Evaluation of hexane and ethyl acetate extracts of the sponge Jaspis diastra collected from Mauritius Waters on HeLa cells.

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Research Paper

Journal of Pharmacy And Pharmacology

Evaluation of hexane and ethyl acetate extracts of the sponge Jaspis diastra collected from Mauritius Waters on HeLa cells Girish Beedesseea*, Avin Ramanjoolooa*, Inés Tiscorniab*, Thierry Cresteilc, Srinivasarao Raghothamad, Deepak Aryae,f, Shashanka Raog, Konkallu Hanumae Gowdg, Mariela Bollati-Fogolinb and Daniel E.P. Mariea a Mauritius Oceanography Institute (MOI), Quatre-Bornes, Mauritius, bCell Biology Unit (CBU), Institut Pasteur de Montevideo (IPMon), Montevideo, Uruguay, cInstitut de Chimie des Substances Naturelles, UPR 2301 CNRS, Gif-sur-Yvette, France, dNMR Research Centre and gMolecular Biophysics Unit, Indian Institute of Science (IISc), eNational Centre for Biological Sciences (NCBS), Bangalore and fManipal University, Manipal, Karnataka, India

Keywords cytotoxic; HeLa cells; Jaspis diastra; Mauritius; sponges Correspondence Daniel E.P. Marie, Mauritius Oceanography Institute, 4th Floor, France Centre, Avenue Victoria, Quatre-Bornes, Mauritius. E-mail: [email protected] Received November 28, 2013 Accepted March 2, 2014 doi: 10.1111/jphp.12256 *These authors contributed equally.

Abstract Objectives Based on previous screening results, the cytotoxic effect of the hexane (JDH) and ethyl acetate extracts (JDE) of the marine sponge Jaspis diastra were evaluated on HeLa cells and the present study aimed at determining their possible mechanism of cell death. Methods Nuclear staining, membrane potential change, flow cytometry analysis of cell cycle distribution and annexin V staining were undertaken to investigate the effects of JDE and JDH. Electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance were used to characterize an isolated bioactive molecule. Key findings JDE displayed an IC50 25 times more significant than the JDH. Flow cytometry analysis revealed JDE induced apoptosis in HeLa cells accompanied by the collapse of mitochondrial membrane potential. Fractionation of JDE resulted in the isolation of the known cytotoxic cyclodepsipeptide, Jaspamide. Conclusions Taking our results together suggest that JDE can be valuable for the development of anticancer drugs, especially for cervical cancer. Further investigations are currently in progress with the aim to determine and isolate other bioactive compounds from this extract.

Introduction Marine organisms have proved to be potential sources of bioactive compounds with therapeutic importance with sponges, bryozoans and tunicates being the most promising organisms for sources of new active compounds.[1–3] Because of the prevalence, ease of collection and ability to synthesize a wide array of structurally diverse compounds, marine sponges have become an important source of biologically active natural products.[4] Marine sponges are the most primitive multicellular animals and contain many pharmacologically important metabolites. Sponges of the genus Jaspis (family, Jaspidae) have been an interesting and important source of biologically active natural products. This genus has received extensive consideration since the discovery of the cyclodepsipeptide Jaspamide in the sponge Jaspis cf. johnstoni and its associated prominent biological

activities including antifungal, antihelminthic, insecticidal and cytotoxic activities.[5–8] Cervical cancer is a common disease in women and the carcinoma of the uterine cervix is one of the highest causes of mortality in female cancer patients around the world and due to inadequate or poor screening, the disease is detected at late stage. Chemical as well as biological agents that induce apoptosis have shown promise in the understanding and management of such cancers.[9] In previous studies, we have evaluated organic extracts from 20 species of marine sponges from Mauritius against nine human cell lines and found the sponge Jaspis diastra to display cytotoxicities ≥ 75%.[10] According to the World Register of Marine Species, the sponge Jaspis diastra is reported only around Madagascar.[11] This is the first study to describe the

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 1317–1327

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Cytotoxicity of the sponge Jaspis diastra

Girish Beedessee et al.

occurrence and the cytotoxic activity of extracts of this sponge from Mauritius Waters with this species being endemic to the Indian Ocean. We found the ethyl acetate (JDE) and hexane extracts (JDH) to possess potent apoptotic effect on HeLa cells and investigations were undertaken to elucidate molecular mechanisms of this apoptotic effect.

Materials and Methods

ATCC, Rockville, MD, USA). Epidermoid carcinoma (KB) cell line was obtained from Dr Thierry Cresteil (ICSN, CNRS, Gif-sur-Yvette, France). Cells were cultured in RPMI-1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 0.1% gentamicin, in a humidified incubator containing 5% CO2 at 37°C and upon reaching 75% confluence were passaged with a solution of 0.25% trypsin-EDTA.

