z 441.3040 [M+H]⁺, calcd. for C₂₇H_4ON₂O₃ m/z 440.3039.

Epoxynepapakistamine-A (2). White crystals; m.p 118-120 ^oC; IR (KBr) n_max cm^-1: 3446, 2945, 1728, 1660, 1150, 847; ¹H-NMR (CDCl₃, 600 MHz): Table 1; ¹³C-NMR (CDCl₃, 125 MHz): Table 1; HR-TOFMS m/z 559.3670 [M+H]⁺, calcd. for C₃₂H_5ON₂O₆ m/z 558.3669.

DISCUSSION   

The ethanolic extract from the milled fresh of Xestospongia sp was concentrated and extracted successively with n-hexane, ethyl acetate and n-butanol. All of the extracts were evaluated for their antiplasmodial activity against P. falcifarum 3D7 in vitro and the ethyl acetate extract exhibited strongest antiplasmodial activity with an IC₅₀ value of 0.41 µg/mL. By using antiplasmodial assay to guide separations, the ethyl acetate fraction was separated by combination of column chromatography on silica gel and octadesylsilane to afford two antiplasmodial steroidal alkaloids, 1 and 2  (Figure 1).

Figure 1. The chemical structures of 1 and 2 isolated from Xestospongia sp.

Figure 2. Selected ¹H-¹H COSY and HMBC Correlations for Compounds 1 and 2.

         Compound 1 was isolated as yellowish solid; m.p 113-115 ^oC; [α]²⁰_D +16.5^o (c = 0.1, CHCl₃). Its molecular formula was determined as C₂₇H_4ON₂O₃ (nine indices of hydrogen deficiency) by HR-TOFMS m/z 441.3040 [M+H]⁺ and NMR spectral data (Table 1). The UV spectrum showed an absorption maximum at 280 nm (log ɛ 5.2), indicating the presence of an enone system (Supratman et al., 2000). Infrared (IR) absorption bands  due to amine, conjugated carbonyl, amide and ether groups were observed at 3440, 1670, 1645 and 1146 cm^-1, respectively.

