Bioactivities from Marine Algae of the Genus Gracilaria

Seaweeds are an important source of bioactive metabolites for the pharmaceutical industry in drug development. Many of these compounds are used to treat diseases like cancer, acquired immune-deficiency syndrome (AIDS), inflammation, pain, arthritis, as well as viral, bacterial, and fungal infections. This paper offers a survey of the literature for Gracilaria algae extracts with biological activity, and identifies avenues for future research. Nineteen species of this genus that were tested for antibacterial, antiviral, antifungal, antihypertensive, cytotoxic, spermicidal, embriotoxic, and anti-inflammatory activities are cited from the 121 references consulted.

These algae also produce important bioactive metabolites like the primary compound with antibiotic activity acrylic acid [43], and the eicosanoids which are derivatives C 20 polyunsaturated fatty acid (PUFA) metabolism through oxidative pathways that originate mainly from arachidonic acid and eicosapentaenoic acids, the precursors of prostaglandins (PGs) [44,45]. Species such as G. asiatica and G. lichenoids contain PGE 2 [46,47]. PGF 2 and 15-keto-PGE 2 were respectively isolated from G. lichenoids and G. asiatica [45]; G. verrucosa contains PGA 2 that appears to be responsible for a gastrointestinal disorder, known as "ogonori" poisoning in Japan [48].
The possibility of finding new molecules from natural products is immeasurable. For this reason the plants and their derivatives are major sources of all drugs, affecting about 30% of pharmaceutical market [71]. According to Newman et al. (2003), between the years 1981 and 2002, 877 new molecules were introduced into the market, with 49% of substances isolated from natural sources followed by semi-synthetic derivatives or synthesized molecules taking the structures of natural origin as models [29].
The search for new effective medicines remains a challenge for scientists. Therefore around the world, many researchers have focused on natural sources for new molecules with algae among the targets of these studies. So in this study we reviewed the literature related to bioactivities for Gracilaria algae.

Results and Discussion
In this review, among the 160 species of Gracilaria already identified taxonomically, only 19 of them had their extracts and fractions chemically tested for toxicity, cytotoxic, spermicidal, antiimplantation, antibacterial, antiviral, antifungal, antiprotozoa, antihypertensive, antioxidant, anti-inflammatory, analgesic, and spasmolytic effects in gastrointestinal tract (Table 1).     These biological studies were mainly developed in Japan and Brazil. This fact is justified by the extensive coastlines and marine biodiversity and is influenced by several factors for the development of these species, such as temperature, radiation, salinity, metal ions and other chemically fundamental components. Australia and Guam have recently become interested in the study of algae and diverse marine species. The consumption of algae has increased in European countries in recent decades with 15 to 20 species of algae being marketed in Italy, France and Greece. In western countries like Venezuela, USA and Canada, the macroalgae are industrially used as a source of hydrocolloids agar, carrageenan and alginate [100]. Carrageenan has been found to be useful in ulcer therapy and alginates are known to prolong the period of activity of certain drugs [8][9][10][11].

Studies of Toxicity
In France, extract studies with ethanol/water draw up from dried entire plant of G. foliifera showed toxicity in humans when treated with oral dose and cytotoxicity studies [75,76]. G. coronopifolia and G. edulis were also toxic to humans [65,49] (See Table 1). Carbohydrate, heparin [97], agar [101], manauealide A, manauealide B [64], manauealide C [102], palmitic, palmitoleic, oleic, lauric and myristic acids [103], steroids and alkaloids malyngamide [104] were found in these species (Figure 1). There is currently a tendency to substitute the use of laboratory animals in toxicological tests with alternative methods to reduce their numbers in experiments, or refine the existing methodology in order to minimize pain and stress [105]. A rapid and effective alternative to realize primary toxicity and biological action screening of compounds is the estimation of the 50% lethal concentration (LC 50 ) through brine shrimp assay using Artemia salina L. [106]. A 90% ethanol extract of G. domingensis had LC 50 of 200 μg/mL against A. salina [74].

Effects on the Nervous System
Studies related to nervous system are important to understanding and treat complex degenerative and behavioral diseases. 90% ethanol extracts from G. corticata, G. edulis and G. verrucosa did not cause central or periphery effects for mice or dogs (50 mg/kg), and did not show analgesic or anticonvulsant activities for mice [75] (Table 1).

Contraception Activity
The researchers have also investigated new molecules with anticonceptive action; the post-coital contraceptive action of marine seaweeds was also evaluated in animals. Methanol: methylene chloride (1:1) extract from G. corticata was orally administered at 500 or 1000 mg/kg/day to female rats from day 1 to day 7 of their pregnancies. Higher doses produced significant post-coital contraceptive activity due to enhanced pre-implantation without any marked side effects. These findings indicate that red marine algae are a potential source for post-coital contraceptive drugs [80]. 90% Ethanol extracts from G. edulis (100 mg/kg) and G. corticata were inactivated before the antiimplantation effect when they tested in pregnant rats [75,80]. Ethanol extracts from shade dried thallus of G. edulis and G. verrucosa were inactive in spermicidal bioassays [75]. Extracts from G. edulis showed 100% inhibition of sperm motility and this effect was related to disruption of the plasma membrane by spermicidal compounds [3] (Table 1).