MTT assay

Chemicals Hexane, methanol, dichloromethane and ethyl acetate were from SDFC Limited (Mumbai, Maharashtra, India). RPMI-1640, fetal bovine serum (FBS), trypsinethylenediaminetetraacetic acid (EDTA) and L-glutamine were from PAA cell culture (Pasching, Austria). Dimethylsulfoxide (DMSO), 5,5′,6,6′-tetrachloro-1,1′,3, 3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1), 3(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33258 and gentamicin were from Sigma (St. Louis, MO, USA). CellTiter 96 Aqueous NonRadioactive Cell Proliferation Assay (MTS) was from Promega (Charbonnières, France).

Species collection and preparation of fractions

Cytotoxicity was determined as previously described with modifications.[12] Briefly, 1 × 104 HeLa cells were seeded in a 96-well plate and incubated for 24 h at 37°C. After this incubation period, cells were treated with different concentration of JDE and JDH extracts (0.05–100 μg/ml) and incubated for 48 h. Control groups received DMSO (final concentration did not exceed 0.5%). After treatment, the conditioned medium was replaced with 200 μl of fresh medium containing MTT (5 mg/ml) and plates were further incubated for 4 h at 37°C. The formazan was dissolved in 100 μl DMSO and the absorbance was measured using a microplate reader at 560 nm and subtracted at 670 nm. Cytotoxicity was determined using ((1 − (ODtreated/ ODcontrol) × 100). Experiments were performed in triplicate.

Clonogenic survival determination

The sponge specimen studied in this work was collected around the island of Mauritius through scuba diving at depth of 15–20 m. Samples were photographed in situ for better species characterization and identification. Samples were characterized and identified with the help of Professor Rob Van Soest as Jaspis diastra. A voucher sample (ZMAPOR21788) was deposited at the Zoological Museum of University of Amsterdam, the Netherlands. Freshly collected sponge specimen was set free of any debris, cut into small pieces and weighted. The sponge (wet mass: 456.67 g) was exhaustively macerated with methanol and dichloromethane (1 : 1). After maceration, the solution was filtered and evaporated to dryness on a rotary vacuum evaporator set at a maximum temperature of 40°C to obtain the crude extract (17.17 g). The latter was dissolved in distilled water and partitioned with n-hexane and ethyl acetate to afford non-polar extract (2.28 g) and semi-polar extract (0.14 g). The extracts were stored at −20°C until use. For all experiments, JDE and JDH were dissolved in DMSO (10 mg/ml) and further diluted in RPMI-1640 medium for the final testing concentration. Control cultures received the carrier solvent (0.5% DMSO).

Total DNA was extracted using Favorprep DNA extraction mini kit according to the supplier’s manual (Favorgen Biotech Corp, Ping-Tung, Taiwan). Briefly, 5 × 105 HeLa cells were cultured overnight in 12-well plate and then treated with 10 and 50 μg/ml of JDE or JDH extracts for 24 h and 48 h. Samples were resolved on a 1.8% agarose gel at 50 V for 120 min using Tris-borate/EDTA electrophoresis buffer and visualized using ethidium bromide stain under UV transillumination.

Cell line and cell medium

Nuclear staining

Human cervical adenocarcinoma cell line (HeLa) was purchased from the American type culture collection (CCL-2,

HeLa cells were exposed to 5 and 25 μg/ml (for JDE) and 25 and 50 μg/ml (for JDH) of extracts, washed with PBS and

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HeLa cells were seeded in a 12-well plate (300/well) and incubated overnight. Cells were then exposed for 24 h to different concentrations of JDE and JDH (0.05–50 μg/ml) after which medium was aspirated and cells were rinsed with phosphate buffered saline (PBS). Fresh medium was added to each well and plate was incubated at 37°C. After 10 days of incubation, the cells were stained with Giemsa stain and colonies with >50 cells were counted under a dissection microscope. Experiments were conducted in triplicate. The plating efficiency of the cells was taken into consideration when calculation the surviving fraction.

DNA fragmentation analysis



fixed with 4% paraformaldehyde for 20 min. Cells were then washed with PBS, stained with Hoechst 33258 for 15 min at room temperature before being observed under a fluorescence microscope (Nikon Eclipse TE2000-S; Melville, NY, USA).


software for acquisition and analysis. For each sample 10 000 counts were analysed, gated on a FSC vs. SSC dot plot and excluding doublets.

