5. SPONGE CRUDE EXTRACTION AND PRIMARY SCREENING

Transcription

1 5. SPONGE CRUDE EXTRACTION AND PRIMARY SCREENING 5.1 Introduction The goal of marine natural products research is to discover and harness the therapeutic potential of compounds bio-synthesized by marine organisms and their symbiotic microorganisms. Marine sponges, tunicates, soft-coral and algae have been shown to contain a wealth of novel compounds with high anti-bacterial, antitumor, anti-viral, anti-inflammatory and analgesic properties (Rudi et al., 2001). Particularly, the sponges are a rich source of marine secondary metabolites and due to their relative abundance, ease of collection, and the structurally diverse classes of natural products they produce they hold good scope (Jenna arruda & Jennifer Carroll, 2009). In addition, the relatively high percentage of symbiotic microorganisms in marine sponges, which can be as high as 60% of the total body mass, produce a variety of chemical profile in relation to environmental and temporal changes. Countless applications of these compounds have been explored and identified including their inhibition of tumor cell growth, anti-viral activity (Hepatitis B) and for the treatment of Alzheimer s disease. Recent years have seen considerable advances in our understanding of natural product biosynthesis coupled with improvements in approaches for natural product isolation, characterization and synthesis. These advancement could be opening the door to a new era in the investigation of natural products in academia and industry (Clardy & Walsh, 2004). With recent technologies for the purification, Tim et al. (2008) have explained a new method for rapid separation of metabolites. The marine invertebrate extracts can be rapidly separated on HP20SS. The method effectively separates the organic constituents from the inorganic salts and can concentrate the active principal components. By creating partially purified libraries, daughter plates can be easily generated for new screening programs. Additionally, identifying compounds directly from the LCMS fractionated material archive facilitates rapid dereplication and purification of active components as compared to returning to the source organism. Integrating the HP20SS library with our automated LCMS 58

2 fractionation protocol has increased the number of projects that we can pursue as well as allowing rapid dereplication. Plants, animals and microorganisms have always been a constant source of new and useful compounds. Modern analytical and isolation techniques have made it considerably easier to isolate and exploit these compounds for useful purposes. Identifying bioactive molecules from complex biomass requires careful selection and execution of relevant bioassays in the various stages of the discovery process of potential leads and targets. To achieve multidisciplinary approach and a higher degree of fundamental research in development of bioassays are necessary to identify and to fully understand the mode of action of bioactive molecules with novel structure-activity relationships from natural sources (Strömstedt et al., 2014). Present attempt deals with the bioassay development using HeLa nuclear extract for a specific cancer target Hidtone deacytylases (HDAC) and crude extract preparation of marine sponge samples, bioassay (HDAC) screening of crude extracts and cell based anticancer assay. 5.2 Materials and methods Based on the microscopic and morphological characteristics the sponges were identified and classified (Chapter-4). The sponge samples were extracted selectively in ethyl acetate to obtain the biologically active compound in medium polar range Ethyl acetate extraction Processed and preserved samples were recovered from -80 C and kept in the dark place for 30 min to attain ambient temperature. For the isolation of bioactive extract, 500 g of sponge sample was minced in a tissue homogenizer (IKA, USA) and extracted with ethyl acetate for three times. The combined Ethyl acetare extract was filtered and concentrated in a rotary vacuum evaporator (Buchi, Switzerland) at room temperature. The completely dried crude extract was defatted with n-hexane for three changes in a separating funnel. Again ethyl acetate fraction was concentrated in a rotary vacuum evaporator and stored in -20 C. The crude ethyl acetate fraction was dissolved in DMSO at concentration of 20 mg/ml stock and used for the bioactivity. 59

3 HDAC assay HeLa Nuclear extract preparation: Nuclear extract was prepared from HeLa cells following the method of Timothy W. Nilsen (2011) with slight modification according to our laboratory. Reagents and solutions I) Hypotonic buffer 10 mm HEPES, ph 7.9 at 4 C 1.5 mm MgCl 2 10 mm KCl Store up to 2 weeks at 4 C Immediately before use 0.2 mm PMSF 0.5 M DTT are to be added. II) Extraction buffer High-salt buffer was prepared with 0.02 M KCl or without KCl. Nuclear extract preparation HeLa cells were grown in T-175 flask at 80% confluence cells that were used in nuclear extract preparation. Culture medium was removed from the flask and the cells scraped into fresh ice cold PBS cells collected in 15 ml centrifuge tube and centrifuged for 10 min at 1850 g (~3000 rpm). Supernatants were discarded and cells were suspended in hypotonic solution and cells allowed to swell on ice 10 min. Cells were transferred to a glass Dounce homogenizer. The cell suspension was homogenized with 10 to 18 up and down strokes using the loose pestle. Cells were transferred to centrifuge tubes and centrifuged to collect the nuclear pellet. Nuclear pellet was collected by centrifuging 15 min at 3300 g (~4000 rpm). Supernatant was removed and nuclear pellet was suspended in extraction buffer and the suspension was sonicated for 30 sec. Nuclear lysate was again centrifuged at rpm for 10 60