          The ¹H-NMR spectrum of 1, displayed the presence of two tertiary methyl signals at δ_H 0.82 and 0.87 (each 3H, s, Me-18 and Me-19), a secondary methyl at [δ_H 1.12 (3H, d, J=6.5 Hz, Me-21), two methyl proton of tigloyl group at δ_H [2.40 (3H, d, J=6.9 Hz, Me-3¢) and 2.53 (3H, d, J=1.2 Hz, Me-5¢)] and a N_b-Me at δ_H 3.26 (3H, s). Proton signals of olefinic protons at δ_H [7.55 (1H, dd, J=6.9, 2.6 Hz, H-2) and 6.34 (1H, dd, J=6.9, 1.2 Hz, H-3¢)], an oxygenated sp³ methine at δ_H 3.10 (1H, dd, J=6.7, 2.4 Hz, H-16) and two N-H protons at δ_H [8.03 (1H, br.s, N_a-H) and 2.10 (1H, br.s, N_b-H) were also observed in the H-NMR spectrum. The ¹³C NMR spectrum showed 27 signals, which are clarified by their chemical shifts and the DEPT spectra, consisting of six methyls, six methylenes, eight methines (including two olefinic carbons at δ_C 125.9 and 131.2), three sp³ quaternary carbons, two sp² quaternary carbons at δ_C 132.1 and 131.5, a carbonyl ketone at δ_C 196.2 and a carbonyl amide at δ_C 168.0. These functionalities accounted as four out of the total nine indices of hydrogen deficiency. The remaining five indices of hydrogen deficiency were consistent to  the pregrane-type steroidal skeleton with a tigloyl amino group, N-methylaminoethane substituent and additional an epoxide ring (Kaulanai et al., 2002; Farabi et al., 2018; Wu et al., 2019).  To clarify the position of functional group in 1, 2D NMR experiments were carried out and the results as shown in Figure 2. In the ¹H-¹H COSY spectrum of 1 displayed correlations in C₁-C₂, C₅-C₆-C₇-C₈-C₉, C₁₁-C₁₂, C₁₄-C₁₅-C₁₆ and C4'-C3' supporting the presence of pregnane-type steroidal structure with a tigloyl group and additional an epoxide ring. The HMBC correlations from tertiary methyl signals at δ_H 0.82 and 0.87 to C-10 (δ_C 38.0), C-1 (δ_C 38.9), C-5 (δ_C 54.2) and C-13 (δ_C 41.6), C-12 (δ_C 32.8), C-14 (δ_C 45.2) was enabled to assign two tertiary methyls were attached at C-10 and C-13, respectively. An olefinic signal at δ_H 7.55 was correlated to C-1 (δ_C 38.9), C-3 (δ_C 132.1) and C-4 (δ_C 196.2), whereas H-5 (δ_H 2.28) was correlated to C-4 (δ_C 196.2), suggest the position of α,β-unsaturated ketone was located at C-2, C-3 and C-4. Another olefinic signal at δ_H 6.34 was correlated to Me-4'(δ_C 12.20), Me-5' (δ_C 13.9) and carbonyl amide (δ_C 168.0), suggested a tigloyl amino group was attached at C-3. An oxygenated proton at δ_H 3.10 was correlated to C-17 (δ_C 72.9) and C-15 (δ_C 27.0), whereas methine proton at δ_H 1.36 was correlated to C-16 (δ_C 59.4), indicated that epoxy ring was located at C-16 and C-17. A methine proton at δ_H 2.51 was mutually coupled to methyl at δ_H 1.12 and were correlated to oxygenated carbon at C-17 (δ_C 72.9), indicated N-methylaminoethane substituent was attached at C-17. The relative stereochemistry of 1 was determined on the basis of coupling constant in the ¹H-NMR spectrum and a biogenetic point of view the occurrence of pregnane-type steroidal structure in natural (Rahman et al., 2002; Farabi et al., 2018).  These observations along with the similarity of spectral data and physicochemical properties between 1 and previously reported (20S)-20-(N-methylamino)-3β-(tigloyl-amino)-5α-pregnan-16α,17α-epoxy-4-one, isolated from the leaves Sarcococca coriacea (Kaulani et al., 2002; Wu et al., 2019), let us identify 1 as (20S)-20-(N-methylamino)-3β-(tigloyl-amino)-5α-pregnan-16α,17α-epoxy-4-one, which isolated from marine sponge Xestospongia sp for the first time.

          Compound 2 was isolated as white crystal; m.p 113-115 ^oC; [α]²⁰_D +20.5^o (c = 0.2, CHCl₃). Its molecular formula was determined as C₃₂H_5ON₂O₆ (nine indices of hydrogen deficiency) by HRTOFMS m/z m/z 559.3670 [M+H]⁺ and NMR spectral data (Table 1). The UV spectrum showed an absorption maximum at 285 nm (log ε 5.1) Shiono et al., 2016, indicating the presence of an enone system. Infrared (IR) absorption bands  due to amine, conjugated carbonyl, amide and ether groups were observed at 3,420, 1,720, 1,670 and 1,146 cm^-1, respectively. NMR spectral of 2 was very similar to those of 1, except the absence of α,β-unsaturated ketone at C-2, C-3 and C-4 at  [δ_H 7.55 (1H, d, J=6.9, 2.6 Hz), δ_C 125.9, 132.1 and 196.2] and appeared the oxygenated carbons at [δ_H 4.53 (1H, d, J=9.3), δ_C 71.7 and 4.52 (1H, dd, J=8.7, 2.4 Hz), δ_C 74.1,)] and two acetyl groups [δ_H 2.05 and 2.00 (each 3H, s), δ_C 20.9, 21.3, δ_C 170.1 and 170.4], suggesting that 2 was an acetyl derivative of 1. An oxygenated proton at δ_H 4.53 was correlated to C-3 (δ_C 50.0), C-1 (δ_C 40.2) and carbonyl ester at C-1'' (δ_C 170.1), whereas another an oxygenated proton at δ_H 4.52 was correlated to
C-3 (δ_C 50.0), C-5 (δ_C 48.7) and carbonyl ester at C-1''' (δ_C 170.4), suggested two acetyl groups was located at C-2 and C-4, respectively.