Isolation of JDC3 Measurement of mitochondrial transmembrane potential We used the mitochondrial-specific cationic dye, JC-1, that undergoes potential dependent accumulation in the mitochondria. Approximately 1 × 104 HeLa cells were treated for various time period in presence of JDE or JDH, washed twice with PBS and incubated with 100 μl JC-1 (final conc. 1 mg/ml) for 30 min at 37°C in the dark. Fluorescence intensity (JC-1 green, Ex = 485, Em = 528; for JC-1 red, Ex = 530, Em = 590) was measured using a microplate reader (Synergy HT, BioTek, Winooski, VT, USA). The ratio of the red to green fluorescence of JC-1 was calculated. Experiments were performed in triplicate.

Annexin V and propidium iodide staining HeLa cells (5 × 104/well) were seeded in 24-well plates and incubated for 24 h. Then, cells were treated with JDE (10 μg/ml), JDH (10 μg/ml) or DMSO (0.1%) for 24 h. Culture media were carefully collected and gently centrifuged to collect floating cells; adherent cells were harvested after addition of trypsin. Mixed with the pellet of floating cells, washed with PBS and stained with fluorescein isothiocynate-labelled annexin V (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Propidium iodide (PI) was added immediately before acquisition to a final concentration of 2 μg/ml. Samples were analysed using a CyAn ADP (Beckman Coulter, Brea, CA, USA) flow cytometer and Summit 4.3 software was employed for acquisition and analysis. For each sample, 10 000 counts, gated on a forward scatter (FSC) vs. side scatter (SSC) dot plot, were acquired.

Selective extractions with hexane and ethyl acetate were repeated with another batch of crude extract of Jaspis diastra and total JDE (0.22 g) was used for column chromatography to isolate JDC3. JDE was fractionated using normal phase silica gel with stepwise elution of solvent system from low polarity to high polarity using hexane, ethyl acetate and methanol in various ratios viz (HexaneEtOAc: 1-0, 9.9-0.1, 9.5 : 0.5, 9 : 1, 8-2, 7-3, 6-4, 1-1, 4-6, 3-7, 2 : 8,0-1 and EtOAc: MeOH: 9.5-0.5, 9 : 1, 8-2, 7-3, 1-1, 0 : 1). Fractions were collected individually, concentrated by evaporation and checked by thin layer chromatography (TLC). Based on TLC analysis, similar fractions were mixed. TLC of the fraction (133-188) revealed one spot indicating the presence of one pure compound (JDC3) was isolated. On addition of hexane to this fraction, white precipitate was observed which was separated, washed with hexane and dried under vacuo. The mass of JDC3 obtained was 58.7 mg.

Mass spectrometry and NMR spectroscopy Electrospray ionization mass spectra were recorded in positive ion mode using HCT-Ultra ETD II ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). JDC3 was dissolved in methanol and analysed in the positive ion mode under conditions of capillary voltage 3 kV, nebulizing gas pressure 10 psi, drying gas flow rate 4 L/min and drying gas temperature 300°C. The data were processed using Esquire data analysis software, version 4.0 (Bruker Daltonics). Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AV700 MHz spectrometer. The 1D and 2D spectra were recorded in CDCl3 at 303 K. All processing was done using BRUKER TOPSPIN software.

Cell cycle analysis HeLa cells (4 × 105/well) were seeded in 6-well plates and incubated for 24 h. Then, cells were treated with JDE, JDH (10 μg/ml) or DMSO (0.1%) for 24–48 h. Culture media were carefully collected and gently centrifuged to harvest floating cells; adherent cells were collected after addition of trypsin, mixed with the pellet of floating cells, washed with PBS and fixed with 70% ethanol and kept at −20° until staining. Cells were rinsed with PBS-EDTA 2 mM, and incubated with RNAse A (50 μg/ml) for 30 min at 37°C. Then, PI (50 μg/ml) was added and incubated for further 30 min in the dark. Samples were analysed using a CyAn ADP (Beckman Coulter) flow cytometer and Summit 4.3

Cytotoxicity of JDC3 KB cells (650 cells/well) were plated in 96-well tissue culture microplates in 200 μl medium and treated 24 h later with 1 μg/ml and 10 μg/ml of JDC3 dissolved in DMSO. Controls received the same volume of DMSO (1% final volume). After 72 h exposure, MTS reagent (Promega) was added and incubated for 3 h at 37°C. Absorbance was monitored at 490 nm and results expressed as the inhibition of cell proliferation calculated as the ratio (1 − (OD490treated/ OD490control) × 100). Experiments were performed in triplicate.