4 min at 4 C. Supernatant was collected and protein estimation was carried out to determine the concentration of protein in the nuclear extract. Nuclear extracts were stored in the -80 C as 100 µg aliquots HDAC assay Initial assay was performed to ascertain the assay conditions and to study the standard compound inhibitory profile. Two standard inhibitors were purchased from Sigma Aldrich, USA. (Suberanilohydroxamic acid (SAHA) and Trichostatin A (TSA)). Fluorometric HDAC assay was performed according to the method published by Dennis wegner et al. (2003) with minor modification. Fluorescence substrate Ac - Arg - Gly Lys (Ac) AMC were purchased from Anaspec, USA. This fluorogenic substrate of HDACs was synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C-terminus of the peptide chain. The assay utilizing this substrate provides a good tool to characterize the HDAC activity. The 50 mm substrate as final concentration was used in the assay. Initially standard compounds with appropriate final concentrations were pre-incubated with 5 µg nuclear extract for 10 min at 37 C and reaction was initiated by adding 50 mm fluorescence substrate and incubated further 30 min at 37 C. The reaction terminated by adding signal developer solution which containing (trypsin (10 mg/ml)/ Trichostatin A 2µM). After 15 min incubation fluorescence intensity was measured using Synergy HT multimode microplate reader (BioTeK, USA.) using excitation at 390 nm and emission at 460 nm. The substrate blank value was eliminated with all readings and percentage inhibition was calculated considering control as 100% activity. Inhibitory concentration 50 (IC 50 ) was calculated using the inhibition curve formula. 61

5 HeLa Nuclear extract Crude EA Extract 250 mm substrate Developer Solution Incubation 10 min Incubation 30 min Incubation 15 min Read at 390 nm & Emi 460 nm Fig. 10 HDAC assay-schematic representation Fig. 11 Concentration curve of fluorogenic peptide Sample preparation Ethyl acetate fraction of study samples were brought to the ambient temperatures. Each fraction was weighed 2.0 mg in a sterile micro centrifuge tube and the fraction was dissolved in 100 µl of DMSO (Concentration equivalent 20 mg/ml). The tubes were vigorously mixed for the complete solubilization. Further dilutions were made using HDAC assay buffer. 5X stock concentration of desired 62

6 test range was prepared. The broader range of analysis it was decided that 1000 and 300 µg as the preliminary screening concentrations. Hence 5X stock concentrations were prepared as 5000 and 1500 µg concentrations Preliminary screening assay HeLa nuclear extract was diluted for the desired reactions (Nuclear extract concentration 5 µg per reaction). Compounds were pre-incubated with nuclear extract 10 min at 37 C with mild shaking. After 10 min the reaction was initiated by adding the 250 mm (5X) concentration of fluorogenic substrate in all wells except substrate blank. The assay plate was further incubated for 30 min at 37 C. The reaction was terminated by adding developer solution (10 mg/ml trypsin and 2 µm Trichostatin A) and the plate was further incubated for 10 min at 37 C. The plate fluorescence was read at excitation 390 nm and emission 460 nm. The RFU (Relative Fluorescence Unit) was compared to untreated control (in this assay control was considered as 100% active). Percentage inhibition of crude extracts was calculated. While performing the screening assay standard compound was used as internal control In vitro anticancer screening Similarly, anticancer assay was carried out using MTT for all the ethyl acetate fractions in three cell lines. The human cancer cell lines HeLa, HT-29 and A549 were purchased from NCCS, Pune (India). They were grown in a RPMI1640 medium supplemented with 10% fetal bovine serum and antibiotics as mentioned earlier. Cytotoxicity (MTT) assay was performed following the method described by Carmichael et al. (1987) and percentage of cell viability was determined by Spectrophotometric determination of accumulated formazan derivative in treated cells at 560 nm in relation to the untreated ones. For the MTT assay, the cells were grown in 25cm 25cm 25cm tissue culture flasks containing DMEM/Hams F12 nutrient mix as culture medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin (GIBCO) and grown at 37 C under a humidified atmosphere of 95% air and 5% CO 2. Cells were regularly passaged and maintained before including for the experiment. When a cell density in a culture flask reached 70-80% confluence, they were trypsinized and seeded in 96-well plates at varying 63

