OPCE-STR.vp Acta Bot. Croat. 70 (1), 81–90, 2011 CODEN: ABCRA 25 ISSN 0365–0588 Biological activity of the red alga Laurencia brandenii ASEER MANILAL1, SUGATHAN SUJITH1, BALU SABARATHNAM1, GEORGE S. KIRAN1, JOSEPH SELVIN1*, CHIPPU SHAKIR1, AARON P. LIPTON2 1 Department of Microbiology, Bharathidasan University, Tiruchirappalli 620 024, India 2 Central Marine Fisheries Research Institute, Vizhinjam 695 521, Thiruvananthapuram, India Abstract – The marine red alga Laurencia brandenii collected from the southwest coast of India (Indian Ocean) was extracted and fractioned using column chromatography. The individual fractions were evaluated in vitro via antimicrobial activity against six species of Microbial Type Culture Collection and three species of clinical human pathogens, antipest activity on Sitophilus oryzae, maggoticidal activity against 2nd instar larvae of Sarcophaga sp. and termiticidal activity against Microtermes obesi. It was found that the fraction eluted using petroleum ether:chloroform (6:4) exhibited broader biological activi- ties. The phyco-constituents of the active fraction were identified by gas chromatography- -mass spectrometry (GC-MS) analysis. The GC-MS profile of the active fraction revealed that the main constituent was octadecadienoic acid (49.75%) followed by n-hexadecanoic acid (14.24%), which might have a functional role in the biological activities. The overall activity profile envisages that these bioactive compounds from L. brandenii could be uti- lized as a renewable natural resource for the development of novel environmental-com- patible formulations for the control of human pathogens, pests, termites and maggots. Keywords: Alga, Laurencia brandenii, antimicrobial activity, antipest activity, maggoti- cidal activity, termiticidal activity, larva, Sarcophaga, octadecadienoic acid, n-hexade- canoic acid Introduction Natural marine products are in great demand due to their prolific biological activities and serve as source for the discovery of novel bioactive compounds (BLUNDEN 2001). Dur- ing the past few decades, over 14,000 novel natural products from marine organisms have been isolated and described (PROKSCH 2002). Such biogenic compounds exhibit unique structural and functional properties that are not found in terrestrial natural products (FENICAL 1982). Among the diverse group of marine flora, seaweeds are the most primitive vegetation, surviving over a large range of environmental conditions and being continu- ACTA BOT. CROAT. 70 (1), 2011 81 * Corresponding author, e-mail: selvinj@rediffmail.com Copyright ® 2011 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:51 Color profile: Disabled Composite 150 lpi at 45 degrees ously exposed to high density of harmful micro and macrofauna. The survival of this sed- entary vegetation has primarily relied on efficient defence mechanism against diverse in- vaders. Seaweeds from varied locales have been evaluated for a wide range of biological activities, e.g., antibacterial (TUNEY et al. 2006), antiviral (SERKEDJIEVA 2004), antifungal (TANG et al. 2002) and antialgal activities (HELLIO et al. 2002). Over 2,400 secondary me- tabolites have been isolated and described from the divisions Rhodophyta, Phaeophyta, and Chlorophyta, many of which have been reported to have excellent biological activity (FAULKNER 2001 and references therein). Of the three groups of seaweeds, the class Rhodophyta produces a plethora of structur- ally diversified novel halogenated natural products, which in fact symbolize the extraordi- nary wealth of biogenic compounds principally for pharmaceutical leads (FAULKNER 2001). Among the red algae, the genus Laurencia is known to produce the largest number and diversity of secondary metabolites, ultimately making it the world’s most chemically complex seaweed genus (PEREIRA et al. 2003). Moreover, the anticandidal, antibacterial (MANILAL et al. 2009a, SHANMUGHAPRIYA et al. 2008), nematicidal and mosquito larvicidal activity, as well as the ichthyotoxicity and brine shrimp cytotoxicity (MANILAL et al. 2009b) of crude extracts of different red algal species from the Indian coast has already been re- ported. Therefore, the present study was aimed to evaluate