Green Algae Halimeda macroloba in Spermonde Archipelago: Phytochemical and In Vitro Antibacterial Studies

Green algae, known as green seaweed, emerged between 900 and 500 billion years ago. These algae belong to the eukaryotic photosynthetic group. One of them is Halimeda from the Udoteaceae family. This plant lives in habitats associated with coral reefs and contains high amounts of calcium carbonate. Therefore, Halimeda is classified as calcified or calcareous algae.1,2 According to several studies, Chlorophyta or green algae compounds active against human, fish, and shrimp pathogenic bacteria. It is one of the marine natural organisms (MNO) which is a diverse source of secondary metabolites. Natural products produced by various species of the genus Halimeda, including Halimeda macroloba, H. opuntia, H. macrophysa, H. gracilis, H. tuna, and H. renschi have been tested as potential antibacterial agent.3-7 For example, 4 new diterpenoids, halimedatrial-tipe compounds (structures 1,3,4,5) (Figure 1) which are potential as antibacterial agents8, have been isolated from several Halimeda species. Halimedatrial has antibacterial activity against a number of marine microorganisms, including Vibrio splendida, V. leiognathi, V. harveyi, Bacillus subtilis, and Staphylococcus aureus. 9,10


INTRODUCTION
Green algae, known as green seaweed, emerged between 900 and 500 billion years ago. These algae belong to the eukaryotic photosynthetic group. One of them is Halimeda from the Udoteaceae family. This plant lives in habitats associated with coral reefs and contains high amounts of calcium carbonate. Therefore, Halimeda is classified as calcified or calcareous algae. 1,2 According to several studies, Chlorophyta or green algae compounds active against human, fish, and shrimp pathogenic bacteria. It is one of the marine natural organisms (MNO) which is a diverse source of secondary metabolites. Natural products produced by various species of the genus Halimeda, including Halimeda macroloba, H. opuntia, H. macrophysa, H. gracilis, H. tuna, and H. renschi have been tested as potential antibacterial agent. [3][4][5][6][7] For example, 4 new diterpenoids, halimedatrial-tipe compounds (structures 1,3,4,5) ( Figure 1) which are potential as antibacterial agents 8 , have been isolated from several Halimeda species. Halimedatrial has antibacterial activity against a number of marine microorganisms, including Vibrio splendida, V. leiognathi, V. harveyi, Bacillus subtilis, and Staphylococcus aureus. 9,10 Halimeda macroloba in Lae-Lae Island, Spermonde Archipelago, grows in complex environmental conditions (relatively high and changing salinity of seawater, high heavy metal content, and susceptibility to the surrounding organisms).
Environmental factors such as temperature, humidity, light intensity, water supply, minerals, and CO 2 , affect the growth and production of secondary metabolites in a plant. 11 In order to survive, as a response to mentioned complex environmental factors, seaweed developed the ability to produce distinctive secondary metabolites.
A preliminary screening of the antimicrobial activity of crude algae extracts can help in the search of new antimicrobial seaweed metabolites. Such results can direct further phytochemical investigation and result in the isolation and structural elucidation of new natural products. For that reason, we have set our goal towards determining the antimicrobial potential of crude extracts of green algae Halimeda macroloba that grows in challenging environmental conditions of Spermonde Archipelago.

Collection of sample
Green algae samples (Halimeda macroloba) were collected by hand (Snorkeling) from Lae-Lae Island, Makassar City, South Sulawesi Province, Indonesia. Samples were washed with seawater, freshwater, and distilled water to clean seaweed from salt, epiphytes, and other impurities. Then the wet samples (1 kg) were dried using a freeze drier. The sample was identified by the Productivity and Water Quality Laboratory, Faculty of Marine Science and Fisheries, Hasanuddin University, South Sulawesi Province, Indonesia.

Extractions
Samples were extracted in various solvents (a selection was based on the difference in their polarity). The extraction of secondary metabolites was carried out using n-hexane, ethyl acetate, and methanol; a ratio of powder extract to solvent was set 1:8 (w:v). 12

Antibacterial analysis
Bacterial species (Vibrio harveyi (M-120), Aeromonas hydrophilla, and Vibrio parahaemolyticus (T-170) (shrimp pathogenic bacteria)) were donated by The Center for Brackish Water Aquaculture, Takalar, and Maros, South Sulawesi Province, Indonesia. The turbidity of the suspension was standardized against 0.5 McFarland using a spectrophotometer at a wavelength of 600 nm. 13 The bacterial inoculum was 10 8 cfu/ml. Antibacterial activity tests included positive, negative control and antibacterial activity tests for seaweed extract. The positive control test was carried out using ciprofloxacin antibiotics, and the negative control test used solvents (DMSO). The antimicrobial activity was determined by using slightly modified agar diffusion method. [14][15] Sterile paper discs with a diameter of 6 mm were soaked in extracts with various concentrations. The concentrations were 4 µg, 3 µg, and 2 µg (25 μl /disc). The samples were incubated for 24 hours at 30°C. The distinct zone around the paper disc is a sign of bacterial activity. Every experiment was conducted in triplicate. Inhibition zones >15 mm were declared as strong, from 8 to 15 mm as moderate, and 1 to 8 mm as weak activities. 16

