The objective of this research was to identify the predominant sargassum species in coastal areas of Pakistan, to explore the pharmacological activities residing in them in them and to study the toxicity of harmful dinoflagellates associated with them. Sargassum species such as s. tenerrimum j. Agardh, s. wightii (Greville) j. Agardh and s. cervicorne Greville were collected during winter, spring, summer and autumn seasons; from six different locations along 1000 km long Pakistan Coast i.e. Sandspit, Buleji, pacha, Jiwani, Pasni and Gawadar. Sargassum species were identified taxonomically and morphologically on the basis of type of holdfast, stem, leaves, vesicles and receptacles. The physical seawater parameters in which the seaweeds grow such as pH, temperature and salinity were taken into consideration and their relative effect on biomass was also observed. The winter and spring season with pH (8-8.02), lower temperature (21-25oc) and having salinity values between 37-42 ppt. appear to favor the growth and overall biomass of all sargassum species particularly, S. tenirrimum which was collected as high as 100 kgm-2.
The sargassum species were dried and crushed to obtain powder which subjected to solvent systems to afford hexane, methanol and butanol extracts. These extracts were evaluated for toxicity, analgesic and anti-inflammatory activies in albino mice and rats, respectively. The results of anti-inflammatory and analgesic activies indicated that butanol extracts from sargassum sppp. Collected during winter and spring seasns causes complete inhibition of rat paw edema induced by carrageenan (1-4 hours of observation), as compared to extracts of summer and autumn. Butanol extracts form the winter collection of sargassum species were highly effective at the dose of 100 mg/kg and the potency order appeared to be s. wightii(60-89.5%)> s. servicorne (56-83.5%)> s. tenerrimum (63-76%). This effect was slightly better than the synthetic drugs like aspirin (36.70%) and ibuprofen (30-58.2%) and was equal to diclofenac (83.3-89 5%). Thus it appears that species of sargassum possess appreciable aniti-inflammatory property but it is governed by multiple factors like season of collection, type species and solvents used for extraction. It is speculated that the anti-inflammatory activity residing in the extracts may be due to its interaction with either the release or synthesis of mediators like histamine, serotonin, kinin and prostaglandins. In this study the statistical connection between different biotic and abiotic factors such as species, nature of solvent and seasons using analysis of variance revealted that type of solvent and seasonal variation have a significant effect on the anti-inflammatory properties of sargassum species.
The results of anti-inflammatory activity at the 3rd hour were further suppoted by the analgestic experiments like acetic acid writhing test. Prostaglandins which are released at 3rd hour in response to tissue injury or inflammation can also activate pain, their inhibition means increased possibility of presence of analgesic activity. Defferent polar and non-polar fractions of sargassum sargassum species induced significant and dose dependent analgesic effect by reducing number of writhes. Unlike, anti-inflammatory activity the analgesic activities seems to be independent of any external or internal factors. At 100 mg/kg dose, maximum reduction in number of wrethes i.e. more than 90% was produced by methanol and butanol extracts of s. wightii, s.tenerrimum and s. cervicorne, which was slightly better than the non-steroidal drugs like aspirin (53.5%) and paracetamol (64%). The minimum (1 mg/kg) and maximum (53 mg/kg) ic50 values were exhibited by s. tenerrimum extract of spring (methanol) and autumn(butanol) collections respectively, which are comparable to standard drugs like aspirin (87.5 mg/kg) and paracetamol (70 mg/kg) used.
Efforts were also made to study bloom forming harmful dinoflagellates associated with the sargassum wightii. Both the epiphytic/benthic dinoflagellates Oxyrrhis marina Dujardin and amphidinium carterae Hulburt were observed successively forming bloom during spring season. This probably is the first report of their occurance in the northern Arabian sea. A. carterae was identified on the basis of distinct cingulum and sulcus, compressed body and pariety chloroplast, wherease 0. marina on the basis of absence of these features.
0. marina and A. carterae were harvested separately (wild cell filtrate) followed by isolation and maintenance as pure cultures (cultured cell filtrate and were also evaluated for their toxicity in brine shrimp (Artemia salina) and albino mice. The wild and cultured cell filtrates of 0. marina (60 to 9x106 cell ml-1) and A. carterae (2.5x102 to 7.2x104 cell ml-1) were non-toxic to brine shrimps. It may be explained due to the primitive nervous system and receptor/signal transduction system which makes it less sensitive to the ciguatera toxins. Interesting, wild cell filtrate (9x106, 4.5x106 cells ml-1) of bloom of 0. Marina toxic to mice and induced symptoms like muscle contraction, increased respiration, immobility and paralysis in extremities within 3 minutes of intraperitoneal administration, followed by 100% mortality within 24 hours. On the other hand cultured cell filtrate (4x104 to 6x104 cells ml-1) neither caused aforementioned behavioral changes nor mortality in mice. The lack of mortality in cultured cell filtrates of 0. marina is hypothesized to be due to absence of toxic prey species in the culture media, which were prevalent in the wild conditions. Moreover, A. carterae also demonstrated similar behavioral symptoms like 0. marina that were reversed within 2-3 hours in mice, without causing any mortality, regardless of its source i.e. wild (7.2x104 cells ml-1) or cultured (4x104 ml-1) cell filtrates, due to either insufficient or less potent toxin(s) produced by the autotrophic A. carterae.
The present study led to the following conclusions: 1) sargassum species grows better during winter and spring seasons. S. tenerrimum and s. cervicorne prefer sandy (sandspit) and rocky (pacha) of sindh coastal areas, respectively, whereas s. wightii was found to grow luxuriantly on rocky beaches (jiwani) of Balochistan coast. 2) The anti-inflammatory activites of winte4r and spring collection of all sargassum species demonstrated significandt inhibition of edema but butanol extract of winter collection of s. wightii was slightly better.3) the anti-inflammatory activity resides in polar solvents and seems to be influenced by the biotic and abiotic factors while analgesic activity was independent of these factors. 4) thus sargassum possess anti-inflammatory and analgesic activities, but the variable results obtained in the present study may explain the controversies found, in literature and folk medicine, over the effect of medicinal plants. 5) The non-toxicity of cell filtrates of dinoflagellates o. marina and A. carterae in brine shrimps and their toxic effect in mice is probably due to the less developed nervous system of brine shrimps. 6) 0. marina induced mortality in mice could be related to the toxins residing in its prey species. 7) The wild and cultured cell filtrates of A. carterae was able to induce behavioral changes in mice but was not potent to cause mortality.