the biological activity of L. brandenii against human pathogens, pests, maggots and termites and to detect the bioactive compounds from L. brandenii that could be useful for the development of novel environ- mental compatible formulations for the control of human pathogens, pests, termites and maggots. To this day, information regarding biological activities of flora and fauna from the southwest coastline of India (Kollam coast) remains scanty. Materials and methods Study area and collection of samples The study area was located along the 45 km long southwest coast of India (Indian Ocean), between latitude 08°54' N and longitude 76°38' E. Seaweed specimens were collected from the intertidal and subtidal habitat of Kollam prefecture (Thirumullavaram) located on the southwest coast. The area is rich in flora and fauna (MANILAL et al. 2009a). Samples were collected during December 2007 to February 2008 when red algal diversity remains domi- nant. Cleaned plant materials were shade-dried under a stream of air for one week to pre- vent photolysis and thermal degradation. The completely dried material was weighed and ground coarsely in a mechanical grinder. In the present study we evaluate the biological po- tencies of a column-purified fraction of the red alga Laurencia brandenii, on the following bioassays: 1) antimicrobial activity on nine species of human pathogens; 2) antipest activ- ity on Sitophilus oryzae; 3) maggoticidal activity on 2nd instar larvae of Sarcophaga sp. and 4) termiticidal activity against Microtermes obesi. Extraction of seaweed bioactives The shade-dried samples (5 kg) were extracted using methanol and purified in column chromatography to yield eleven fractions (F1-F11). The individual fractions were evaluat- 82 ACTA BOT. CROAT. 70 (1), 2011 MANILAL A., SUJITH S., SABARATHNAM B., KIRAN G. S., SELVIN J., SHAKIR C., LIPTON A. P. U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:51 Color profile: Disabled Composite 150 lpi at 45 degrees ed for biological activities (data not shown). The fraction eluted using petroleum ether: chloroform (6:4), fraction F8, showed a wide spectrum of activity in all the bioassays. For the extraction of bioactive compounds, 100 g of the dried seaweed powder was weighed and immersed in a flask containing 1000 mL of methanol (purity grade 99%) and placed at 35 °C in a shaker at 120 rpm for 7 days for the extraction of active ingredients. The algal material was re-extracted with methanol in a 1 L capacity round bottom flask in a water bath at 60 °C for 3 h. The individual crude mixture was pooled and filtered using paper fil- ter fitted with a Buchner funnel using suction pressure followed by centrifugation (Eppen- dorf) at 6000 � g for 5 min at 20 °C. The supernatant was collected in a round-bottomed flask and the remaining solvent was concentrated up to 15–20 mL in a rotary vacuum evap- orator (Yamato). The residue collected was evaporated to dry completely in a vacuum des- iccator and stored in the refrigerator. Chromatography of Laurencia brandenii The methanolic extract of Laurencia brandenii (200 g) was applied in a silica gel (60–120 mesh) column developed with petroleum ether and eluted with petroleum ether and chloroform (9:1 to 1:9 and 100% chloroform) followed by chloroform and methanol (9:1 to 1:9 and 100% methanol) and yielded eleven fractions. The individual fractions were screened for biological activity (data not shown). The fraction that was eluted using petro- leum ether: chloroform (6:4), which exhibited activity was used for Hewlett Packard gas chromatography-mass spectrometry (GC-MS) analysis. Peak identification was carried out by comparison of the mass spectra with those available in the NIST Version 2 (2005). Test microorganisms To characterize the antimicrobial activity, the algal fractions were evaluated against a battery of human pathogenic bacteria. Antimicrobial activity (inhibition area) was deter- mined against six species of type cultures from Microbial Type Culture Collection (MTCC) of human Gram-positive pathogens, such as Staphylococcus aureus (MTCC 2940), Bacil- lus subtilis (MTCC 1306), Micrococcus luteus (MTCC 106), Rhodococcus rhodochrous (MTCC 265), and the