Anti-Inflammatory and Antioxidant Activities
The anti-inflammatory activity of seaweeds has been studied. Polysaccharide fractions from G. verrucosa at a dose of 4.0 mg/animal were orally and intraperitoneally administered to mice and showed immunopotentiating activity stimulating phagocytosis [82]. Methanol extract and polysaccharide fractions from G. verrucosa were also antioxidant [82,83]. Aqueous extract from G. textorii at a dose of 100 µg/mL did not inhibit platelet aggregation induced by adenosine diphosphate, arachidonic acid or collagen [81]. G. verrucosa, G. asiatica, G. lichenoides and others species contain PGE 2 [47,85], that have physiological effects including hyperthermia, hypotension, smooth muscle dilatation, hyperalgesia and gastric secretion inhibition [114,115] (Table 1).

Antibiotic Activity
Extracts or ingredients from various algae have shown antibacterial activity in vitro against gram-positive and gram-negative bacteria [117]. The agar disc diffusion method for antibacterial susceptibility was used for evaluation and 6 mm discs were impregnated with 20 µL of the extracts and placed in inoculated Muller Hinton agar. Antibacterial activity from chloroform extract of G. edulis (Gmelin) Silva was tested against bacterial strains of Vibrio cholera, Staphylococcus aureus, Shigella dysenteriae, Shigella bodii, Salmonella paratyphi, Pseudomonas aeruginosa and Klebsiella pneumonia ( Table 1). We observed higher activity for G. edulis extract than S. aureus extract [12]. Yet it was inactive for Sporotrichum schenckii, Candida albicans and Cryptococcus neoformans [75]. In the present investigation, the chemical compounds isolated from the species were steroids (carotenoids, β-cryptoxanthin and β-carotene) [118] and carbohydrates [84,85,119] (Figure 4).  Mahasneh et al. (1995) demonstrated activity of organic extracts from algae against multi-resistant bacteria to antibiotics [120]. Ethanol extract from G. debilis showed antibacterial activity against S. aureus but was inactive against Mycobacterium smegmatis [92].
95% ethanol extract from whole dried G. cervicornis algae was active against S. aureus at a concentration of 5.0 mg/mL [89]. Methanol extract from fresh G. corticata was active against Bacillus subtilis, Bacillus megaterium, S. aureus and Streptococcus viridians [91].
G. corticata and G. pygmea did not inhibit the growth of Aspergillus niger, Fusarium solani, Alternaria solani, or Penicillium funiculosum [91]. Petroleum ether, chloroform and methanol extracts from this seaweed at a concentration of 1.0 µg/units proved to be inactive on the inhibition of penicillinase enzyme [87]. From this specie, stearic lipids and capric acids were isolated [121] ( Figure 5). Ethanol extracts from G. domigensis and G. sjoestedii showed antibacterial activity against E. coli and S. aureus. Ethanol extracts from G. debilis, G. domingensis and G. sjoestedii were active against Candida albicans shown by agar plate method [92]; Chloroform, ether and methanol extracts from G. tikvahiae were inactive [93]. The growth of Neurospora crassa was not inhibited by extracts from G. sjoestedii and G. debilisi; ethanol extract from G. domigensis was active against Mycobacterium smegmatis and Neurospora crassa [92]. G. domigensis has as chemical constituents, polysaccharide CT-1 [122], palmitic acid and steroids (stigmasterol, sitosterol, campesterol, cholest-7-en-3-β-ol and brassicasterol) [52] (Figure 6).  Some studies highlighting antiparasitic activity of seaweeds also were verified. 90 % ethanol extract from G. corticata and G. edulis were tested against Entamoeba histolytica and Plasmodium berghei and were not active [75].

Antivirial Activity
Extracts from G. bursa-pastoris and Gracilaria sp were inactive against the Herpes simplex 1 virus (HSV) and the human immunodeficiency virus (HIV) when evaluated in cell cultures [96]. Granin BP and citrullinyl-arginine proteins were isolated from these extracts [123,124]. Methanol extract from dried G. pacifica at a concentration of 200.0 µg/mL was active against Sindbis virus, but was not effective against H. simplex 1 when tested at a concentration of 400 µg/mL. Extracts and compounds obtained from Gracilaria sp with anti-HIV activity are also active against other retroviruses such as HSV. However, the pharmacodynamic mechanisms of the antiretroviral activity are still unknown because bioactive compounds from seaweed poorly investigated [9] (Table 1

Material and Methods
In this article, some reports about bioactivity of Gracilaria algae were reviewed in the specialized literature published up to January 2011. The search was carried out using data banks such as; Biological Abstracts, AlgaeBase, SciFinder Scholar, Pubmed and NAPRALERT (acronym for Natural Products ALERT-University of Illinois in Chicago, USA).

Conclusions
Algae are abundant in the oceans and represent a rich source of as yet unknown secondary metabolites. In this review, we found only a few studies with complete chemical profiles and pharmacological potential of the Gracilaria species. Most studies raised concerns about antimicrobial activity against Staphylococcus, Streptococcus, Candida and Herpes genus. Others referenced the cytotoxicity bioassays in which these algae species were not active, but they produce various types of prostaglandins and others substances that can be toxic to humans such as gastrointestinal disorders and lethality caused by G. verrucosa and G. edulis, respectively. To research new drugs it is necessary to evaluate other bioassay models to preserve the safety, efficacy and quality of the end products. In Brazil, there is a great need for toxicological, pharmacological, preclinical and clinical studies, as recommended by the RDC 48/2004.
Finally, we conclude that algae of the Gracilaria genus are a potential source for synthesis of new natural medicines. It is important to taxonomically classify and standardize extractions, while identifying the active compounds to attenuate possible environmental interference that could undermine the pharmacochemical profile, and thus generate different pharmacologic effects. In addition, it is important to sensitize corporate researchers and financial agencies to support this cause.