Preparation Of Crude Extracts Using Methanol As Solvent Biology Essay

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In this study, the treatment of the sponge samples prior to extraction process is significant as it affects the bioactive compounds that could be extracted from the samples. The fresh sponge samples were dried in an oven at a temperature not lower than 40oC to prevent the oxidation of active enzymes and not higher than 60oC to avoid the high temperature from damaging the biogenic compounds. In addition, the drying process reduces the water content in tissues, thus preventing the degradation by enzyme and fungal infection (Fennell et al., 2004). In this study, Haliclona sp. samples were dried at 45oC in an oven until the weight become constant. After that, the dried samples were ground into fine powdered form to provide a higher surface to volume ratio facilitating the penetration of the extraction solvent.

In this study, the dried samples were used in the extraction process to obtain the crude extract of Haliclona sp. instead of fresh samples. This is due to the high water content in the fresh samples may interfere with the extraction process and cause improper separation during partitioning (Wright, 1998). Furthermore, time is another critical factor affecting the extraction process. This was proved with the difficulty in dealing with the fresh samples if there happened to be any time delay between the sample collection and processing (Kueh, 2007). The dried samples are more advantageous as they allow storage for a long period before analysis. To obtain the crude extract, there are several traditional ways of extracting the bioactive compounds including using the Soxhlet extractor, magnetic stirrer, boiling, maceration, grinding and heat reflux (Yang et al., 2008). In this study, the maceration method was used due to its low cost and ease in extracting the secondary metabolites from marine sponges.

In general, the extraction method depends largely on the type of extraction solvents employed, energy input, and agitation to improve the chemical solubility and efficiency of mass transfer (Yang et al., 2010). The major factor affecting the extraction process is the type of organic solvents used. According to Kandhasamy and Arunachalam (2008), the choice of organic solvents used for extraction affects the bioactive compounds present in the crude extract and its activity. Likewise, the solubility and polarity of bioactive compounds affect the effectiveness of solvent used for extraction. In this research, methanol was chosen as the solvent used for the extraction process. This is due to high polarity properties of methanol which is a good solvent for extracting wide variety of bioactive compounds from the marine sponges. According to Silva et al. (1998), the alcoholic solvent can penetrate the cell membranes effectively which permits the extraction of high amounts of intracellular components. Investigation of antimicrobial activity of marine sponge Haliclona exigua revealed that the methanol extract of the sponge samples showed promising antifungal activity against several species of fungi (Lakshmi et al., 2010). This result indicates that methanol is a good solvent for extracting bioactive compounds from natural sources. Besides, several studies regarding the effectiveness of extraction method revealed that the methanolic extract exhibited greater antimicrobial activity as compared with other solvents such as n-hexane and benzene (Rosell and Srivastava, 1987). In this research, of the 20.0 g of dried Haliclona sp., 1.298 g of crude extract was acquired and has contributed to the high yield percentage of methanol which is 6.4% indicating that methanol is an effective solvent for extraction.

5.2 Screening of antimicrobial activity of Haliclona sp. methanolic crude extract

In this study, the methanolic extract preparation was used to test on a list of test organisms including 12 bacteria, 6 fungi and 2 yeasts. The purpose of this test is to determine the antimicrobial activity of Haliclona sp. and its potential to be recovered and developed as a new antimicrobial drug. According to the previous study by Monks et al. (2002), the organic extract from Haliclona aff tubifera demonstrated significant antibacterial activity against B. subtilis whereby the strongest activity was exhibited and moderate antibacterial activity was observed on E. coli. However, this result opposed with the screening result of Haliclona sp. extract from local Penang area whereby the methanolic crude extract exhibited no antibacterial activity on E. coli. This variation in the antibacterial activity could be due to several extrinsic factors. For instance, method of extraction, choice of solvent used, seasons of the sponge samples collection, geographical location of sponge samples, temperature, nutrient availability are among the extrinsic factors contributing to this variation (Kandhasamy and Arunachalam, 2008).