Gram-negative bacteria Escherichia coli (MTCC 739) and Pseudo- monas aeruginosa (MTCC 2453), as well as three species of clinical isolates, Vibrio cholerae, Salmonella typhi, and Streptococcus pneumoniae. The resistant patterns of these human pathogenic isolates were confirmed using selective antibiotics: streptomycin, oxy- tetracycline, ampicillin and erythromycin, in a preliminary experiment. Antimicrobial assay The antibacterial assay was carried out following the methodology of SELVIN and LIPTON (2004). Anti-pest activity Pesticidal activity of the algal fraction was evaluated by a modified area preference test (MCDONALD et al. 1970) using the common rice weevil Sitophilus oryzae (L.). The organ- ism was collected from infested rice obtained from a local market and reared in glass bot- tles under standard insectaria conditions at ambient temperature (28 � 3 °C) and the relative humidity 70 � 5%. A uniformly saturated filter paper disc of different concentrations of al- BIOACTIVITY OF LAURENCIA BRANDENII 83 ACTA BOT. CROAT. 70 (1), 2011 U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:51 Color profile: Disabled Composite 150 lpi at 45 degrees gal extracts (2, 4, 6, 8 and 10 mg cm–2) was pasted in the bottom of each petri dish. Ten un- known-sex insects of same age were released in the petri dish and kept in a dark room. Mor- tality was observed after 24 h of exposure. All the experiments performed in the present study were repeated six times to validate the findings statistically. The mortality percentage was converted into probit scale to determine the LD50 values. Maggoticidal activity For the determination of maggoticidal activity, 2nd instar maggots of the flesh fly, Sarcophaga sp. (Miegen) were used as test organism. A stock colony of the insect was es- tablished using mature flies collected from local slaughter house. A couple of adults were released into a wide-mouth glass chamber covered with a copper mesh screen and a piece of chicken meat was placed for the oviposition. The whole setup was left for 72 h with con- stant monitoring. Ten successfully hatched larvae were picked using a blend end probe and placed in a 40 mm disposable petri dish filled with boiled egg yolk uniformly mixed sea- weed extracts (20, 40, 60, 80 mg g–1). Petri dishes containing no seaweed extracts and methanol were used as positive and negative control. Since this was the active feeding stage of the maggots, they could consume the appropriate quantity of the algal fraction mixed with boiled egg yolk, so the effect of the algal extracts could be evaluated effec- tively. Mortality rate of treated maggots was recorded after 24 and 48 h of exposure. Based on the percent mortality, the LD50 value was determined using probit scale (WARDLAW 1985). Termiticidal activity The test termite Microtermes obesi (Holmgren) worker was collected from the thickets of Bharathidasan University. The collected organisms were maintained for three days on a degraded log in a glass chamber at 30 °C ± 2 °C, 75 ± 5% relative humidity and a 12:12 h photoperiod. Ten worker termites were transferred to a 30 mm Petri dish uniformly lined with different concentration of algal fractions which dried overnight to remove the solvent completely. The Petri dishes were covered and kept in a dark cabinet maintained at 25 °C for 24 h. The survivors were counted under mild light and the percentage of survivors was calculated. All the experiments were repeated six times to validate the findings statistically. Results In antimicrobial assay, the algal fraction exhibited a growth inhibition range of 213 mm2 to 87 mm2 against the nine species of Microbial Type Culture Collection and clinical isolates (Fig.1). The results of the antipest activity indicated that the algal fraction was toxic to Sitophilus oryzae which produce an LD50 value of 3.7 mg cm–2 (Fig. 2). Maggoticidal assay showed the algal fraction was active against the 2nd instar maggots of Sarcophaga with an LD50 of 43 mg g–1 (Fig. 3). Concerning the termiticidal assay, the ac- tive fraction of L. brandenii was found to be highly lethal at 2.6 mg mL–1 causing 50% mor- tality at a 6 h exposure (Fig. 4). The fraction F8 exhibited biological activity and proceeded to GC-MS analysis, to show the relative percentage of identified active compounds in Laurencia brandenii (Tab. 1). The main phycoconstituents of the active fraction were octadecadienoic acid (49.75%) followed by n-hexadecanoic acid (14.24%). 