      These observations along with the similarity of spectral data and physicochemical properties between 1 and previously reported (20S)-20-(N-methylamino)-3β-(tigloyl-amino)-5α-pregnan-16α,17α-epoxy-2β,4β-di-O-acetate, isolated from the leaves Sarcococca coriacea (Kaulani et al., 2002; Wu et al., 2019), let us identify 2 as (20S)-20-(N-methylamino)-3β-(tigloyl-amino)-5α-pregnan-16α,17α-epoxy-2β,4β-di-O-acetate, which also isolated from marine sponge Xestospongia sp for the first time.

           The antiplasmodial activity of the two isolated compounds 1 and 2 against P. falciparum was conducted according to the method described in previous paper (Frӧhlich et al., 2016) and were used an artemisinin (5.20 x 10^-6 µM) as a positive control (Ravikumar et al., 2012; Fröhlich et al., 2016). Compounds 1 and 2 showed strong antiplasmodial activity with IC₅₀ values of 0.013 and 0.158 µM, respectively, suggesting the presence of acetyl groups can decrease antiplasmodial activity, in contras with the presence of an enone group can increase antiplasmodial activity. Both compounds, (20S)-20-(N-methylamino)-3β-(tigloyl-amino)-5α-pregnan-16α,17α-epoxy-4-one (1) and (20S)-20-(N-methylamino)-3β-(tigloyl-amino)-5α-pregnan-16α,17α-epoxy-2β,4β-di-O-acetate (2) have been isolated as a potent cholinesterase inhibiting compounds from Sarcococca coriacea (Kaulani et al., 2002), but the antiplasmodial activity was shown in this study for the first time.

Table 1.   NMR Data (600 MHz for ¹H and 150 MHz for ¹³C, in CDCl₃) for 1 and 2.

 CONCLUSIONS

Two antiplasmodial steroidal alkaloids have been isolated from marine sponge, Xestospongia sp and identified by spectroscopic methods as epoxysarcovagenine-D (1) and  epoxyepapakistamine-A (2). Compounds 1 showed stronger antiplasmodial activity than compound 2 with an IC₅₀ value of 0.013 µM, suggesting the presence of acetyl groups can decrease antiplasmodial activity, whereas the presence an enone group can increase antiplasmodial activity.

ACKNOWLEDGMENTS 

This investigation was financially supported by the Domestic Postgraduate Education Scholarship (BPPDN Grant) from Directorate General of Higher Education, Ministry of Research, Technology and Higher Education, Indonesia (Period: 2014-2017 granted for Murtihapsari). We are extremely grateful to Regis Hocde, Laurent Pouyaud, Amir M. Suruwaky and the Team of Expedition Lengguru 2014 (LIPI, Politeknik KP Sorong, IRD-France) who kindly helped authors during sampling marine sponges in Kaimana. Authors also express thank to Jonathan M. Marbun for the kind support during antiplasmodial  assay work at the Eijkman Institute for Molecular Biology in Jakarta.

REFERENCES

Aguinaga, N.M., Mercado, I.E.S., and Williams, P. 2010. Xestosaprol D and E from Indonesian marine sponge Xestospongia sp. Tetrahedron Letters. 51(4): 751-753. https://doi.org/10.1016/j.tetlet.2009.11.132

Armstrong, R.A., Dingh, H., Torres, J., Nemeth, R.S., Can, A., Roman, C., Eustice, R., Riggs, L., and Moliner, G.G. 2006. Characterizing the deep insular shelf coral reef habitat of the Hind Bank Marine conservation district (US virgin Islands) using the seabed autonomous underwater vehicle. Continental Shelf Research. 26(2): 194-205. https://doi.org/10.1016/j.csr.2005.10.004