The temperature has been proposed as the main factor affecting this variation. This is supported by the production of ecthyonine in Microciona prolifera which decreases in cold months. This compound plays role in the biochemical ecology of sponge, and its variation could result from changes in the environmental conditions (Betancourt-Lozano et al., 1998). Bakus et al. (1986), in their studies of sponges along the Mexican coasts suggested that there is a seasonal variation in the activity levels of the antimicrobial properties of sponges with supportive data showing that sponges exhibited antibiotic activity during the warm season as opposed in the cold season. Thus, in the research of antimicrobial activity of marine sponges, standardization of the parameters involved is critical in the findings of potential antimicrobial agents.

In this study, the disc diffusion method was employed to study and determine the antimicrobial activity of Haliclona sp. This method is chosen as it is a relatively robust and easy technique to perform and it gives rise to high reproducibility (Barker and Kehoe, 1995). Besides, the disc diffusion methodology yields a quantitative result (zone of inhibition) and a qualitative interpretive category (susceptible or resistant). It is a cost effective method which commonly provides qualitative results 24 hours sooner than the standard broth dilution methods (Espinel-Ingroff, 2007). Furthermore, lacking the requirements for specialized equipment is another advantage of this method (Matar et al., 2003). In this study, the spread plate method was employed. This is due to the cell density from this method was greater, leading in turn to relatively smaller zone sizes (Barker and Kehoe, 1995). Initially, the inoculum was spread evenly on the surface of the agar (Acar and Goldstein, 1986). After inoculation, the plates were left to dry for 15 minutes at room temperature with the lid ajar. A multidisc was then placed onto the agar using sterile forceps and pressed down gently to ensure contact. However, many factors such as agar source, inoculum size, disc potency, incubation temperature and the length of incubation can influence the diameter of zone size (Acar and Goldstein, 1986).

Based on the screening results, the methanolic crude extract exhibited significant antimicrobial activity. Besides, the results also revealed that the bioactive compounds of Haliclona sp. exhibited antimicrobial activity only on the unicellular prokaryotes, but not on the eukaryotic organisms. From the results, the methanolic extract exhibited significant antimicrobial activity by inhibiting 6 species of bacteria including 4 Gram-positive and 2 Gram-negative species. This result is in agreement with the previous research done by Ely et al. (2004) where the methanolic extracts of nine marine sponges (Porifera) and two seaweeds collected from the south east coast of India demonstrated significant antimicrobial activity against one or more of the test organisms. In this study, methanol was used as the solvent to dissolve the crude extract paste. Thus, a negative control with methanol was included so as to detect the solvent effects. However, the screening results revealed that methanol did not inhibit the growth of all of the test organisms. Hence, methanol is a suitable organic solvent to be used as the solvent to dissolve the crude extract of Haliclona sp. in this study.

The methanolic crude extract of Haliclona sp. exhibited significant antimicrobial activity against both the Gram-positive and Gram-negative bacteria. This revealed that it possessed a broad spectrum of activity. From the screening results, the crude extract of Haliclona sp. was observed to be exhibiting stronger antimicrobial activity on the Gram-positive bacteria as compared to the Gram-negative bacteria. This was due to the differences in the cell wall structure and composition between the Gram-positive and Gram-negative bacteria. For the Gram-negative bacteria, their cell wall possesses an outer membrane which constitutes the outer surface of their cell wall. This outer membrane acts as a coarse sieve which exerts little control over the movement of substances such as antibiotics into the cell (Black, 2008). Furthermore, the presence of peptidoglycan or murein layers on the cell wall of Gram-negative bacteria also prevents the entry of bioactive compounds into the cells (Tortora et al., 2001). Thus, the Gram-negative bacteria were less susceptible to the crude extract of Haliclona sp. compared to the Gram-positive bacteria which were more susceptible exhibited stronger antimicrobial activity in this study.