84 ACTA BOT. CROAT. 70 (1), 2011 MANILAL A., SUJITH S., SABARATHNAM B., KIRAN G. S., SELVIN J., SHAKIR C., LIPTON A. P. U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:51 Color profile: Disabled Composite 150 lpi at 45 degrees ACTA BOT. CROAT. 70 (1), 2011 85 BIOACTIVITY OF LAURENCIA BRANDENII 0 50 100 150 200 250 SA BS ML RR EC PA VC ST SP Test organisms A re a o f In h ib it io n (m m ) 2 Fig. 1. Antibacterial potentials of Laurencia brandenii against Microbial Type Culture Collection and clinical isolates. BS – Bavillus subtilis, EC – Escherichia coli, ML – Micrococcus luteus, RR – Rhodococcus rhodochrous, PA – Pseudomonas aeruginosa, SA – Staphylococcus aureus, SP – Streptococcus pneumoniae, ST – Salmonella typhi, VC – Vibrio cholerae. 0 20 40 60 80 100 2 4 6 8 Concentration (mg cm )–2 M o rt a li ty (% ) LD 3.7 mg cm50 –2 Fig. 2. Antipest activity of Laurencia brandenii against Sitopzhilus oryzae 0 20 40 60 80 100 20 40 60 80 Concentration (mg g )–1 M o rt a li ty (% ) LD 43 mg g50 –1 Fig. 3. Maggoticidal activity of Laurencia brandenii against Sarcophaga albiceps larvae U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:51 Color profile: Disabled Composite 150 lpi at 45 degrees 86 ACTA BOT. CROAT. 70 (1), 2011 MANILAL A., SUJITH S., SABARATHNAM B., KIRAN G. S., SELVIN J., SHAKIR C., LIPTON A. P. Concentration (mg mL )–1 M o rt a li ty (% ) 0 20 40 60 80 100 1 2 3 4 LD 2.6 mg50 mL –1 Fig. 4. Termiticidal activity of Laurencia brandenii against Microtermes obesi Tab. 1. GC-MS data of active fraction of Laurencia brandenii No RT Name of the Compound Molecular Formula Molecular Weight Peak Area % 1 5.01 Cyclohexasiloxane – dodecomethyl C12H36O6Si6 444 1.07 2 8.20 Trisiloxane 1,1,1,5,5,5-hexamethyl-3,3- -bis[(trimethyl silyl)oxy]- C12H36O4Si5 384 0.89 3 9.36 3,5-Dimethyl-5-hexan-3-ol C18H6O 128 0.11 4 11.64 3,5-Dimethyl-5-hexan-3-ol C18H6O 128 0.11 5 13.59 4-Dodecanol C12H26O 186 0.08 6 16.04 2-Decanone. 5,9-dimethyl- C12H24O 184 0.13 7 18.65 n-Hexadecanoic acid C16H32O2 256 14.24 8 20.90 9-Dodecanoic acid, methyl ester,(E)- C13H24O2 212 0.48 9 21.11 Oxalic acid, allyl hexadecyl ester C21H38O4 354 0.40 10 21.85 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280 49.75 11 24.33 Cyclohexanecarboxylic acid, decyl ester C17H32O2 268 2.70 12 25.96 9,12-Octadecadienoyl chloride (Z,Z)- C18H31ClO 298 3.50 13 28.26 Oxalic acid, allyl pentadecyl ester C20H36O4 340 1.23 14 28.75 cis- 9,10-Epoxyoctadecan-1-ol C18H36O2 284 0.61 15 29.79 Heptanoic acid, 9-decen-1-yl ester C17H32O2 268 0.65 16 30.63 9-Octadecenal C18H34O 266 5.91 17 34.00 9,12-Octadecadienoic acid (Z,Z)-, phenylmethyl ester C25H38O2 370 0.44 18 34.51 Oxalic acid, allyl hexadecyl ester C21H38O4 354 1.11 19 37.64 Oxirane, tetradecyl- C16H32O 240 3.49 20 38.61 1,2-15,16-Diepoxyhexadecane C16H30O2 254 3.01 21 40.00 7,11-Hexadecadienal C16H28O 236 5.99 22 40.40 Thunbergol C20H34O 290 4.11 U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:51 Color profile: Disabled Composite 150 lpi at 45 degrees Discussion Data obtained from the antimicrobial activity of the column purified fraction (F8) of Laurencia brandenii exhibited considerable activity against all the tested microbial type culture collection and clinical isolates. The magnitude of inhibition against organisms was on the decreasing order of Bacillus subtilis (213 mm2) > Micrococcus luteus (194 mm2) > Staphylococcus aureus (158 mm2) > Rhodococcus rhodochrous (132 mm2) > Pseudomo- nas aeruginosa (118 mm2) > Escherichia coli (103 mm2). A high activity range was ex- tended against one of the Microbial Type Culture Collection human pathogens, B. subtilis with 213 mm2, whereas the activity was moderate against E. coli 103 mm2. Regarding the clinical isolates, Salmonella typhi was found to be the most resistant bacteria, producing an inhibition area of 64 mm2 whereas the fraction was moderately able to inhibit the growth of Streptococcus pneumoniae (87 mm2). The bactericidal potency of