Among the many chemical classes present in plant species, alkaloids stand out as one of major importance in the development of new drugs, because they possess a wide variety of chemical structures and have been identified as responsible for many of the pharmacological properties of medicinal plants [1–5]. Some plant families have the genetic capability of producing more than one alkaloid, reflected in the structural diversity of these compounds [6–8].
Indole compounds constitute an extensive family of chemicals found in bacteria, plants and animals. In general, these compounds are related to the metabolism of tryptophan and present substitutions in different positions of the indole ring [6,9]. Indolic compounds with significant and complex physiological roles include those related to melatonin and serotonin (5-hydroxytryptamine). Indole alkaloids possess an indole ring in its structure, constituting a versatile heterocyclic discovery in 1866 ; this extensive group of alkaloids received more attention after the isolation of reserpine from Rauwolfia serpentina Benth., an alkaloid that changed the history of pathologies conditions as diverse as schizophrenia and hypertension. Indolic alkaloids include various plant-derived medicinal products, including the well-known anti-tumor vinblastine, vincristine, vincamin isolated from Catharanthus roseus  and camptothecin, is a monoterpene indole alkaloid isolated from the Chinese tree Camptotheca acuminate Decne. (Icacinaceae) which displays potent antitumour activity .
The knowledge about the chemical composition of marine organisms is an essential element for assessing chemotaxonomic, chemical ecology, and natural products studies, including that directed towards evaluating the pharmacological roles [12–20]. Recently, we initiated a program to investigate the hypothesis that Caulerpa species produce secondary metabolites with possible antinociceptive and anti-inflamatory actions. In our preliminary investigation of the crude methanolic extract and phases from C. racemosa we had observed an antinociceptive effect in animal models. However, pharmacological and chemical studies are continuing in order to characterize the mechanism(s) responsible(s) for the antinociceptive action and also to identify other active principles present in Caulerpa racemosa .
The present study was conducted to characterize caulerpin (1, Figure 1), an alkaloid isolated from the lipoid extracts of Caulerpa racemosa collected in the Northeast of Brazil. Caulerpin comes from a family of bisindole natural products, and has an extra eight-member ring between the two indole rings which are incorporated directly with the carbonyl group. This alkaloid show a variety of important biological activities already described in the literature, among which it is important to mention the antitumor , growth regulator  and the plant root growth stimulant properties , but its in vivo antinociceptive and antiinflamatory activities weren’t reported yet. In addition, caulerpin may also be classified as a compound of low toxicity . However, to date there are a few investigations supporting the pharmacological properties of this seaweed. Thus, this study was intended to evaluate the antinociceptive and anti-inflammatory activities of caulerpin from Caulerpa racemosa in animal models.
2. Results and Discussion
Three different animal models were used in this study to investigate the antinociceptive potential of caulerpin. These methods were selected to ensure both centrally and peripherally mediated effects were investigated. The acetic acid-induced abdominal constriction and the hot-plate methods analyzed peripheral and central activity, respectively, while the formalin test investigated both. In addition, the ear edema induced by capsaicin and the peritonitis induced by carrageenan in mice were used to examine the anti-inflammatory activity of this compound. The acetic acid-induced writhing is a visceral pain model and widely used for the evaluation of peripheral antinociceptive activity . The intraperitoneal administration of an agent that irritates the serous membranes cause a stereotypical behavior in mice which is characterized by abdominal contractions, movements of the body as a whole, twisting of the dorsum abdominal muscles, and a reduction in the motor activity and coordination . The results depicted in Figure 2 shows that caulerpin, given 40 min before, has produced an inhibition of the acetic acid-induced abdominal constrictions in mice. The IC50 calculated with 95% confidence interval) for caulerpin was 0.0945 μmol (0.0103–1.0984) (n = 10) and for dypirone it was 0.0426 μmol (0.0092–0.1972) (n = 10). The dates were expressed as % inhibition was compared to the respective acetic acid control group. The mean of contortions of the control group was 42.8 ± 1.9.
Acetic acid causes an increase in the peritoneal fluid level of prostaglandins (PGE2 and PGF2α) as well as lipooxygenase products, involving in part peritoneal receptors and inflammatory pain by inducing capillary permeability [26,28]. Collier et al.  postulated that acetic acid acts indirectly by inducing the release of endogenous mediators, which stimulate the nociceptive neurons. The most important transmission pathways for inflammatory pain are that comprising peripheral polymodal nociceptors sensitive to protons, such as acid sensitive ion channels and to algogen substances, such as bradykinin and cytokines. Although the writhing test has poor specificity (e.g., anticholinergic, tricyclic antidepressants and antihistaminic and other agents show activity in this test), it is a very sensitive method of screening the antinociceptive effects of compounds [29–31].
