Historically, C. splendens has been used in African folk medicine for treating a number of diseases and medical conditions. However, despite the widespread traditional use of C. splendens in tropical countries, little is known regarding the active components responsible for its therapeutic properties. Previously, antipyretic and anti-inflammatory effects of methanolic extracts from C. splendens have been described , whereas the immunomodulatory activities of high-molecular weight polysaccharide fractions from this plant are unknown. In the present work, we provide evidence demonstrating that C. splendens polysaccharides have potent immunomodulatory properties in vitro and in vivo and suggest that this may contribute, at least in part, to the therapeutic potential of C. splendens-derived medicines.
Our ethnobotanical survey and literature review indicate that a decoction in water of the leaves is the mode of extraction most commonly used to prepare remedies from C. splendens in African folk medicine. Based on this observation, we isolated eight polysaccharide fractions from the hot-water extract of C. splendens leaves and provided initial structural and pharmacological characterization. Average molecular weights of the fractions were in the range of 5.5 to 60.6 kDa. We found that the high-molecular weight sub-fractions CSP-AU1 (mol. weight 38.5 kDa) and CSP-NU1 (mol. weight 60.6 kDa) were quite potent in biological tests, whereas the low-molecular weight sub-fraction CSP-AU2 was inactive or minimally active in the same assays. Indeed, sub-fractions CSP-AU1 and CSP-NU1 contained potent immunomodulatory activity, as demonstrated by their ability to induce NO production in macrophage J774.A1 cells and cytokines by monocyte/macrophages and human PBMCs. These cytokines included of IL-1α, IL-1β, IL-6, TNF, IL-10, and GM-CSF. In addition, we found that CSP-AU1 and CSP-NU1 induced increased levels of certain serum cytokines in vivo, including the anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines IL-6, TNF, MCP-1, MIP-1α, and MIP-1β. In contrast, the low-molecular weight sub-fraction CSP-AU2 was minimally active in vivo (Figure 10).
A common feature of plant polysaccharides that modulate macrophage functions may be higher molecular weight, as we found previously that immunomodulatory activity positively correlated with increased molecular weight of various plant polysaccharides [23–25, 36]. Although the immunologic effects of Clerodendrum polysaccharides following oral administration were not studied in the present work, numerous dietary polysaccharides do appear to elicit diverse immunomodulatory effects in various animal tissues, including the blood, gastrointestinal tract and spleen [37–42]. Note, however, that our recent studies suggest that nasal application of some plant polysaccharides may be even more effective than oral administration for stimulation of innate immunity . In any case, further studies are now in progress to determine the therapeutic potential of Clerodendrum polysaccharide sub-fraction CSP-AU1 and its structural features conferring biological activity.
Active and inactive polysaccharide fractions contained polyphenols, which were present over a broad range. Polyphenols have been previously noted in polysaccharide fractions of various plants (e.g. [42, 44]), and it has been reported that polysaccharides are able to bind to polyphenols by intermolecular interactions , changing the molecular conformation of carbohydrates. Therefore, the presence of polyphenols in polysaccharide fractions must be considered. However, activities of the fractions did not correlate with polyphenol content in our biological studies. Indeed, one of the most active fractions (CSP-AU1) contained a relatively high level of polyphenols (7.6%), whereas other polyphenol-enriched fractions (CSP-AB, 9.3% polyphenols; CSP-AU2, 8.8% polyphenols) were inactive or had low activity in most biological assays. Finally, the highly active sub-fraction CSP-NU1 had the lowest polyphenol content (0.2%).