the algal fraction was high against the clinical pathogen, Vibrio cholerae to the extent of 113 mm2 at 37 °C. In the present study, algal fraction of L. brandenii showed maximum antibacterial activity against the gram positive bacteria, whereas the activity was found to be moderate against gram negative bacteria. The resistant mechanism of gram negative bacteria could be due to the permeability provided by the cell wall or to the membrane accumulation tactics (ADWAN and ABU-HASAN 1998). Antimicrobial potentials of red algae have already been reported from different locales, but their efficacy against clinical and human pathogens have scarcely been investigated. Concerning the prevalence of microbial resistance to conven- tional antibiotics, our study provides a new insight into the development of novel phyto- therapeutics for the management of infectious diseases. Data pertaining to the susceptibil- ity of gram positive strains to algal extracts were reported by many authors (PESANDO and CARAM 1984, ROSETT and SRIVASTAVA 1987). Our result is in accordance with the antibacte- rial activity of Laurencia spp. reported from various geographic regions (DE NYS et al. 1996, HELLIO et al. 2001, TASKIN et al. 2007) against marine, human and fish bacterial pathogens (VAIRAPPAN et al. 2001, 2003; BANSEMIR et al. 2006). Based on the present find- ings L. brandenii could be utilized as renewable bioresource for the development of poten- tial antimicrobials. In the present study, the algal fraction of Laurencia brandenii exhibited considerable effects on the rice pest, Sitophilus oryzae showing an LD50 value of 3.7 mg after 24 h, sig- nificantly higher than that of solvent control, clearly demonstrating the direct contact toxic- ity as the rationale. The mode of action of this seaweed might be due to the shutdown of dif- ferent biosynthetic routes of the pest’s metabolic pathways. These insecticidal compounds can inhibit the feeding behaviour and growth regulators, disrupting the endocrinological balance of the insects (BALANDRIN et al. 1985). Marine flora and fauna with their broad chemical diversity are still an untapped resource for the development of new agro-chemical agents (CROMBIE 1990) and insecticides (SAXENA 1987). The findings of the present study indicate that red alga Laurencia could be employed as a biological grain protectant to com- bat invading pests. The maggoticidal activity of Laurencia brandenii was studied by feeding the maggots on an artificial diet containing different concentrations of the active fraction of the sea- weed. It showed only a moderate toxicity effect on maggots. Response varied with differ- ent concentrations of the algal fraction i.e., the toxicity of the extracts varies with an in- crease in concentration. Maximum mortality (100%) was recorded at 60 mg g–1, whereas at ACTA BOT. CROAT. 70 (1), 2011 87 BIOACTIVITY OF LAURENCIA BRANDENII U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:52 Color profile: Disabled Composite 150 lpi at 45 degrees 20 mg g–1 extract showed a decrease in activity. The results indicate that the L. brandenii fraction contains compounds that are toxic to maggots since the LD50 value is moderately low. The literature proved that the present study would be the first report on the maggo- ticidal property of seaweeds. The mortalities of the larvae were 100% and 50% after 24 h of exposure to 60 and 43 mg g–1 of algal extract respectively. At higher concentrations, the seaweed fraction was an annoyance to maggots, which showed feed aversion, avoidance, and repellence. The present findings proved that a direct feeding trial assay would be useful to prove the maggoticidal property of seaweed. According to this finding, L. brandenii could be used for developing a novel natural veterinary grade maggoticides, since these natural products would likely be biodegradable, thus posing no threat to the environment. The inexorable use of synthetic termiticides is a major threat to the natural environment, and as a result there is an urgent need to explore and utilize naturally occurring products for combating harmful termites (CARTER 1976). Historically, plants are well-known for their var- ious bioactivities. The