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Statistical analysis

(a) 100

JDE JDH

80 % Inhibition

GraphPad Prism Software version 5.00 (San Diego, CA, USA) was employed for statistical analysis wherever appropriate. Experiments were performed in triplicates, and data are expressed as mean ± standard deviation. Data were analysed by Kruskal–Wallis test and the significant differences between groups were analysed by Dunnett’s test. Differences were considered statistically significant if P < 0.05.

Result and Discussion

60 40 20

Mechanism of action 0

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–2

–1

0 1 Log [conc]

2

3

(b) 70 60 Surviving fraction (%)

Treatment for cervical cancer has so far been unsatisfactory and there is urgent need to look for novel therapies that can help in controlling this malignancy. Cervical cancer epidemiology has been associated with human papillomavirus, oncogene expression (e.g. c-Myc, Ha-Ras and Erb-2) or upregulation of pro-inflammatory cytokines.[13–16] One such avenue has been to look at the anticancer potential of natural resources. The field of natural products chemistry has mostly been associated with isolation of compounds from terrestrial sources. However, the search for novel molecules from marine organisms has intensified since the 1970s with the acknowledgement of the ocean as an untapped resource with novel compounds.[17,18] Among these marine organisms, sponges are considered to be very attractive sources of molecules.[19] Marine natural products are known to possess a wide variety of biological activity and some of these compounds are thought to play a role in chemical defence of the organism from which they are isolated.[20] From our initial screening programme, we identified the sponge Jaspis diastra as an interesting candidate for further evaluation since it displayed a cytotoxicity ≥75% at 50 μg/ml on various cell lines namely HL-60, HeLa, KB and Mia-Paca.[10] In this regard, detailed investigations were undertaken here to define the mechanism by which the JDE and JDH of this sponge induce apoptosis on HeLa cells. Apoptosis is characterized by distinct morphological features such as cell shrinkage, chromatin condensation, membrane blebbing, DNA fragmentation and finally the formation of apoptotic bodies.[21] HeLa cells were exposed to different concentrations of JDE and JDH during 48 h and the percentage of growth inhibition was analysed using MTT assay. The IC50 (50% of growth inhibition) for JDH was calculated to be 25 ± 1 μg/ml while JDE showed an IC50 of 1 ± 0.5 μg/ml (Figure 1a). Furthermore, colony formation inhibition induced by JDE and JDH was assessed on cells treated during 24 h (Figure 1b). Control cells showed a surviving fraction of 98 ± 21% while with increasing JDE and JDH concentration, the survival capacity of HeLa cells decreased. Thus, a dose-dependent colony-forming inhibitory effect was observed for both extracts. The morphologi-

JDH JDE

50 40 30 20 10 0

0.05

0.5

1 5 10 Extract concentration (μg/ml)

50

Figure 1 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assay of human cervical adenocarcinoma cell line (HeLa) cells. (a) Inhibition of HeLa cells growth by ethyl acetate extract (JDE) and hexane extract (JDH). Cells were seeded onto 96-well plate, treated with the extracts at different concentrations; and percentage inhibition was determined by MTT assay. (b) Inhibition of HeLa cells colony formation by the extracts from the sponge Jaspis diastra. Cells were seeded onto 6-well plate (300/well) and treated with the extracts at different concentrations. The colony number was counted under dissection microscope. Dose-dependent colony formation inhibition was found. Results are expressed as mean values ± standard deviation (SD).

cal observations revealed apoptotic changes including chromatin condensation and fragmentation (Figure 2). The results demonstrated that 25 μg/ml JDE induced apoptosis while a high concentration of JDH (50 μg/ml) was needed to achieve similar effect. In addition, fragmentation of DNA strands was another noticeable feature, which was detected using agarose gel electrophoresis. The results indicated that JDE could cause more DNA fragmentation than JDH when treated with 10 and 50 μg/ml JDE/JDH for 24 and 48 h in a concentration- and time-dependent manner (Figure 3). ΔΨm collapse is known to play a crucial role in mediating apoptosis as it allows the release of apoptotic mediators




(a)

(b)

(d)

(c)

(e)

Figure 2 Changes in the morphology of human cervical adenocarcinoma cell line (HeLa) cells treated with ethyl acetate extract (JDE) and hexane extract (JDH) of Jaspis sp. under a fluorescence microscope. Morphological changes in untreated cells (a), JDE-treated (B-5 μg/ml and C-25 μg/ml) and JDH-treated (D-25 μg/ml and E-50 μg/ml) cells were examined by staining with Hoechst 33258. Control cells showed round and homogeneous nuclei while apoptotic cells displayed condensation and fragmentation of nuclei in the treated cells (the arrow point).