In the research for new antimicrobial drugs, an important factor that affects the findings is the inoculum size. The variations in the size of inoculum are significantly influencing the diameter of inhibition zones (Cook et al., 1990). Hence, inoculum standardization is required in order to obtain accurate results and also reproducibility of the screening test. Besides, the molten agar poured into the Petri dish on a level forming a uniform depth of 4 mm is important. This is due to the depth deeper than 4 mm may give false resistance results and excessively small inhibition zones whereas depth less than 4 mm may give false susceptibility results associated with excessively large zones of inhibition (Jorgensen and Turnidge, 2007). Thus, standardization is required during the preparation of media used for screening test in order to obtain accurate results.

5.3 Determination of minimal inhibitory concentration (MIC)

By definition, minimal inhibitory concentration (MIC) is the lowest concentration of an antimicrobial drug that will inhibit the visible growth of a microorganism after overnight incubation (Cook et al., 1990). MIC is employed as the most basic laboratory measurement of the activity of an antimicrobial agent against a microorganism. Thus, MIC is the fundamental measurement that forms the basis for most of the susceptibility testing methods (Jorgensen and Turnidge, 2007).

In this study, the MIC values of Haliclona sp. methanolic crude extract on Methicillin-resistant S. aureus (MRSA), B. spizizenii, B. subtilis, B. cereus, A. anitratus and Erwinia sp. were determined using the broth dilution method. The reason for employing the broth dilution method in this research was due to its standardization and reliability which is useful for research purpose to test the susceptibility of antibacterial drugs (Jorgensen and Turnidge, 2007). Furthermore, it is a method which is technically easy to perform and relatively inexpensive. Broth dilution method for inhibitory determination is also strongly recommended by Clinical and Laboratory Standards Institute (CLSI) to assess the microbial growth or its inhibition (NCCLS, 2003). In this study, the disc diffusion method was not used as a previous work performed by Rios et al. (1988) proposing that the disc diffusion method only allows the diffusion of polar compounds with small or medium molecular size. In addition, the diameter of inhibition zone is affected by the rate of diffusion of extract through the agar medium as well as the size and hydrophilicity of the extract (Berger et al., 1993). Likewise, a study conducted by Klancnik et al. (2010) also suggested that the disc diffusion method is not a reliable method for screening the antimicrobial activity of plant extracts on the basis of the high MIC values obtained. This is due to the disc diffusion assay is not suited to the natural antimicrobial compounds that are hydrophobic in nature which prevents the uniform diffusion through the agar media (Mann and Markham, 1998).

In this study, the broth dilution method was chosen for determination of MIC values. However, the MIC measurements using the broth dilution method are influenced by several factors including the medium composition, inoculum size, the duration of incubation and the presence of resistant subpopulations of organism (Jorgensen and Turnidge, 2007). Thus, all the parameters must be standardized in order to obtain accurate measurements. In this research, the nutrient broth was employed because it is a general purpose growth medium that supplies the necessary ingredients for the growth of microorganisms and it is commonly used to cultivate the non-fastidious microorganisms (Black, 2008).

Nevertheless, the antibiotic resistant strains may lead to erroneously high reading of MIC values for the broth dilution method. The research done by Gaudreau (1997) exhibited that the MIC values for the resistant strains of Campylobacter jejuni and Campylobacter coli are 8 to 256 times higher than the susceptible strains. This erroneously high reading of MIC was due to the subjectivity of reading end points in the broth dilution method. According to Sauboulle and Hoeprich (1978), a slight growth resulting from the subpopulations which were partially inhibited and a longer period required for the expression of antimicrobial activity by the extract are the contribution factors to this erroneously high reading of MIC.

In this study, the MIC values for all the test organisms including MRSA, B. spizizenii, B. subtilis, B. cereus, A. anitratus and Erwinia sp. were different. Thus, this suggested that the susceptibility of the Haliclona sp. methanolic crude extract was vary for different test organisms. Eventually, the MIC values obtained using the broth dilution method were used as a standard to determine the growth profile as it is the best method to establish the real potency of a pure compound (Rios et al., 1988).