In the hot plate test (Table 1), the treatment with morphine (15 μmol/kg, s.c.) caused a marked increase in the latency time of the animals at 60–150 (12.8 ± 0.4 s, 10.3 ± 0.8 s; 9.7 ± 0.7 s; 9.7 ± 0.9 s) and caulerpin (100 μmol/kg, p.o.) caused a marked increase in the latency time of the animals at the times lecture 90–150 (4.6 ± 0.6, 3.8 ± 0.5, 4.0 ± 0.5).
The hot-plate test is commonly used to assess narcotic analgesia . Although the central and peripheral analgesics respond by inhibiting the number of contractions provoked by chemical pain stimuli, only the central analgesics increase the time of response in the hot plate test . These observations tend to suggest that caulerpin may possess centrally- and peripherally-mediated antinociceptive properties. The peripheral antinociceptive effect of caulerpin may be mediated via inhibition of cycloxygenases and/or lipoxygenases (and other inflammatory mediators), while its central antinociceptive action may be due its possible action as partial agonist of adrenergic, serotoninergic, cholinergic and dopaminergic receptors [34,35]. Proposed mechanisms of this effect include interation with serotoninergic receptors, because structural similarity between serotonin and caulerpin .
In the present study, administration of caulerpin (p.o.) did not cause motor impairment, as evaluated by forced locomotion in the rotarod test, contrary to diazepam (i.p.) that induced a decrease of the fall latency and increase the number of fall on the rotarod assay. Thus, the possibility that the antinociceptive effect of the compounds tested is due to any degree of motor impairment or sedation is improbable.
Table 2 shows the effects of caulerpin (100 μmol/kg; p.o.) and diazepam (35.1 μmol/kg, i.p.) on the rotarod test from 0.5–1 h. The compound tested did not significantly alter either the fall latency or number of falls when compared with the saline group.
The formalin test in mice confirmed an antinociceptive effect (Figures 3A and 3B). Caulerpin (100 μmol/kg, p.o.) caused a significant inhibition of both neurogenic (28.5%) and inflammatory (55.7%) phases of formalin-induced licking. The treatment with indomethacin (100 μmol/kg, p.o.) was able to inhibit the second phase by 50.3%.
The formalin test is believed to represent a more valid model for clinical pain . The formalin test is a very useful method, not only for assessing antinociceptive drugs, but also helping in the elucidation of the action mechanism. The neurogenic phase is probably a direct result of stimulation in the paw and reflects centrally mediated pain with release of substance P while the late phase is due to the release of histamine, serotonin, bradikynin and prostaglandins . Drugs that act primarily on the central nervous system, such as narcotics, inhibit both phases equally while peripherally acting drugs such as anti-inflammatory non-steroidal (NSAID) and anti-inflammatory steroidal only inhibit the late phase [27,33]. Caulerpin was able to block both phases of the formalin response although the effect was more pronounced in the second phase.
To investigate the anti-inflammatory activity we did the model of capsaicin-induced ear edema in mice, a well-characterized and largely used model of inflammation. Topical application of capsaicin was observed to cause a significant rise in plasma extravasations when compared to control ear in the same animal treated with a vehicle. This was significantly reduced by pretreatment with caulerpin, which had shown 55.8% of the inhibition (Figure 4).
2.1. Common Carageenan Sources
Commercial carrageenans are extracted from the carrageenophyte red seaweed genera Kappaphycus, Gigartina, Eucheuma, Chondrus, and Hypnea, in which the carrageenans comprise up to 50% of the dry weight . κ-Carrageenan is mostly extracted from Kappaphycus alvarezii, known in the trade as Eucheuma cottonii, while ι-carrageeman is predominantly produced from Eucheuma denticulatum, also known as Eucheuma spinosum. λ-Carrageenan is obtained from seaweeds within the Gigartina and Chondrus genera, which as sporophytic plants produce λ-carrageenan while they make a κ/ι-hybrid as gametophytic plants [4,5]. Southeast Asia and Tanzania are the main producers of seaweed derived carrageenans from Kappaphycus alvarezii and Eucheuma spinosum .
Table 2. Summary of seaweed sources, hydrocolloid carbohydrate products, chemical structures (main structural units), and applications of the seaweed derived hydrocolloids carrageenans, agars, and alginates.