such as cytochrome c and apoptosis-inducing factor (AIF) into the cytoplasm. In turn, cytochrome c and AIF directly or indirectly activate members of the caspase family, considered as death effector molecules.[22] We examined the mitochondrial membrane potential in HeLa treated cells and noticed a significant decrease in both JDE- and JDHtreated cells (Figure 4). A drastic drop was noticed within the first 6 h of exposure to 10 μg/ml JDE. However, JDE achieved this decrease at a much lower concentration (0.5, 5 and 10 μg/ml) when compared with JDH (1, 10 and 50 μg/

ml). This was evidence for the alterations of permeability of the outer membrane of the mitochondria. The redistribution of membrane phosphatidylserine was investigated by simultaneously staining HeLa cells with annexin V and PI to discriminate between the apoptotic and necrotic cells.[23] Both compounds induced a significant raise in AV (+)/PI (–) cells, generally associated with early apoptotic events, and AV (+)/PI (+), related to late apoptotic and necrotic events (Figure 5). After 24 h of treatment with 10 μg/ml of both extracts, around 40–50% of


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L


JDE

80

JDH

% Cells

60

*

* 40 20

*

*

0 NT

JDH

JDE 10 μg/mL

Figure 5 Annexin V (AV)/propidium Iodide (PI) staining of human cervical adenocarcinoma cell line (HeLa) cells treated with ethyl acetate extract (JDE) and hexane extract (JDH) (10 μg/ml) for 24 h. Both extracts induced a significant increase in the percentages of AV (+)/PI (−) cells (grey bars) and in AV (+)/PI (+) cells (black bars). Results are indicated as median ± standard deviation of one experiment performed in duplicate. NT: non-treated cells.

Figure 3 DNA fragmentation analysis of ethyl acetate extract (JDE) and hexane extract (JDH)-treated cervical adenocarcinoma cell line (HeLa) cells. Cells were treated with JDE and JDH; total DNA was extracted and samples were analysed on 1.8% agarose gel. Lane 1: DNA ladder, lane 2: DMSO control, lanes 3–4: 10 and 50 μg/ml JDE (24 h), lanes 5–6: 10 and 50 μg/ml JDE (48 h), lanes 8–9: 50 and 10 μg/ml JDH (24 h), lanes 10–11: 50 and 10 μg/ml JDH (48 h).

JDE

180 Relative Fluorescence unit (590/528)

0.1% DMSO

160

0.5 μg/ml

140

5 μg/ml

120

10 μg/ml

100 80 60 40 20 0 6h

Relative Fluorescence unit (590/528)

180

12 h

18 h

JDH

160

1 μg/ml

140

10 μg/ml

120

50 μg/ml

100 80 60 40 20 0 6h

12 h

18 h

Figure 4 Effect of the ethyl acetate extract (JDE) and hexane extract (JDH) of Jaspis diastra on dissipation of mitochondrial transmembrane potential in human cervical adenocarcinoma cell line (HeLa) cells. Cells were treated for 6–18 h with 0.5, 5 and 10 μg/ml of JDE (left); 1, 10 and 50 μg/ml of JDH (right).

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(a) 100

24 h Sub G1

80 % Cells

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G1 S

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G2/M 40 * 20

*

0 Control

JDH 10 μg/mL

(b) 100

48 h

80 % Cells

JDE 10 μg/mL

*

60

*

40

*

20

*

0 Control

JDH 10 μg/mL

JDE 10 μg/mL

Figure 6 Ethyl acetate extract (JDE) and hexane extract (JDH) induce DNA fragmentation (sub-G1 peak). Human cervical adenocarcinoma cell line (HeLa) cells were treated with JDE or JDH at 10 μg/ml for 24 (a) and 48 (b) h. Columns indicate % of cells in different phases of cell cycle (Sub-G1, G1, S and G2/M). Flow cytometry analysis was performed and a significant increase in sub-G1 peak was observed, especially for JDE. Results are indicated as median ± standard deviation of one experiment performed in triplicate.