5.4 Determination of minimal bactericidal concentration (MBC)

By definition, the minimal bactericidal concentration (MBC) is the lowest concentration of drug that kills at least 99.9% of cells in the original inoculum (Levison, 2000). In this study, the MBC values recorded for B. subtilis, B. cereus, A. anitratus and Erwinia sp. were significantly higher than MRSA and B. spizizenii. From the results of MBC, it suggested that the methanolic crude extract of Haliclona sp. was more effective in killing the bacterial cells of MRSA and B. spizizenii compared to B. subtilis, B. cereus, A. anitratus and Erwinia sp. This result also revealed that the test organisms possessed different susceptibility to the extract.

According to Levison (2000), if the minimal concentration of a drug that prevented turbidity has lowered the bacterial density from 1 x 105 cells/ml to 1 x 102 cells/ml, the MIC that prevented the turbidity is also considered as the MBC. In this study, the results indicated that the MBC values obtained were significantly higher than the MIC values. Thus, this result suggested that higher concentration of methanolic extract was required in order to kill the bacterial cells instead of inhibiting their growth. Generally, the MBC value for bactericidal drugs is usually the same as or not more than fourfold of the MIC value. Hence, the methanolic extract can be said as bactericidal on all the test organisms including MRSA, B. spizizenii, B. subtilis, B. cereus, A. anitratus, and Erwinia sp.

5.5 The effects of addition of extract on the growth of bacteria

5.5.1 Growth profiles for the Gram-positive bacteria

For the study of growth profile of bacteria, the standard plate count method was employed to enumerate the bacterial cells present in the samples. Black (2000) described that the plate count method is generally based on the assumption in which a single cell divide exponentially forming a discrete colony. In this study, the accuracy of serial dilution and plate count method depend significantly on the homogeneous dispersal of the organisms in each dilution to ensure even distribution and good growth of the organisms. This method was employed to determine the growth profiles of MRSA, B. spizizenii, B. subtilis, and B. cereus in this study due to its ability to enumerate only the viable bacterial cells present in the sample as compared to the optical density measurement which is unable to differentiate between the dead and living bacterial cells. This is particularly important as it provides the information about the effects of Haliclona sp. extract on the bacterial cells, whether it is bacteriostatic or bactericidal (Kueh, 2007).

Based on the results, the addition of Haliclona sp. methanolic crude extract has significantly inhibited the growth of all the Gram-positive bacteria in contrast to the control which is not treated with the extract. These results were in agreement with the growth profile pattern obtained by several local investigators (Darah et al., 2006; Sasidharan, 2006). Based on the growth profiles of all the 4 species of Gram-positive bacteria including MRSA, B. spizizenii, B. subtilis, and B. cereus, general pattern of growth phases with lag phase, exponential phase, stationary phase and death phase were observed. From the results, the colony forming unit (CFU) obtained was observed to be decreasing at each period of intervals with the increase in the concentration of methanolic extract. This is due to the inhibition of bacterial growth by the bioactive compounds present in the extract. The effectiveness of the Haliclona sp. extract as an antibacterial drug was increased by increasing the concentration of extract. From the results, the bacterial cells were killed at the concentration of extract that is bactericidal (2MIC).