7 cell number according to the size and shape of the cell between 5000 and cells per well in 100 µl and incubated for 24 h at CO 2 incubator. Compounds were added as 2X concentration to the cell in 100 µl volume and the concentration range were: 100, 10, 1, 0.1, 0.01µg/ml. The plates were further incubated for 48 hours in the CO 2 incubator. MTT solution was composed of 3-4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) at 5 mg/ml in phosphate buffered saline (1.5 mm KH 2 PO 4, 6.5 mm Na 2 HPO 4, 137 mm NaCl, 2.7 mm KCl; ph 7.4), from this solution 50 µl was pipetted out into each well. The plate was further incubated for 2h 30min and the medium was carefully decanted. The formazan crystals were air dried in dark place and dissolved in 100 µl DMSO and the plates were mildly shaked at room temperature and the OD was measured using Synergy H4 microplate reader at 570nm. From the optical densities the percentage growths were calculated using the following formulae, if T is greater than or equal to T 0, percentage growth = 100 x [(T-T 0 )/(C-T 0 )] and if T is less than T 0, percentage growth = 100 x [(T- T 0 )/T 0 )], Where T is optical density of test, C is the optical density of control and T 0 is the optical density at time zero. From the percentage growth a dose response curve was generated and GI 50 values were interpolated from the growth curves. 5.3 Results The concentrated fractions were appeared as dark brown or black in colour. Approximately, 500 g of each marine sponge was applied for the ethyl acetate fractionation. The final recovered crude extract were used for the HDAC assay (Table 15). Table. 15 Quantity of ethyl acetate fraction Sponge name Weight of ethyl acetate fraction (g) Cliona viridis 7.31 Halichondria glabrata Mycale trincomaliensis 5.84 Cliona quadrata 4.24 Psammaplysilla purpurea 1.41 Heteronema erecta 7.21 Jaspis penetrans 16.3 Spirastrella inconstans Sigmadocia petrosioides

8 % i n h i b i t io n HDAC assay with standard compounds The initial assay was performed with standard compounds (SAHA & TSA) to validate the assay performance. IC 50 assay has been performed with 6 semilogarithmic concentration starting form SAHA 3000, 1000, 300, 100, 30 and 10 nm; TSA 30, 10, 3, 1, 0.3 and 0.1 nm. It was observed a concentration dependent inhibition of HDAC activity. Standard compounds accounted IC 50 of 89 nm for SAHA and 3.0 nm for TSA. IC50 Curve of SAHA nm- IC Conc. (nm) Fig. 12a IC 50 determination of SAHA and TSA in HeLa Nuclear extract based assay 3 nm- IC Fig. 12b IC 50 determination of SAHA and TSA in HeLa Nuclear extract based assay 65

9 Preliminary screening Preliminary screening was carried out with all 9 sponge samples. The results were revealed that sponge Jaspis penetrans exhibited potent inhibition of HDAC enzyme. At 1000 µg concentration it showed 70±4.68% inhibition and with 300 µg the recorded inhibition is 45±7.25% results are shown in the Table. 16 and Fig. 13. Hence, sponge Jaspis penetrans fraction was identified as active fraction. Fig. 13. Percentage inhibition of HDAC activity Table. 16 (a & b) Percentage inhibition of HDAC activity against the sponge samples. (a) Test conc. Preliminary screening of marine sponge in HDAC assay Conc. (µg/ml) Cliona viridis Halichondria glabrata Mycale trincomaliensis Cliona quadrata Psammaplysilla purpurea ±0.87 8± ± ±15 16± ± ±28.8 0±0.0 15±7.4 15±1.22 (b) Test conc. Preliminary screening of marine sponge in HDAC assay Conc.(µg/ ml) Heteronema erecta Jaspis penetrans Spirastrella inconstans Sigmadocia petrosioides ± ± ±5.81 9± ±7.4 45±7.25 8±1.83 5±