cells were in early apoptosis revealing both JDE and JDH to be strong inducers of apoptosis. To determine JDE/JDHinduced apoptosis in HeLa cells, we investigated the effect on the nuclear changes by flow cytometry. As compared with control, the percentage of cells in the sub-G1 peak increased after the exposure to extracts; however, this increased accumulation of cells in the sub-G1 phase was much more evident in JDE-treated cells (24 h: 19%; 48 h: 28%) than JDH-treated cells (Figure 6). The increase in sub-G1 population in our results indicates the induction of apoptosis, as sub-G1 peak is reported to be a quantitative indicator of apoptosis.[24] Fractionation of the JDE from the sponge Jaspis diastra resulted in the isolation of the cyclodepsipeptide, Jaspamide, which has been shown to be active against 36 human solid tumour cell cultures.[8] The isolation of new natural jaspamide derivatives has provided insights in our understanding of their structures and activity. Jaspamide is known to bind to F-actin, promotes actin polymerization under non-polymerizing conditions and lower the critical concentration of actin assembly in vitro.[25] The cytotoxicity of JDC3 (Jaspamide) from JDE was evaluated on KB cells and found to be 96 ± 1% at 10 μg/ml and 96 ± 1% at 1 μg/ml.

Elucidation of JDC3 The electrospray ionization mass spectrometry (ESI-MS) spectrum of JDC3 showed a pseudomolecular ion peak at m/z 709.280 (MH+), which corresponded to that of Jaspamide (Figure 7a), the parent compound.[7] The complete structure of JDC3 was elucidated based on 1H and 13 C-NMR, total correlation spectroscopy (TOCSY), heteronuclear multiple-quantum correlation spectroscopy (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral data and were found to be Jaspamide. Jaspamide is a 19-membered ring made up of four moieties, namely an alanine moiety, an abrine (N-methyl tryptophan) moiety, a β-tyrosine moiety and a polypropionate moiety (11-carbon hydroxyl acid containing four methyl groups on alternating carbons).[5,7,26,27] The presence of each of the four moieties in JDC3 was confirmed by 1D and 2D NMR as follows: (1) The alanine moiety showed 1H-NMR signals at δH 4.71 (quin, 6.7) for H-15, 0.75 δH (d, 6.7) for Me-16 and δH 6.59 (1H, d, 6.7 Hz) for the NH-Ala. 13CNMR signals at δC 174.0, δC 46.0 and δC 17.7 ppm were assigned to C-14, C-15 and C-16, respectively. Moreover, TOCSY and HMBC experiments (Table 1) confirmed the


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×104

2.5 731.382

Intens. [a.u.]

(a)

2.0

709.280

1.0

747.384

1.5

0.5

0.0 720

710

740

730

(b)

750

760

770

m/z

OH 30 β-Tyrosine O

27 β HN

21 18 19

β

24 26 HN

9

11

Tryptophan

O

α

12

7 O

α N

33

O

5

Br 17 Alanine β 16

34

14 15 α

Polypropionate NH

3

35

1 O

36

Figure 7 (a) Electrospray ionization mass spectrometry (ESI-MS) spectrum of JDC3; (b) Key heteronuclear multiple-bond correlation (HMBC) correlations of JDC3 (Jaspamide).

presence of the alanine moiety. (2) The indole moiety of the 2-bromoabrine (N-methyl tryptophan) moiety showed 1 H-NMR resonances at δH 8.19 (1H, s), δH 7.24 (1H, d, 8.0 Hz), δH 7.11 (1H, t, 8.0 Hz), δH 7.15 (1H, t, 8.1 Hz) and δH 7.55 (1H, d, 7.8 Hz) that were assigned to NH-Trp and H-21 to H-24, respectively.[7,27] 13C-NMR signals observed at δC 168.5, δC 55.4, δC 23.2, δC 110.8, δC 132.1, δC 110.5, δC 122.6, δC 120.3, δC 118.2, δC 136.1 and δC 109.0 ppm were assigned to C-12, C-13 and C-18 to C-26, respectively. The 1324

presence of the methyl group (Me-17) of the tryptophan moiety was confirmed by HMBC correlation of the crosspeak between Me-17 and H-13. (3) The p-substituted phenol of the β-Tyrosine moiety was confirmed based on 13 C-NMR signals at δC 155.1 and δC 132.1 ppm and were ascribed to C-30 and C-27, respectively. The presence of the proton attached to NH-Tyr was confirmed by 1H-NMR signal at δH 7.50 (1H, d, 8.8 Hz). Carbon atoms C-9, C-10 and C-11 of β-tyrosine moiety were confirmed based on



Table 1 C 1 2 3 4 5 6 7

1D and 2D NMR data (700 MHz, CDCl3) of JDC3 δC (ppm) (type)a 174.7 (C = O) 39.7 (CH) 40.7 (CH2) 133.7b (C) 127.7 (CH) 29.2 (CH) 43.4 (CH2)

8 9 10

70.7 (CH) 170.3 (C = O) 40.1 (CH2)