At the concentration of half MIC, the growth profiles of MRSA, B. spizizenii, B. subtilis, and B. cereus generally exhibited the normal growth phases. However, the growth of bacteria generally ceased and the stationary phase eventually reached earlier and was relatively shorter than the control. The bacterial count declined gradually following the short stationary phase and entered the death phase. This result revealed that the extract significantly inhibited the growth of all the 4 species of the Gram-positive bacteria. However, the inhibition effect was significantly vary for different species of bacteria with the strongest inhibitory activity was observed on MRSA, followed by B. spizizenii, B. subtilis, and B. cereus respectively with decreasing inhibitory activities. Nevertheless, this concentration was not sufficient to exhibit significant antibacterial activity of the extract due to the high CFU count was obtained at the end of the incubation period. At the concentration of MIC, the growth of bacteria did not exhibit distinct growth phases as shown in the control. Thus, it suggested that the extract exhibited significant antibacterial activity on all the Gram-positive bacteria at this concentration. According to a pharmacodynamics study by Levison (2000), the growth of bacteria at the concentration of MIC must be less than ten-fold increase in the bacterial density after 24 hours of incubation period. This statement supported that the MIC values obtained from the broth dilution method for MRSA, B. spizizenii, B. subtilis, and B. cereus in this study were relevant due to the low CFU count recorded after 24 hours of exposure to the extract.

At the concentration of 2MIC, the extract was observed to be bactericidal. In general, the growth profiles of MRSA, B. spizizenii, B. subtilis, and B. cereus at this concentration exhibited decreasing trend associated with low CFU count which decreased gradually at each exposure intervals. Thus, the result indicated that higher concentration of extract which is more than the MIC value is required to kill the bacterial cells. At this concentration, the extract was effective to cause the lysis of the bacterial cells. The MBC values obtained by the broth dilution method were relevant because the extract has significantly lowered the bacterial density to less than 1 x 102 cells/ml (Levison, 2000).

In this study, the generation time of the extract treated culture was a good evidence of the antibacterial activity exhibited by the methanolic crude extract of Haliclona sp. From the results, the addition of the extract has significantly prolonged the generation time of MRSA, B. spizizenii, B. subtilis, and B. cereus. At the MIC concentration, the generation time for the bacterial cells was relatively longer than that of the control. Thus, this result revealed that a longer period of time was needed for a bacterium to produce two identical cells by binary fission after addition of the extract (Black, 2008). This condition is good for the application of the extract as an antibacterial agent.

5.5.2 Growth profiles for the Gram-negative bacteria

Based on the results, the addition of Haliclona sp. methanolic extract has significantly inhibited the growth of the Gram-negative bacteria in contrast to the control which is not treated with the extract. However, the effects of inhibition were significantly lower and weaker than that of the Gram-positive bacteria. Based on the growth profiles of A. anitratus and Erwinia sp., general pattern of growth phases with lag phase, exponential phase, stationary phase and death phase were observed. However, the length of each phase was varying significantly compared to the Gram-positive bacteria. From the results, the colony forming unit (CFU) obtained was observed to be decreasing at each period of intervals with the increase in the concentration of methanolic crude extract. Likewise, the bacterial cells were observed to be killed at the concentration of extract that is bactericidal (2MIC).

At the concentration of half MIC, the growth profiles of A. anitratus and Erwinia sp. generally exhibited the normal growth phases as exhibited in the Gram-positive bacteria. However, the growth of bacteria ceased slowly and the stationary phase was relatively shorter and indistinctive than the control. The bacterial count declined in slow fashion entering the death phase. This result revealed that the extract inhibited the growth of both the Gram-negative bacteria, but the inhibition effects were much lower and weaker than that of the Gram-positive bacteria. Nevertheless, this concentration was not sufficient to exhibit significant antibacterial activity of the extract due to the high CFU count was obtained at the end of the incubation period. At the concentration of MIC, the growth of bacteria did not exhibit distinct growth phases as shown in the control. Thus, it suggested that the extract exhibited significant antibacterial activity on the Gram-negative bacteria at this concentration. The growth of bacteria at the concentration of MIC is less than ten-fold increase in the bacterial density after 24 hours of incubation period. This revealed that the MIC values obtained from the broth dilution method for A. anitratus and Erwinia sp. in this study were relevant due to the low CFU count after 24 hours of exposure to the extract.