Phytochemical test
A phytochemical identification test was used to determine the chemical content in a material qualitatively. Slightly modified previously described tests were used to determine the presence of alkaloid, flavonoid, steroid, and terpenoid compounds in the extracts. 17

RESULTS AND DISCUSSION
Results of the phytochemical tests (Table 1) showed that the crude extracts have several classes of compounds. They were dominated by steroid/terpenoid compounds. A positive Wegner test suggested the presence of alkaloids. Although, the outcome of two other diagnostic tests for alkaloids (Meyer and Dragendorf) was negative. These results are consistent with the results of the previous studies, which stated that steroids and terpenoids are found in Halimeda sp. 5,8 Many representatives of these classes of compounds are strong antimicrobials. Thus, it was reasonable to expect that herein studied Halimeda extract will have at least some antimicrobial potential. The result of the antibacterial activity screening of four crude extracts against Gram-negative bacteria is summarized in Table 2  the highest antimicrobial activity. It inhibited the growth of two Gramnegative bacteria. Aeromonas hydrophilla with the inhibition zone of 8.27 mm at a concentration of 4 µg, was the most susceptible organism. Inhibition of the growth of Vibrio harveyi was slightly weaker at a concentration of 4 µg. It was 8.23 mm. The value of the inhibition zones belonged to the category of weak. 16 As previously shown, metabolites of green algae Halimeda macroloba exhibit a broad spectrum of antibacterial activity. 3 Halimeda sp. showed to inhibit the growth of Gram-positive bacteria Bacillus cereus and Staphylococcus aureus. 7 While Halimeda opuntia metabolites are active against Escherichia coli and Staphylococcus aureus bacteria. The results are inhibition of 21 mm and 19 mm using 70% ethanol solvent. 6 Other related studies to Halimeda, showed that methanolic extracts of H. macrophysa, H. gracilis, H. opuntia, and H. renschi inhibited growth of four types of bacteria. 5 Extracts of another species H. tuna were tested against 10 types of human pathogenic bacterial strains. The results of this study showed that methanolic extract had higher antimicrobial potential, compared to ethanol and chloroform ones. 4 The detected antibacterial activity of Halimeda extracts might be the outcome of the presence of terpenoid compounds. For example, compounds halimedatrial (1), halomedatriol triacetate (2), halimedalactone (3), halimedatetraacetate (4), and Bis-nor-Dianpenoid (5) have been isolated from several species of Halimeda sp. (Figure  1). These species have activity against bacteria, i.e. Vibrio sp., Bacillus subtilis, and Staphylococcus aureus. 10 Halimedatrial (diterpenoid trialdehyde derivative), and halimedatetraacetate (tetraacetate diterpenoid derivative), secondary metabolites that are mostly found in Halimeda algae, are reported as antibacterial agents against some marine microorganisms. 9 Although crude extracts of Halimeda macroloba show only weak bacterial growth inhibition, it does confirm that some of its constituents are potential strong antimicrobials. Isolation and structure elucidation of pure constituents and their subsequent antimicrobial screening is needed to locate exact carriers of the detected activity. Thus, here presented data can be used as a starting point and illustrates potential of secondary metabolites from macroalgae in Lae-Lae Island as potential antibacterial agents against shrimp pathogenic bacteria. They might be even used in the future as alternatives to conventional antibiotics in aquaculture.

CONCLUSION
Halimeda macroloba crude extracts (3 different solvents) showed weak antibacterial activities against the tested organisms (3 Gram-negative bacteria). Ethyl acetate crude extract had the highest activity (disc diffusion method). The fast phytochemical test was used to determine the presence of certain classes of compound in the studied extracts and suggested that the extracts contained steroids, terpenoids, and alkaloids. However, further studies (isolation and structural elucidation) are needed to locate exact carriers of the observed antimicrobial activity. Extracts  N-hexane  Ethyl acetate  Methanol  1  Wegner  +  +  +  2  Meyer  ---3  Dragendorf  ---4  FeCl3  ---5 Lieberman-Buchard ++ ++ ++

ABOUT AUTHORS
Lulu Adilla Latifah is a Doctoral Student at the Department of Fisheries Science, Faculty of Marine Sciences and Fisheries, Hasanuddin University, Indonesia. Lulu's current research focuses on studies of secondary metabolite compounds from marine natural products as antibacterial or anticancer drugs, especially marine sponge and macroalgae.
Nunuk Hariani Soekamto is a lecturer at the Department of Chemistry, Faculty of Mathematics and Natural Sciences, Hasanuddin University, Indonesia. She is a Professor in organic chemistry and interested in working in the field of natural product chemistry.