10 In vitro anticancer screening MTT assay was carried out for all sponge samples against with the cancer cell lines (HeLa, A59 and HT-29) to determine the GI 50 values of the fractions. Data revealed that the marine sponge, Jaspis penetrans exhibited growth inhibition of all the three investigated cell lines. It accounted GI 50 of 1.01±0.96, 0.57±0.65 and 1.32±0.37 in A549, HeLa and HT-29 respectively. Similarly, sponges Cliona viridis and Psammaplysilla purpurea have exhibited moderate cell growth inhibition (Table 17). It clearly evidence that the marine sponge, Jaspis penetrans ethyl acetate fraction exhibited potent HDAC inhibition and anticancer activity. Hence, this fraction was selected for further studies. Table. 17 Anticancer activity of marine sponges in MTT assay Sponge name GI 50 (µg) (Cancer cell lines) A549 HeLa HT-29 Cliona viridis 12.43± ± ±2.4 Halichondria glabrata >100 >100 >100 Mycale trincomaliensis >100 >100 >100 Cliona quadrata 22.39± ± ±2.01 Psammaplysilla purpurea 10.05± ± ±1.6 Heteronema erecta 79.81± Jaspis penetrans 1.01±0.96* 0.57±0.65* 1.32±0.37* Spirastrella inconstans >100 >100 >100 Sigmadocia petrosioides >100 >100 > Discussion Marine natural products offer an abundant source of pharmacologically active agents with great chemical diversity and complexity, and hence the potential to produce valuable therapeutic entities. Marine natural product extracts present several problems with respect to modern drug discovery programs. The first and foremost problem encountered with marine invertebrate extracts from sponges and tunicates is the presence of large quantities of inorganic salts (Tim et al., 2008). Additionally, the chemical diversity found in one sponge may represent several different classes of bioactive molecules 67

11 that exhibit different and sometimes opposing pharmacological activities. In many cases, the presence of a major non-selective compound can mask the activity of minor selective compounds. Minor compounds in many cases are present in crude extracts at concentrations that are below detection thresholds. From a discovery standpoint, these problems can be addressed to a certain point through the use of pre-fractionation strategies (Appleton et al., 2007; Harvey, 2007; Koehn, 2005 & 2008). The complexity of the natural product some time may not allow us to purify the compounds. In such instance pre-fractionation strategies to eliminate the nonactive compounds at the initial stage. Similar approach was performed in the initial stage as inexpensive assay with two testing concentration such that more number of compounds were screened. Prior to performing a large number of separations, tests were performed to standardize our extracts. Primarily, it was hypothesized that there was little need to dry and weigh individual extracts prior to fractionation. Our hypothesis was based on the fact that the mixture of compounds present in each extract could not be predetermined; therefore, the concentration of individual components would be independent of weight. Additionally, if the fractionation step concentrated organics, it would increase the hit rate above what we observed for pre-weighed extracts that were submitted to High throughput screen (HTS) (Tim et al., 2008). The important aspect of pre-separation stage of any natural product. Our views is before we make separation efforts it is important to obtain the activity and conforming the activity of the fraction. Otherwise it would cause economical and time loses. Strategy of developing simple inexpensive assay models would be of great supportive in eliminate the inactive fraction. The assay should be simple and robust. A simple fluorescence based assay was established and it was very helpful in screening the initial extracts and fractions of the test samples. It seems that compared to other 8 species of sponges, the Jaspis penetrans extract contained very good bioactive metabolites vis-à-vis anticancer compound. The marine sponge, Jaspis penetrans fraction as active fraction in the primary HDAC assay and subsequently its anticancer property was confirmed in 68

12 the MTT assay. Cell based proliferation assays can be used as common anti-cancer assay to select the crude extract or fraction. The introduction of high-throughput screening and the miniaturization of assays have created a need to optimize natural product samples to better suit these new technologies. Furthermore, natural product programs are faced with an ever shortening time period from hit detection to lead characterization (Wagenaar, 2008) Koehn (2008) explained the impact of HTS technologies in the natural product discovery. He added that natural products have been de-emphasized as high throughput screening resources in the recent past, in part because of difficulties in obtaining high quality natural products screening libraries, or in applying modern screening assays to these libraries. In addition, natural products programs based on screening of extract libraries, bioassay-guided isolation, structure elucidation and subsequent production scale-up are challenged to meet the rapid cycle times that are characteristic of the modern HTS approach. Fortunately, new technologies in mass spectrometry, NMR and other spectroscopic techniques can greatly facilitate the first components of the process - namely the efficient creation of high-quality natural products libraries, bimolecular target or cell-based screening, and early hit characterization. The success of any high throughput screening campaign is dependent on the quality of the chemical library. The construction and maintenance of a high quality natural products library, whether based on microbial, plant, marine or other sources is a costly endeavor. The library itself may be composed of samples that are themselves mixtures - such as crude extracts, semi-pure mixtures or single purified natural products. Each of these library designs carries with it distinctive advantages and disadvantages. Crude extract libraries have lower resource requirements for sample preparation, but high requirements for identification of the bioactive constituents. Pre-fractionated libraries can be an effective strategy to alleviate interferences encountered with crude libraries, and may shorten the time needed to identify the active principle. Strategy of establishing target based preliminary assay is a very attractive way forward for the natural product researchers. The narrowed down sponge extract based on the anticancer activity was further characterized to obtain pure compound and the evaluation of its anticancer effect subsequently. 69