At the concentration of 2MIC, the extract was observed to be bactericidal. In general, the growth profiles of A. anitratus and Erwinia sp. at this concentration exhibited decreasing trend associated with low CFU count which decreased gradually at each exposure intervals. Thus, the result indicated that higher concentration of extract which is more than the MIC value is required to kill the bacterial cells.

From the results, the addition of the extract has significantly prolonged the generation time of A. anitratus and Erwinia sp. At the MIC concentration, the generation time for the bacterial cells was relatively longer than that of the control. Thus, this result revealed that a longer period of time was needed for a bacterium to produce two identical cells by binary fission after addition of the extract (Black, 2008).

In this study, the main reason for the differences in the bacterial susceptibility to the extract of Haliclona sp. could be due to the cell wall properties of the Gram-negative bacteria. In the Gram-negative bacteria, there is an outer membrane surrounding their cell wall which restricts the diffusion of compounds through its lipopolysaccharide covering, as previously reported (Vaara, 1992). In addition, the periplasmatic space contains enzymes which are capable of breaking down foreign molecules introduced from the outside (Vaara, 1992). Thus, this outer membrane of the Gram-negative bacteria serves as a protective cover that prevents the bioactive compounds present in the extract from inhibiting the growth of these organisms.

5.6 SEM (Scanning Electron Microscopy) observation of the structural degeneration of the extract treated bacterial cells

The morphology of the Gram-positive MRSA was observed under the scanning electron microscope (SEM) to study the effect of addition of the methanolic crude extract on the structures of bacterial cells. SEM is a very useful tool employed to create images of the surface of microorganisms and it also allows the study of external structure of the cell (Black, 2008). In this study, the cells of MRSA exhibited a very distinct morphological difference after treated with 100 mg/ml of the methanolic crude extract. However, the control cells which were not treated with the extract were observed to be showing normal coccal-shaped structure.

After 12 hours of exposure to the extract, the morphological structure of MRSA exhibited a distinct difference as compared to the control cells. The bacterial cells were observed to be crumpled and shrunk. The extract treated cells became crumpled together due to the secretion of sticky mucus and the normal cellular functions were corrupted leading to the shrinkage in the bacterial cells.

After 24 hours of exposure to the extract, there was formation of pores or cavities on the surface of the bacterial cells. This indicated that the extract kill the cells of MRSA by forming pores to increase the permeability of the cell wall or by disintegration of the cell membrane which will affect the metabolism and physiological activities of the cells (Sasidharan, 2006). This is due to the cell wall of the Gram-positive MRSA primarily consists a single type of molecules that is less rigid than the cell wall of the Gram-negative bacteria (Tuney et al., 2006). From the SEM micrograph, it revealed that the target of the methanolic crude extract is on the cell wall due to the formation of cavities. Furthermore, according to Kueh (2007), the lost of cellular materials and osmolarity control caused the surface of the bacterial cells became crumpled.

After 36 hours of exposure to the extract, the structure of the bacterial cells were collapsed making the cells lost the coccal shape and the complete invagination of the cell wall was observed. From the result, it suggested that the cells without a normal and sturdy cell wall structure burst when exposed to low osmotic pressure (Black, 2008). According to Sasidharan et al. (2008), the complete invagination of the cell wall indicated the lost of cellular materials and organelle from the cells' cytoplasm.

According to the research by Koprivnjak (2002), the microbial cell wall is the target of the extract due to the presence of polyanionic bioactive compounds. The integrity of the cell wall is mainly depending on the Ca2+ and Mg2+ ions which ionically link the polysaccharide side chains. The interaction between these polyanionic groups and the divalent cations in the cell wall of bacteria significantly disrupted the integrity of the cell wall and subsequently causes the cell lysis. Furthermore, a study by Blondelle (1996) also suggested that the interaction between the bioactive compounds with the sialic acid or lipid layer in the microbial cell wall will cause cell lysis due to the lipid packing in the cell wall is disrupted by the interaction.

