**1. Introduction**

Current epidemiological data indicate an increase in immunological diseases. This has stimulated the search for a class of molecules, generally called immunomodulatory molecules, capable of increasing or suppressing the immune response in immune-mediated diseases [1].

Research on natural compounds that can modulate the immune response has become a focus in the experimental field, since such compounds have potential applications in the areas of immunopharmacology and oncotherapy. Di fferent authors have reported that polysaccharides obtained from plants, fungi, and seaweed are able to modify various cellular processes and consequently possess a

variety of bioactivities, particularly potent effects on immune function [2–4]. Thus, immunostimulatory compounds, such as sulfated polysaccharides (SPs), have potential applications in the treatment of infections, immunodeficiencies, and cancer [5].

In this context, macrophages have been used as a study model in the identification of compounds with immunomodulatory properties [6,7]. Together with neutrophils, these cells constitute the body's first line of defense [8]. Through their antigen-presenting ability, they also play an important role in adaptive immunity [9]. Activation of macrophages is a key event in innate and adaptive immunity and is essential for defense mechanisms. Once activated by foreign agents, these cells can phagocytose and kill microorganisms and tumor cells, as well as produce molecules that recruit and activate other cells to the site of infection. In response to this stimulus, macrophages increase the production of reactive nitrogen intermediates such as nitric oxide (NO), reactive oxygen species (ROS), and proinflammatory cytokines such as TNF-α and IL-6 [10]. Therefore, macrophages are often used to evaluate the immunomodulatory effects of bioactive compounds from natural sources [11,12].

Although an increasing number of studies on the immunomodulatory activity of SPs from green seaweeds are being conducted, the extent of their activities varies greatly among different species of seaweed, and the structure correlations remain undefined [13].

The SPs of green seaweeds consist mainly of galactose, xylose, arabinose, mannose, rhamnose, glucuronic acid, and/or glucose [14]. The proportion of these monosaccharides seems to differ with the genus of the seaweed; for example, in seaweeds of the genera *Monostroma* and *Ulva*, it is more common to find rhamnose [15], whereas homo- and heterogalactans are common in the seaweeds of the genus *Caulerpa* [16], which are known to synthesize polysaccharides with immunomodulatory activities. Xylogalactans from *Caulerpa lentillifera* were found to exhibit a potent immunomodulatory effect by stimulating phagocytosis and increasing NO and cytokine secretion in RAW 264.7 macrophages [17]. In another work, Sun et al. [18] found that xylogalactomannans increased cell proliferation, phagocytosis, NO secretion, and alkaline phosphatase activity in macrophages. Galactans of *C. cupressoides* var. *lycopodium* showed antinociceptive and anti-inflammatory effects in vivo by reducing leukocyte migration in the peritoneal cavity of rats [19], while galactans of *Caulerpa mexicana* showed antinociceptive effect and decreased paw edema and myeloperoxidase activity in mice [20]. Ribeiro et al. [21] observed that *Caulerpa racemosa* SPs also exhibited antinociceptive and anti-inflammatory activities in an in vivo mouse model.

Less information is available on the SPs of the macroalga *C. cupressoides* var. *flabellata* and their immunomodulatory effects. This species synthesizes SPs that have antioxidant, anticoagulant, and antiproliferative activities in vitro [22]. Costa et al. [23] obtained four SPs populations with different characteristics regarding molecular weight and sulfate content. Monosaccharide composition analysis showed that galactose was the main constituent for all fractions; although glucose, mannose, xylose, rhamnose, and fucose were found, their proportions were different in each fraction. Subsequently, Barbosa et al. [24] investigated the immunostimulatory potential of these four polysaccharide fractions to determine their capacity to stimulate the production of different inflammatory mediators in the RAW 264.7 cell line. Of these, the so-called CCB-F1.0 fraction had a potent effect on the production of NO, ROS, and the cytokines TNF-α and IL-6. Although the SPs of the CCB-F1.0 fraction were determined to be composed of 76.47% total sugars and 17.95% sulfate, have a molecular weight of 155 kDa, and consist of galactose, mannose, and xylose in the ratio of 1.0:0.1:0.6 [23], further structural details of the polysaccharides of this fraction were not determined. Therefore, the present work aimed to purify the SPs of the CCB-F1.0 fraction of *C. cupressoides* var. *flabellata* and to characterize them structurally using nuclear magnetic resonance spectroscopy. In addition, to confirm that CCB-F1.0 immunomodulatory activity came from these molecules, the effects of SPs on some immunostimulating mediators (NO, ROS, and cytokines) using RAW 264.7 murine macrophages cells were investigated.

#### **2. Results and Discussion**

#### *2.1. Purification of SPs*

Initially, to obtain the SPs-rich extracts, proteolysis and methanol precipitation steps were performed. After these procedures, the SPs were fractioned with increasing volumes of propanone until the fraction CCB-F1.0 was obtained as described by Costa et al. [23]. Then, the SPs of this fraction were purified by ion exchange chromatography and eluted in a stepwise NaCl gradient (0.25–1.0 M). As shown in Figure 1, the elution of CCB-F1.0 (50 mg) generated three peaks in the chromatogram, corresponding to the molarities of 0.525 M (15.5 mg), 0.675 M (4.2 mg), and 0.9 M (1.8 mg), respectively. Based on this elution profile, the SPs obtained were labeled SP1, SP2, and SP3. Furthermore, SP1, SP2, and SP3 yielded 72%; 19.5%, and 8.5% (w/w) of the eluted SPs present in CCB-F1.0, respectively. Because of the lower yield obtained in the SP3 purification, the following analyses were not performed with this material.

**Figure 1.** Stepwise elution profile of SPs from *C. cupressoides* on a HiTrap DEAE FF column.

Physicochemical analysis showed that SP1 and SP2 were composed mainly of polysaccharides and low quantity of protein, ~0.25% and 0.20%, respectively. In addition, both SPs were composed mainly of galactose, sulfate, and traces of mannose, as seen in Table 1.

**Table 1.** Chemical composition of CCB-F1.0 and purified sulfated polysaccharides from *Caulerpa cupressoides*.


Gal = galactose; Man = mannose; Xyl = xylose, SO4 = sulfate, n.d. = not detected.

SP1 and SP2 were further analyzed by gel permeation chromatography (GPC) in a Sephadex® G-100 column to determine their homogeneity, as seen in Figure S1. The chromatograms of the *Caulerpa* SPs showed a single peak. Furthermore, the chromatogram obtained from GPC was used to calculate the apparent molecular weight using a regression equation determined using different molecular weight standards. Thus, the molecular weight of SP1 and SP2 was found to be 125 and 135 kDa, respectively. These values were similar to those demonstrated by Costa el al. [23] in the studies of SPs in the CCB-F1.0 fraction.