We found that both of the Diaion-bound fractions, CSP-AB and CSP-NB, generally were inactive or had low biological activity. Sugar analysis showed that these fractions contained much less galacturonic acid but more neutral sugars (glucose and mannose) than the Diaion-unbound fractions CSP-AU and CSP-AU. Moreover, analysis of the fractions using the Yariv reagent demonstrated that both CSP-AB and CSP-NB tested negative for the presence of type II arabinogalactan. Conversely, the most potent immunomodulatory properties were associated with the fractions that exhibited a positive reaction for type II arabinogalactan. Thus, native type II arabinogalactans could be the main structures responsible for immunomodulatory properties of the C. splendens-derived polysaccharides, although presence of type I arabinogalactan in the fractions could not be excluded. In any case, it appears that Diaion HP-20 resin could be used to enrich bioactive C. splendens polysaccharides by removing inactive material that bound to the resin . Note, we also evaluated if the inactive Diaion-bound material might possibly have inhibitory activity by assessing its ability to inhibit LPS-induced cytokine production but found that this material was inactive, having no effect on LPS induced cytokine production (data not shown). Future chemical and physical analysis of these fractions will be necessary for characterization of Diaion-bound and unbound polysaccharide fractions and definition of the polysaccharide structures responsible for immunomodulatory activities.
Previously it was proposed that selective immunostimulatory and anti-inflammatory properties of citrus polysaccharides could be attributed to different degrees of methyl esterification [47–49]. In the present study, we found that the polysaccharide fraction with the most potent immunomodulatory activity (CSP-AU1) had a low degree of methyl esterification of uronic acid residues. Thus, free carboxylic groups and associated electrical charge may enhance the bioavailability and binding potential of CSP-AU1 polysaccharides to different cell receptors.
Clerodendrum polysaccharides and recently characterized polysaccharides from Alchornea cordofolia seem to be unique, as compared to LPS and many other plant polysaccharides, in their ability to induce GM-CSF production by MonoMac-6 cells and PBMCs. Indeed, previously characterized polysaccharide fractions A-I from A. tripartite, T-I from T. vulgare, C-I from O. polyacanta, and even high concentrations of LPS (up to 10 μg/ml) were unable to stimulate GM-CSF production by human PBMCs, whereas CSP-AU, CSP-AU1, CSP-NU1, and CSP-NU2 all significantly induced production of this cytokine by MonoMac-6 cells and PBMCs.
Because Con A stimulated GM-CSF production by human PBMCs (Figure 5), we considered whether the GM-CSF-inducing activity of Clerodendrum polysaccharide fractions might correlate with hemagglutinating activity but found that the Clerodendrum polysaccharides, as well as polysaccharides isolated from A. cordofolia, had no hemagglutinating activity (up to 1 mg/ml). On the other hand, we found that polysaccharide CP-AU isolated from Combretum racemosum had relatively high hemagglutinating activity but minimally stimulated GM-CSF production by human PBMCs and only at the highest tested concentration (0.5 mg/ml) . Thus, plant polysaccharide structures responsible for GM-CSF stimulation and hemagglutination appear to be distinct from each other.
Differences in intracellular signaling pathways were observed between Clerodendrum polysaccharides and previously reported immunomodulatory botanical polysaccharides. For example, polysaccharides from algae Capsosiphon fulvescens induced ERK1/2, but not p38 , polysaccharide SP1 from Caulerpa lentillifera increased the phosphorylation of p38 , whereas polysaccharide AP-1 from Platycodon grandiflorum activated phosphorylation of all three MAPKs . Here, we demonstrated that CSP-AU1 induces phosphorylation of Akt2, Akt3, GSK-3α/β, p38β/γ/δ, p70S6K1, RSK2, and mTOR, but not ERK1/2 in human peripheral blood mononuclear cells. Thus, it appears that various immunomodulatory polysaccharides exert their biological activities through different modes of action.