Ashok, P., Ganguly, S., and Murugesan, S. 2014. Manzamine alkaloids: isolation, cytotoxicity, antimalarial activity and SAR studies. Drug Discovery Today. 19(11): 1781-1791. https://doi.org/10.1016/j.drudis.2014.06.010

Bagavan, A., Rahuman, A.A., Kaushik, N.K., and Sahal, D. 2011. In vitro antimalarial activity of medicinal plant extracts against Plasmodium falciparum. Parasitology Research. 108(1): 15-22. https://doi.org/10.1007/s00436-010-2034-4

Bell, J.J., and Smith, D. 2004. Ecology of sponge assemblages (Porifera) in the Wakatobi region, south-east Sulawesi, Indonesia: richness and abundance. Journal of the Marine Biological Association of the United Kingdom. 84(3): 581-591. https://doi.org/10.1017/S0025315404009580h

Calcul, L., Longeon, A., Mourabit, A.A., Guyot, M., and Bourguet, K.M.I. 2003. Novel alkaloid of the aaptamine class from an Indonesian marine sponge of the genus Xestospongia. Tetrahedron. 59(34): 6539-6544. https://doi.org/10.1016/S0040-4020(03)01069-X 

Dai, J., Sorribas, A., Yoshida, W.Y., Kelly, M., and Williams, P.G. 2010. Xestosaprols from Indonesian Marine Sponge Xestospongia sp. Journal of Natural Products. 73(6): 1188-1191. https://doi.org/10.1021/np100203x

Darumas, U., Chavanich, S., and Suwanborirux, K. 2007. Distribution patterns of the renieramycin-producing sponge, Xestospongia sp., and its association with other reef organisms in the Gulf of Thailand. Zoological Studies. 46(6): 695-704.

Farabi, K., Harneti, D., Nurlelasari., Maharani, R., Hidayat, A.T., Awang, K., Supratman, U., and Shiono, Y. 2018. New cytotoxic pregnane-type steroid from the stem bark of Aglaia elliptica (Meliaceae). Record of Natural Products. 12(2): 121-127. https://doi.org/10.25135/rnp.21.17.07.118

Frӧhlich, T., Karagӧz, A.C., Reiter, C., and Tsogoeva, S.B. 2016. Artemisinin-derived dimers: potent antimalarial and anti-cancer agents. Journal of Medicinal Chemistry. 59: 7360-7388. https://doi.org/10.1021/acs.jmedchem.5b01380

Fromont, J. 1991. Descriptions of species of the Petrosida (Porifera: Demospongiae) occurring in the tropical waters of the Great Barrier Reef. Beagle Records Northern Territory Museum of Art and Sciences. 8: 73-96.

Girard, E., Jaimovitch, E., Vergne, C., Almourabit, A., Laurent D., and Molgo, J. 2004. In rencontres en toxinologie. Envenimations, intoxications. Lavoisier. 141–152.

Kerr, R.G., and Borges, M.K. 1994. Biochemical and morphological heterogeneity in the Caribbean sponge Xestospongia muta (Pterosida: Petrosiidae). Sponges in Time and Space. Proceedings of the 4^th International Porifera Congress. 4: 65-73.

Kaulani, S.K., Choudhary, M.I., Khalid, A., Manandhar, M.D., Shaheen, F., Rahman, A-ur., and Gewali, M.B. 2002. New cholinesterase inhibiting steroidal alkaloids from the leaves of Sarcococca coriacea of Nepalese origin. Chemical and Pharmaceutical Bulletin. 50(11): 1423-1426. https://doi.org/10.1248/cpb.50.1423

Kritsanapuntu, S.N., Chaitanawisuti, T., Yeemin, S., and Putchakan, S. 2001. First investigation on biodiversity of marine sponges associated with reef coral habitats in the eastern Gulf of Thailand. Asian Marine Biology. 18: 105-115.