In this study, the morphology of the Gram-negative A. anitratus was also observed under the scanning electron microscope (SEM) to study the effect of addition of the methanolic crude extract on the structures of bacterial cells. The cells of A. anitratus exhibited a distinct morphological difference after treated with 100 mg/ml of the extract. However, the control cells which were not treated with the extract were observed to be showing normal rod-shaped structure.

After 12 hours of exposure to the extract, the morphological structures of A. anitratus were observed to be crumpled and shrunk in contrast with the control cells which are in normal rod shape. The extract treated cells became crumpled together due to the secretion of sticky mucus and the normal cellular functions were corrupted leading to the shrinkage in the bacterial cells.

After 24 hours of exposure to the extract, there was formation of cavities on the surface of the bacterial cells which was an indication that the extract kill the cells of A. anitratus by forming pores to increase the permeability of the cell wall or by disintegration of the cell membrane which will affect the metabolism and physiological activities of the cells. From the electron micrograph, it revealed that the target of the methanolic crude extract is on the cell wall due to the formation of cavities.

After 36 hours of exposure to the extract, the structures of the bacterial cells were totally collapsed making the cells lost their rod shape and the cell wall was completely invaginated. From the result, it suggested that the cells without a normal and sturdy cell wall structure burst when exposed to low osmotic pressure (Black, 2008). According to Sasidharan et al. (2010), the complete invagination of the cell wall indicated the lost of cellular materials and organelle from the cells' cytoplasm.

5.7 Determination of in vivo toxicity using the brine shrimp lethality assay

The brine shrimp lethality assay was proposed by Michael et al. (1956). The brine shrimp lethality assay is a useful tool for the preliminary assessment of toxicity. and it has been employed for the detection of fungal toxins (Harwig and Scott, 1971), plant extract toxins (McLauglin et al., 1991), heavy metals (Martínez et al., 1998), cyanobacteria toxin (Jaki et al., 1999) and also pesticides (Barahona and Sánchez-Fortún, 1999). In this study, the modified method of Carballo et al. (2002) was employed to study the toxicity of the extract because the brine shrimp lethality assay is more sensitive in detecting the toxicity of the extract compared to other bioassay. Furthermore, the assay was relatively easy to perform and did not require the use of any special equipment. However, there are several factors which critically affect the outcomes of the toxicity test including the water hardness, pH, temperature, chemical formulation, age and the developmental stage of Artemia salina (Barahona, 2006). Thus, these parameters were standardized in this study in order to obtain accurate results and prevent erroneous outcomes.

In this study, the main objective of the brine shrimp lethality assay is to determine the LC50 (50% lethal concentration) values for both the acute and chronic toxicities. By definition, LC50 is the concentration that kills half of the sample population in a specific period of exposure. In general, the acute toxicity describes the adverse effects of a substance in a short period of exposure time that is usually shorter than 10 hours. In contrary, the chronic toxicity describes the adverse effects of a substance over a long period of exposure time (Sasidharan et al., 2010). Eventually, this study is very significant in determining the potential of Haliclona sp. to be developed as a safe antimicrobial drug without causing any toxic effects. In this study, 1.5% DMSO diluted with 3.8% artificial sea water was used as the negative control. From the results, 1.5% DMSO did not cause any lethality of A. salina for both the acute and chronic toxicities. Thus, this revealed that DMSO with the concentration less than 1.5% was suitable to be employed as the solvent to dissolve the extract.

Based on the results of brine shrimp lethality assay, the LC50 values for both the acute and chronic toxicities were significantly different. Most investigators came up to an agreement that there was a tendency for the LC50 to decrease with longer exposure duration to the toxic compounds (Barahona, 1996; Carballo et al., 2002). In this study, the LC50 values recorded for the acute toxicity was 2066 µg/ml while for the chronic toxicity was 314 µg/ml. According to Simionatto et al. (2005), the crude extract with the LC50 value more than 1.0 mg/ml is interpreted and considered as not toxic to A. salina. In this study, the LC50 value for the acute toxicity was higher than 1.0 mg/ml indicating that it is not toxic, whereas for the chronic toxicity the LC50 value recorded was lower than 1.0 mg/ml. Thus, these results suggested that the crude extract possessed significant toxicity to A. salina.