11 12 13 14 15 16 17 18

48.9 (CH) 168.5 (C = O) 55.4 (CH) 174.0 (C = O) 46.0 (CH) 17.7 (CH3) 30.8 (CH3) 23.2 (CH2)

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 NH-Ala NH-Tyr NH-Trp


110.8 (C) 132.1 (C) 110.5 (CH) 122.6 (CH) 120.3 (CH) 118.2 (CH) 136.1 (C) 109.0 (C) 132.1b (C) 127.4 (CH) 115.5 (CH) 155.1 (CH) 115.5 (CH) 127.4 (CH) 19.1 (CH3) 22.0 (CH3) 18.6 (CH3) 20.4 (CH3) – – –

δH (ppm) mult. (J Hz) – 2.48 (m) 2.37 (A) (dd, 15.9, 11.5) 1.88 (B) (d, 16.0) – 4.75 (d, 9.3) 2.24 (m) 1.31 (A) (m) 1.11 (B) (d, 6.9)C 4.62 (m) – 2.68 (A) (dd, 4.8, 15.1) 2.62 (B) (dd, 5.7, 15.1) 5.27 (m) – 5.81 (dd, 6.3, 10.3) – 4.71 (quin, 6.7) 0.75 (d, 6.7) 2.98 (s) 3.36 (A) (dd, 6.3, 15.3) 3.23 (B) (dd, 10.4, 15.3) – – 7.24 (d, 8.0) 7.11 (t, 8.0) 7.15 (t, 8.1) 7.55 (d, 7.8) – – – 7.00 (d, 8.5) 6.70 (d, 8.5) – 6.70 (d, 8.5) 7.00 (d, 8.5) 1.06 (d, 6.4) 0.82 (d. 6.7) 1.56 (d, 6.3) 1.11 (d, 6.9)C 6.59 (d, 6.7) 7.50 (d, 8.8) 8.19 (s)

HSQC

TOCSY

– H-2 H-3A, B

H-8 – H-10A, B

– H-3 (A & B), H-5, Me-35, Me-36 H-2, H-5, H-3B, H-35, H-36; H-2, H-5, H-3A, H-35, H-36 – H-2, H-3 (A & B), H-6, H-7, H-8, Me-33, Me-34, Me-35, Me-36 H-5, H-7, H-8, Me-33, Me-34, Me-35, Me-36 H-5, H-6, H-7B, H-8, H-33, H-34, H-36 H-5, H-6, H-7A, H-8, H-33,H-34 H-5, H-6, H-7, H-33, H-34, H-36 – H-11

H-11 – H-13 – H-15 H-16 H-17 H-18A, B

H-10 (A & B), NH-Tyr – H-18 (A & B) – H-16, H-17 H-15, H-17 H-16, H-15 H-13

– – H-21 H-22 H-23 H-24 – – – H-28 H-29 – H-31 H-32 H-33 H-34 H-35 H-36 – – –

– – H-22, H-23, H-24 H-21, H-23, H-24 H-21, H-22, H-24 H-21, H-22, H-23 – – – H-29 H-28 – H-32 H-31 H-5, H-6, H-7, H-8, H-34 H-5, H-6, H-7, H-8, H-33, H-35, H-36 H-2, H-3 (A & B), H-5, H-6, H-7, H-34, H-36 H-2, H-3 (A & B), H-5, H-6, H-7, H-8, H-34, H-35 H-15, H-16 H-10 (A & B), H-11 –

– H-5 H-6 H-7A, B

1

H and 13C assignments aided by TOCSY, HSQC and HMBC experiments. Carbon type assignments based on DEPT experiment. bCarbon assignments can be interchangeable. cProton assignments observed are same based on Heteronuclear single quantum coherence (HSQC) experiment. a

C-NMR signals at δC 170.3, δC 40.1 and δC 48.9 ppm, respectively. (4) The four methyl groups of the polypropionate moiety showed 1H-NMR signals at δH 1.06 (3H, d, 6.4 Hz), δH 0.82 (3H, d, 6.7 Hz), δH 1.56 (3H, d, 6.3 Hz) and δH 1.11 (3H, d, 6.9 Hz) and were ascribed to methyl protons of Me-33 to Me-36, respectively. 13C-NMR signals observed at δC 19.1, δC 22.0, δC 18.6 and δC 20.4 ppm also confirmed the presence of Me-33 to Me-36. Moreover, 13

HMBC correlations showed cross-peaks between Me-36 and C-1, C-2, C3; Me-35 and C-3, C-4, C-5; Me-34 and C-5, C-6, C-7; Me-33 and C-6 and C-7 (Fig. 7b). An E-C(H) = C(Me)-array was confirmed by 13-NMR signals at δC 133.7, δC 127.7, δC 18.6 ppm for C-4, C-5 and C-35.[5] Other carbon atoms namely C-1 to C-3 and C-6 to C-8 were confirmed by 13C-NMR signals at δC 174.7, δC 39.7, δC 40.7, δC 29.2, δC 43.4 and δC 70.7 ppm, respectively.