toxicity of plants in termites has been reported since 1947 (WOLCOTT 1947). The bioactive compounds have evolved in plants for defense against phytophagous insects. The sesquiterpenes from Cryptomeria japonica reported to possesses termiticidal ac- tivity (ARIHARA et al. 2004). Similarly, essential oils from Melaleuca species can suppress the growth of termites (SAKASEGAWA et al. 2003). The termiticidal assay represents a rapid, inexpensive and simple bioassay for testing seaweed bioactivity. In the present study, a fatal effect was observed when 3 mg mL–1 con- centration of algal fraction of L. brandenii was applied to the petri dish, causing 90% mor- tality at a 6 h exposure. The mortality rate for the positive control experiment was much lower (<10) while that of negative control gave a mortality of 15%. The study showed that the termite population declined drastically relative to the concentration and time of expo- sure. The termiticidal potential of L. brandenii has not been investigated or reported in the literature. The tested seaweed fraction may be a valuable source of biorational compounds and could be employed for the development of novel natural termiticide. Spectral data by GC-MS showed that a mixture of fatty acids characterised the chemis- try of the active fraction. In all, 22 peaks with different retention times were observed. It is possible that bioactive compounds primarily consisting of 9, 12-Octadecadienoic acid (Z, Z) – (49.75 %) may be involved in the biological activity. The fatty acid composition of the active fraction revealed that the main acid was octadecadienoic acid (49.75 %) followed by n-hexadecanoic acid (14.24 %). Seaweeds exhibit a high level of fatty acid diversity and many of them possess potential bioactivity. Therefore in the present study, the biological activity of L. brandenii might be attributed due to the presence of the fatty acid octadeca- dienoic acid (49.75 %) in a high percentage. Acknowledgements A. M. is grateful to the Council of Scientific and Industrial Research, New Delhi for the award of Senior Research Fellowship. Thanks are extending to professors M. Jayakumari and C. K. Thankachi, Faculty of Zoology, Sree Narayana College, Kollam, Kerala for iden- tification of insects. This work was funded by the Department of Biotechnology, Govern- ment of India (BT/PR8064/ AAQ/03/290/2006). 88 ACTA BOT. CROAT. 70 (1), 2011 MANILAL A., SUJITH S., SABARATHNAM B., KIRAN G. S., SELVIN J., SHAKIR C., LIPTON A. P. U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:52 Color profile: Disabled Composite 150 lpi at 45 degrees References ADWAN, K., ABU-HASAN, N., 1998: Gentamicin resistance in clinical strains of Enterobac- teriaceae associated with reduced gentamicin uptake. Folia Microbiologica 43, 438–440. ARIHARA, S., UMEYAMA, A., BANDO, S., IMOTO, S., ONO, M., YOSHIKAWA, K., 2004: Three new sesquiterpenes from the black heartwood of Cryptomeria japonica. Chemical and Pharmaceutical Bulletin 52, 463–465. BALANDRIN, M. F., KLOCKE, J. A.,WURTELE, E. S., BOLLINGER,W. H., 1985: Natural plant chemicals: Sources of industrial and medicinal materials. Science 228, 1154–1160. BANSEMIR, A., BLUME, M., SCHRÖDER, S., LINDEQUIST, U., 2006: Screening of cultivated seaweeds for antibacterial activity against fish pathogenic bacteria. Aquaculture 252, 79–84. BLUNDEN, G., 2001: Biologically active compounds from marine organisms. Phytotherapy Research 15, 89. CARTER, F. L., 1976: Responses of subterranean termites to wood extractives. Material and Organisms 11, 357–364. CROMBIE, L., 1990: Natural models for the design of insect control compounds: In: Crombie, L., (ed.), Recent advances in the chemistry of insect control II. 23–51. The Royal Society of Chemistry Special Publication. Cambridge. DE NYS, R., LEYA, T., MAXIMILIEN, R., ASFAR, A., NAIR, P. S. R., STEINBERG, P. D., 1996: The need for standardised broad scale bioassay testing: a case study using the red algae Laurencia rigida. Biofouling 10, 213–224 FAULKNER, D. J., 2001: Marine natural products. Natural Product Reports 18, 1–49. FENICAL, W., 1982: Natural products chemistry in the marine environment. Science 215, 923–928. HELLIO, C., BERGE, J. P., BEAUPOIL, C., LE