Laurent, D., Valerie J., Arnaud P., Martine K., Dominique D., Sophie S., Olivier L., Nicolas L., Maryvonne F., Frederic A., et al. 2006. Antimalarial potential of xestoquinone, a protein kinase inhibitor from a vanuatu marine sponge Xestospongia sp. Bioorganic & Medicinal Chemistry. 14(13): 4477-4482. https://doi.org/10.1016/j.bmc.2006.02.026

Lambros, C., and Vanderberg, J.P.J. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. The Journal of Parasitology. 65(3): 418-420. https://doi.org/10.2307/3280287 

Moon, S.S., MacMillan, J.B., Olmstead, M.M., Ta, T.A., and Molinski, T.F. 2012. (+)-7S-Hydroxyxestospongin A from the marine sponge Xestospongia sp. and absolute configuration of (+)-xestospongin D. Journal of Natural Products. 65(3): 249-254. https://doi.org/10.1021/np010427z

Murtihapsari, M., Salam, S., Kurnia, D., Darwati, D., Kadarusman, K., Abdullah, F.F., Herlina, T., Husna, M.H.,  Awang, K., Shiono^, Y., et al. 2019. A New Antiplasmodial Sterol from Indonesian marine sponge, Xestospongia sp. Natural Product Research. 18: 1-8. https://doi.org/10.1080/14786419.2019.1611815

Rahman, A-ur., Ul-Haq, Z., Khalid, A., and Choudhary, I.M., 2002. Pregnane-type steroidal alkaloids of Sarcococca saligna: a new class of cholinesterase inhibitors. Helvetica Chimica Acta. 85(2): 678-688. https:// doi.org/10.1002/1522-2675(200202)85:2<678::AID-HLCA678>3.0.CO; 2-2

Ravikumar, S., Inbaneson, S.J., and Suganthi, P. 2012. In vitro antiplasmodial activity of chosen terrestrial medicinal plants against Plasmodium falciparum. Asian Pacific Journal of Tropical Disease. 2(7): S252-180-256.

Renga, B., Mencarelli, A., D’Amare, C., Cipriani, S., D’Auria, M.V., Sepe, V., Chini, M.G., Monti, M.C., Bifulco, G., Zampella, A., et al. 2012. Discovery that theonellaasterol a marine sponge sterol is a highly selective FXR antogonist that protects against liver injury in cholestatis. PLOS ONE. 7(1): 1-12. https://doi.org/10.1371/journal.pone.0030443

Shiono, Y., Miyazani, N., Murayama, T., Koseki, T., Harizon, Katja, D.G., Supratman, U., Nakata, J., Kakihara, Y., Seaki, M., et al. 2016. GSK-3ß inhibitory activities of novel dichloresorcinol derivatives from Cosmospora vilior isolated from a mangrove plant. Phytochemestry Letters. 18: 122-127. https://doi.org/10.1016/j.phytol.2016.09.007

Singh, A., and Rosenthal, P.J. 2001. Comparison of efficacies of cysteine protease inhibitors against five strains of Plasmodium falciparum. Antimicrobial Agents and Chemotherapy. 45(3): 949-951. https://doi.org/10.1128/AAC.45.3.949-951.2001

Supratman, U., Fujita, T., Akiyama, K., and Hayashi, H. 2000. New insecticidal bufadienolide, bryophyllin C, from the leaves of Kalanchoe pinnata. Bioscience, Biotechnology, and Biochemistry. 64(6): 1310-1312. https://doi.org/10.1271/bbb.64.1310

Williams, D.E., Lassota, P.  and Andersen, R.J. 1998. Motuporamines A-C, cytotoxic alkaloids isolated from the marine sponge Xestospongia exigua (Kirkpatrick). The Journal of Organic Chemistry. 63(14): 4838-4841. https://doi.org/10.1021/jo980355p

Wu, J.C., Huo, S.J., and Du, J. 2019. 4-dehydroxyepisarcovagine A, a new steroidal alkaloid from Sarcococca pruniformis Lindl. Natural Product Research. 33(2): 169-173. https://doi.org/10.1080/14786419.2018.14400226