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The connections of the β-tyrosine moiety with N-methyl tryptophan moiety, N-methyl tryptophan moiety with alanine moiety and alanine moiety with polypropionate moiety were confirmed by HMBC correlations of crosspeaks between NH-Tyr and C-12; N (Me-17)-Trp and C-14; NH-Ala and C-1, respectively. The ester linkage between the polypropionate moiety and β-tyrosine moiety was confirmed by 13C-NMR signals at δC 170.3 ppm for the carbonyl carbon atom C-9 of the ester. Therefore, based on 1D and 2D NMR spectral and ESI-MS data, it was concluded that JDC3 is Jaspamide.

Conclusion The present study showed that the JDE of the sponge Jaspis diastra has better potential to inhibit growth of cancer cells and could induce apoptosis in human cancer cells. According to these results, it is suggested that this extract can contain other compounds that can be valuable for the development of anticancer drugs. Further investigations

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Declarations Conflict of interest The Authors declare that they have no conflicts of interest to disclose.

Acknowledgements G.B. wishes to acknowledge Professor Padmanabhan Balaram (MBU, IISc) for his kind laboratory support and the Western Indian Ocean Marine Science Association (WIOMSA) for financial support. The authors wish to thank Professor Rob W.M. van Soest for sponge identification. Our sincere thanks to the National Coast Guard (NCG) for providing security during fieldwork. We also acknowledge the continuous support of Mr Chettanand Samyan and Mr Prakash Mussai for sample collection.

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Characterization and antioxidant properties of six Algerian propolis extracts: ethyl acetate extracts inhibit myeloperoxidase activity.

Apoptosis-Inducing Activity of Marine Sponge Haliclona sp. Extracts Collected from Kosrae in Nonsmall Cell Lung Cancer A549 Cells.

In vitro antitumor activity of stellettin B, a triterpene from marine sponge Jaspis stellifera, on human glioblastoma cancer SF295 cells.

Insecticidal Activity of Ethyl Acetate Extracts from Culture Filtrates of Mangrove Fungal Endophytes.

Antitumor Activities of Ethyl Acetate Extracts from Selaginella doederleinii Hieron In Vitro and In Vivo and Its Possible Mechanism.

A possible anti-inflammatory mechanism of ethyl acetate extracts of Teucrium stocksianum Bioss.

Ethyl acetate extracts of Fructus Ligustri Lucide induce cell apoptosis in human neuroglioma cell.

Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins.

Effect of total hydroalcholic extract of Nigella sativa and its n-hexane and ethyl acetate fractions on ACHN and GP-293 cell lines.

Antiproliferative activity of methanolic extracts from two green algae, Enteromorpha intestinalis and Rizoclonium riparium on HeLa cells.

Antitumor and apoptotic activities of the chemical constituents from the ethyl acetate extract of Artemisia indica.

[Seasonal and geographical variation range of antifungal activity of sponge extracts from the Moroccan Atlantic coasts].

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Metabolism of ethyl acetate in the rat: hydrolysis of ethyl alcohol in vitro and in vivo.

Therapeutic effect of ethyl acetate extract from Asparagus cochinchinensis on phthalic anhydride-induced skin inflammation.

Investigating the effectiveness of pictorial health warnings in Mauritius: findings from the ITC Mauritius survey.

Quantitative variation of bioactive phyto compounds in ethyl acetate and methanol extracts of Pergularia daemia (Forsk.) Chiov.

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Occupational allergic contact dermatitis from ethyl chloro oximido acetate.

Synergistic Anti-bacterial Effects of Phellinus baumii Ethyl Acetate Extracts and β-Lactam Antimicrobial Agents Against Methicillin-Resistant Staphylococcus aureus.

Ethyl acetate extract from marine sponge Hyattella cribriformis exhibit potent anticancer activity by promoting tubulin polymerization as evidenced mitotic arrest and induction of apoptosis.

In vitro antitumor activity of the ethyl acetate extract of Potentilla chinensis in osteosarcoma cancer cells.

Juglans mandshurica Maxim extracts exhibit antitumor activity on HeLa cells in vitro.

Identification of Neuroactive Constituents of the Ethyl Acetate Fraction from Cyperi Rhizoma Using Bioactivity-Guided Fractionation.