2.2. Carrageenan Chemical Structure
Carrageenans are hydrophilic sulfated linear galactans that mainly consist of d-galactopyranose units bound together with alternating α-1,3 and β-1,4 linkages. This base structure is consistent in the three main commercially used carrageenans, κ-, ι-, and λ-carrageenan, Table 2. The presence of 4-linked 3,6-anhydro-α-d-galactopyranose varies among the different carrageenans, as do the substitutions with sulfates, which are ester-linked to C2, C4, or C6 of the galactopyranose units, depending on the specific carrageenan: κ-, ι-, or λ-carrageenan. κ-Carrageenan has one sulfate ester, while ι-and λ-carrageenan contain two and three sulfates per dimer, respectively, Table 2. In addition, the galactopyranose units may also be methylated or substituted with e.g., monosaccharide residues, such as d-xylose, 4-O-methyl-l-galactose, and d-glucuronic acid [12,13]. Acid hydrolysis, infrared spectroscopy, and NMR analyses of commercial carrageenan typically show sulfate content of 25%–30% for κ-carrageenan, 28%–30% for ι-carrageenan, and 32%–39% for λ-carrageenan, although large differences can occur [7,14,15]. The differences in sulfate levels are explained by the fact that carrageenans are very heterogeneous carbohydrates, with structural differences coexisting within the specific type of carrageenan depending on the algal source, life-stage, and extraction method . In addition, naturally occurring carrageenans contain traces of their biosynthetic precursors, μ- and ν-carrageenan, adding further to the complexity of these polysaccharides, Figure 1 . Likewise, hybrid carrageenans exist, representing a mixture of the different carrageenan repeating units .
Figure 1. Conversion of the pre cursors μ- and ν-carrageenan into κ- and ι-carrageenan.
Figure 1. Conversion of the pre cursors μ- and ν-carrageenan into κ- and ι-carrageenan.
2.3. Physico-Chemical Properties of Carrageenans
Carrageenans are soluble in water, but the solubility depends on the content of hydrophilic sulfates, which lowers the solubility temperature, and the presence of potential associated cations, such as sodium, potassium, calcium, and magnesium, which promote cation-dependent aggregation between carrageenan helices . Another factor affecting the physico-chemical properties in relation to viscosity and gelation is the presence of anhydro-bridges: κ- and ι-carrageenans have 3,6-anhydro-galactopyranose units, while λ-carrageenan is composed exclusively of α-1,3 galactopyranose and β-1,4 galactopyranose, Table 2.
The presence of anhydro-bridges in κ- and ι-carrageeenan is proposed to be a result of elimination of a sulfate ester present on their respective precursors, i.e., in μ- and ν-carrageenan, and subsequent spontaneous anhydro-bridge formation in the desulfated monomer residue, Figure 1. The removal of the sulfate esters in µ- and ν-carrageenan reduces the hydrophilicity of the sugar residue and inverts the chair conformation from 1C4 to 4C1, Figure 1. The conformation change allows the polysaccharide to undergo conformational transitions which are conducive to the gelation properties of the anhydro-bridge containing carrageenans .
The thermo-reversible gel formation is proposed to occur in a two-step mechanism, dependent on temperature and gel-inducing agents. At high temperatures, i.e., above 75–80 °C, the carrageenans exist as random coil structures as a result of electrostatic repulsions between adjacent polymer chains. Upon cooling, the polymeric chains change conformation to helix structure. Further cooling and presence of cations (K+, Ca2+, Na2+) lead to aggregation of the helical dimers and formation of a stable three dimensional network, which forms through intermolecular interactions between the carrageenan chains [18,19]. The molecular details of carrageenan gelation are still uncertain. The formation of double helices prior to gelation is not fully proven, and, in principle, the formation of a duplex via chain-chain interactions may not necessarily be an unequivocal evidence for double helix formation. Nevertheless, based on the available literature data and theoretical explanations, we interpret that for the stiff κ-carrageenan gels to form, the cations, typically potassium for κ-carrageenan, function to stabilize the junction zones between the two helixes by binding to the negatively charged sulfate groups without hindering cross-linking of the two helices, Figure 2. According to this model, calcium, typically for ι-carrageenan, analogously function to cross-link the two helices through ionic salt bridges . The charged sulfate esters on the other side of the monomer though, present on ι-carrageenan, encourage an extensive conformation via a repulsion effect of the negative SO3− groups and inhibit gelation while promoting viscosity in the solution . The differences in sulfate position, their proportion, and the presence of anhydro-bridges, thus, give the carrageenans distinctive gel profiles: κ-carrageenan forming strong and rigid gels, ι-carrageenan forming soft gels, and λ-carrageenan that does not gel, but still provides elevated viscosity in solution, due to a structure that does not allow helix formation, Table 2. Is has to be emphasized that natural carrageenans are heterogenous, i.e., have heteropolymeric structures. In practice, the rheological properties of carrageenans reflect that hybrid structures exist.