In the brine shrimp lethality assay, the age of the shrimps has an influence on their sensitivity towards the toxins, a fact that was observed by Hlywka et al. (1997). In this study, for reasons of comparability and repeatability, the larvae of A. salina were employed in the brine shrimp lethality assay exactly 24 hours after hydration of the cysts. This is due to most of the eggs have already hatched within this period of incubation. Furthermore, aeration supply is another vital factor influencing the sensitivity of the shrimps towards the toxins. Thus, it is important for the shrimps to be shaken during the length of the assay. Apparently, the larvae are able to remain close to the surface of solution where there is ample oxygen supply with the solution in permanent motion (Hartl and Humpf, 2000).

In the brine shrimp lethality assay, there may be variability in the sensitivity of the shrimps towards the toxicants. Therefore, a numbers of factors including the temperature of incubation and hatching, the moment of larvae harvest, the period of time between the harvest and the onset of the bioassay and the temperature, aeration supply, light, pH as well as the salinity of the medium during the bioassay have to be well maintained in order to avoid the variability in the sensitivity of the shrimps (Sorgeloos et al., 1978).

According to the study by Barahona (2006), the larvae of A. salina aged 48 hours were used due to their high sensitivity to toxic compounds. In this study, the 48 hours old larvae of A. salina which are in their second and third instar larval stage were selected for the toxicity test. This is due to the higher sensitivity of larvae at this stage compared to the first instar stage larvae (Soares and Calow, 1993). Besides, more reproducible results were obtained by using the larvae in the second and third instar larval stage.

During this study, the living and dead nauplii of A. salina were observed under the inverted light microscope. It was observed that the dead nauplii have shrunken in their size and the lining of their organs seemed to be ruptured and unclear as compared to the living nauplii. There was also none of any internal or external movements being observed for these dead nauplii. These observations could be due to the effects of toxication of the nauplii by the extract of Haliclona sp. Due to this significant toxicity exhibited by the crude extract, thus further analysis and extensive studies should be carried on the extract of Haliclona sp. in order to explore the possibility of using it for pharmacological purpose and also to ensure its suitability to be developed as a safe antimicrobial drug.

CHAPTER 6.0 CONCLUSION AND FUTURE RESEARCH

In this study, the main aim was to study and extract the antimicrobial compounds from the local marine soft sponge, Haliclona sp. From the screening test, the methanolic crude extract of Haliclona sp. exhibited significant antibacterial activity by inhibiting 6 species of test bacteria including 4 Gram-positive and 2 Gram-negative bacteria. Thus, this revealed that the extract possess a broad spectrum of activity and showed a defined potential to be developed as an effective antibacterial drug. However, it was a relatively inactive antifungal and antiyeasts agent due to no inhibitory activity was observed on both fungi and yeasts.

On the basis of the good and significant antibacterial activity exhibited by the methanolic crude extract of Haliclona sp., the in vivo toxicity of the extract was assessed through the brine shrimp lethality assay. The LC50 values obtained for the acute toxicity was higher than 1.0 mg/ml whereas for chronic toxicity the LC50 value was lower than 1.0 mg/ml. However, these results suggested the significant toxicity of the methanolic crude extract of Haliclona sp.

In conclusion, the findings of this study revealed the potential of Haliclona sp. as a source of antibacterial agent from the natural sources which is capable of curbing the threats of antibiotic resistance and emergence of new infectious agents worldwide. Thus, the future research should be focused mainly on the studies and isolation of bioactive compounds that possess significant antimicrobial activity from natural sources to be developed as a safe, effective and potential antimicrobial drug.

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