GAL, Y., BOURGOUGNON, N., 2002: Screening of marine algal extracts for anti-settlement activities microalgae and macroalgae. Bio- fouling 18, 205–215. HELLIO, C., DE LA BROISE, D., DUFOSSE, L., LE GAL, Y., BOURGOUGNON, N., 2001: Inhibi- tion of marine bacteria by extracts of macroalgae: potential use for environmentally friendly antifouling paints. Marine Environmental Research 52, 231–247. MANILAL, A., SUJITH, S., SELVIN, J., KIRAN, G. S., SHAKIR, C., GANDHIMATHI, R., PANIKKAR, M. V. N., 2009a: Biopotentials of seaweeds collected from southwest coast of India. Journal of Marine Science and Technology 17, 67–73. MANILAL, A., SUJITH, S., SELVIN, J., KIRAN, G. S., SHAKIR, C., GANDHIMATHI, R., LIPTON, A. P., 2009b: Antimicrobial potential and seasonality of red algae collected from the southwest coast of India tested against shrimp, human and phytopathogens. Annals of Microbiology 59, 207–219. MCDONALD, L. L., GUY, R. H., SPEIRS, R. D., 1970: Preliminary evaluation of new candi- date materials as toxicant, repellents and attractants against stored product pests I. Mar- keting Research Report 882. Agricultural Research Service, U.S. Department of Agri- culture, Washington DC, 1–8. PEREIRA, R. C., DA GAMA, B. A. P., TEIXEIRA, V. L., YONESHIGUE-VALENTIN, Y., 2003: Eco- logical roles of natural products of the Brazilian red seaweed Laurencia obtusa. Brazil- ian Journal of Biology 63, 665–672. ACTA BOT. CROAT. 70 (1), 2011 89 BIOACTIVITY OF LAURENCIA BRANDENII U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:52 Color profile: Disabled Composite 150 lpi at 45 degrees PESANDO, D., CARAM, B., 1984: Screening of marine algae from the French Mediterranean coast for antibacterial and antifungal activity. Botanica Marina 27, 381–386. PROKSCH, P., EDRADA, R. A., EBEL, R., 2002: Drugs from the seas-current status and micro- biological implications. Applied Microbiology and Biotechnology 59, 125–134. ROSETT, R. G., SRIVASTAVA, L. M., 1987: Fatty acids as antimicrobial substances in brown algae. Hydrobiologia 151/152, 47–475. SAKASEGAWA, M., HORI, K., YATAKAI, M., 2003: Composition and antitermite activities of essential oils from Melaleuca species. Journal of Wood Science 49, 181–187. SAXENA, R. C., 1987: Antifeedants in tropical pest management. Insect Science and Its Ap- plication 8, 731–736. SELVIN, J., LIPTON, A. P., 2004: Biopotentials of Ulva fasciata and Hypnea musiformis collected from the peninsular coast of India. Journal of Marine Science and Technology 12, 1–6. SERKEDJIEVA, J., 2004: Antiviral activity of the red marine algae Ceramium rubrum. Phytotherapy Research 18,480–483. SHANMUGHAPRIYA, S., MANILAL, A., SUJITH, S., SELVIN, J., KIRAN, G. S., NATARAJASEENIVASAN K., 2008: Antimicrobial activity of seaweeds extracts against multiresistant pathogens. Annals of Microbiology 58, 535–541. TANG, H. F., YI, Y. H., YAO, X. S., XU, Q. Z., ZHANG, S. Y., LIN, H. W., 2002: Bioactive ste- roids from the brown algae Sargassum carpophyllum. Journal of Asian Natural Product Research 4, 95–105. TASKIN, E., OZTURK, M., TASKIN, E., KURT, O., 2007: Antibacterial activities of some ma- rine algae from the Aegean Sea (Turkey). African Journal of Biotechnology 6, 2746–2751. TUNEY, I., CADIRCI, B. H., UNAL, D., SUKATAR, A., 2006: Antimicrobial activities of the ex- tracts of marine algae from the coast of Urla (Izmir, Turkey). Turkish Journal of Biol- ogy 30, 171–175. VAIRAPPAN, C. S., 2003: Potent antibacterial activity of halogenated metabolites from Ma- laysian red algae, Laurencia majuscule (Rhodomelaceae, Ceramiales). Biomolecular Engineering 20, 255–259. VAIRAPPAN, C. S., SUZUKI, M., ABE, T., MASUA, M., 2001: Halogenated metabolites with an- tibacterial activity from the Okinawan Laurencia species. Phytochemistry 58, 517–523. WARDLAW, A. C., 1985: Practical statistics for experimental biologists. John Wiley and Sons, Chichester. WOLCOTT, G. N., 1947: Permanence of termite repellents. Journal of Economic Entomol- ogy 40, 124–129. 90 ACTA BOT. CROAT. 70 (1), 2011 MANILAL A., SUJITH S., SABARATHNAM B., KIRAN G. S., SELVIN J., SHAKIR C., LIPTON A. P. U:\ACTA BOTANICA\Acta-Botan 1-11\Manilal.vp 28. o ujak 2011 16:13:52 Color profile: Disabled Composite 150 lpi at 45 degrees