Plant polysaccharides can modulate leukocyte activity via different receptors, including complement receptor 3 (CR3), scavenger receptor, dectin-1, mannose receptor, galectin 3, and TLR4 (reviewed in ). For example, polysaccharides ASP and G1-4A, which were isolated from Acanthopanax senticosus and Tinospora cordifolia, respectively, activated macrophages and B cells by interacting with TLR4 [54, 55]. TLR4 was also found to be one of the cellular receptors mediating TNF secretion induced by ZPF1, polysaccharide isolated from Dioscorea batatas. Likewise, polysaccharide CWSP from Chlorella pyrenoidosa induced cytokine secretion in macrophages via TLR4-mediated signaling pathways . In the present work, we found that the Clerodendrum polysaccharide sub-fraction CSP-AU1 induced secretion of TNF in human PBMCs mainly via TLR4, but binding affinity for this TLR agonist was much lower than that of E.coli-derived LPS. Indeed, a 10-fold higher concentration of LPS-RS over LPS was necessary to completely inhibit LPS-induced TNF production in human PBMC. In comparison, a much lower relative concentration of LPS-RS was needed to inhibit CSP-AU1 activity. Thus, the pharmacological properties of such low-affinity TLR4/MD-2 ligands may be different than those of bacterial LPS. Indeed, although the profiles of pro-inflammatory mediators (NO and most of the cytokines studied here) for in vitro stimulatory activity by bacterial LPS and CSP-AU were fairly similar (Figures 1, 2, 3, and 6), the profile was quite different for production of GM-CSF (Figure 4). Because several plant polysaccharides were recently found having therapeutic effects in different models of autoimmune diseases, such as arthritis [31, 34], lupus erythematosus-like syndrome [30, 33], autoallergic mouse model of Sjogren’s syndrome , asthma , and EAE , we suggest that such natural low-affinity TLR4 agonists may have clinically beneficial effects on autoimmune diseases by their induction of immunosuppression during chronic administration .
Although medical application of hot-water extracts from C. splendens for the treatment of autoimmune diseases has not been reported to date, in the present work we studied therapeutic effects of CSP-AU1 in a mouse model of EAE. We found that chronic i.p. administration of CSP-AU1 induced clinically beneficial effects in EAE, supporting the immunomodulatory properties of Clerodendrum polysaccharides in vivo. Our data showed that CSP-AU1 treatment of mice with EAE delayed disease onset and reduced the clinical severity of EAE. Only one plant polysaccharide, ginsan from Panax ginseng, has been tested to date in this animal model of multiple sclerosis . Similar to our results, these authors observed reduced EAE severity. The polysaccharide ginsan also has been reported to exhibit an anti-allergic reaction in an ovalbumin-induced murine asthma model . Although various plant polysaccharides have been reported to have therapeutic activity in some other models of autoimmune diseases [30, 31, 33, 34, 58], mechanisms of immune suppression by these high-molecular weight molecules in autoimmune disease remain unresolved. Because different plant polysaccharides (e.g., ) and CSP-AU1 activate cells, presumably via TLR4, it is possible that chronic stimulation of the innate immune system via TLR4 could lead to the induction of immunosuppression . Indeed, Vaknin et al. demonstrated that repetitive administration of LPS was sufficient to induce bystander T-cell immunosuppression. Moreover, LPS-stimulated dendritic cells induced tolerance against established collagen-induced arthritis .
In attempts to determine possible immunomodulatory mechanisms of EAE delay by CSP-AU1, we evaluated cytokine production by MOG-peptide reactivated cells in culture, after isolation from CSP-AU1- and PBS-treated EAE mice, and found that the beneficial effect of CSP-AU1 administration was accompanied by reduced IFN-γ, IL-13, IL-17, TNF, GM-CSF and increased TGF-β production. Indeed, a review of the literature regarding immune mechanisms of multiple sclerosis and EAE showed that these cytokines play an important role in progression of EAE disease. In EAE, increased number of IL-17- and IFN-γ-producing cells in the spinal cord resulted from peripheral expansion of these cells after immunization with myelin-derived antigen (reviewed in ). During the EAE disease effector phase, GM-CSF was reported to sustain neuroinflammation via myeloid cells that infiltrated the central nervous system . TNF produced by myeloid cells accelerated the onset of EAE disease by regulation of chemokine expression in the CNS, driving the recruitment of inflammatory cells into the target organ . On the other hand, we recently found that regulatory T cells confer protection against EAE via TGF-β . Thus, the higher level of TGF-β production by splenic and lymph node lymphocytes from CSP-AU1-treated EAE mice could be related to the beneficial therapeutic effects of this polysaccharide fraction.