Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 https://doi.org/10.1186/s12970-019-0281-z RESEARCH ARTICLE Open Access Effects of a polysaccharide-based multi- ingredient supplement on salivary immunity in non-elite marathon runners Emma Roca1* , Elisabet Cantó2, Lexa Nescolarde1,3, Lidia Perea2, Antoni Bayes-Genis4,5,6, Oriol Sibila7 and Silvia Vidal2 Abstract Background: Extreme exercise may alter the innate immune system. Glycans are involved in several biological processes including immune system regulation. However, limited data regarding the impact of glycan supplementation on immunological parameters after strenuous exercise are available. We aimed to determine the impact of a standardized polysaccharide-based multi-ingredient supplement, Advanced Ambrotose© complex powder (AA) on salivary secretory Immunoglobulin A (sIgA) and pro- and anti-inflammatory protein levels before and after a marathon in non-elite runners. Methods: Forty-one male marathon runners who completed the 42.195 km of the 2016 Barcelona marathon were randomly assigned to two study groups. Of them, n = 20 (48%) received the AA supplement for 15 days prior the race (AA group) and n = 21 (52%) did not receive any AA supplement (non-AA group). Saliva and blood samples were collected the day before the marathon and two days after the end of the race. Salivary IgA, pro-inflammatory chemokines (Gro-alpha, Gro-beta, MCP-1) and anti-inflammatory proteins (Angiogenin, ACRP, Siglec 5) were determined using commercially ELISA kits in saliva supernatant. Biochemical parameters, including C-reactive protein, cardiac biomarkers, and blood hemogram were also evaluated. Results: Marathon runners who did not receive the AA supplement experienced a decrease of salivary sIgA and pro-inflammatory chemokines (Gro-alpha and Gro-beta) after the race, while runners with AA supplementation showed lower levels of anti-inflammatory chemokines (Angiogenin). Gro-alpha and Gro-beta salivary levels were lower before the race in the AA group and correlated with blood leukocytes and platelets. Conclusions: Changes in salivary sIgA and inflammatory chemokines, especially Gro-alfa and Gro-beta, were observed in marathon runners supplemented with AA prior to the race. These findings suggested that AA may have a positive effect on immune response after a strenuous exercise. Keywords: Glycans, Dietary plant-derived polydisperse polysaccharide supplementation, Salivary sIgA, Inflammatory chemokines, Marathon, Strenuous exercise * Correspondence: info@emmaroca.com 1 Research Centre for Biomedical Engineering, Universitat Politècnica de Catalunya, Jordi Girona, 1-3, 08034 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 2 of 8 Background xylose, glucose, n-acetyl-glucosamine, n-acetyl-neuraminic In recent years, there has been a significant increase of acid, and n-acetyl-galactosamine) that may regulate im- participants in ultra-endurance races such as marathons munity. In controlled human trials, polysaccharide intake and ultramarathons. In the United States alone, mara- enhanced the immune system biomarkers in the blood of thon runners have increased from 25,000 in 1976 to al- healthy adults [22]. The effect of dietary supplements with most 503,000 in 2010 [18]. Competing in very strenuous polysaccharides on retired professional football players events imposes severe metabolic stress and causes acute supported and optimized their quality of life [25]. How- responses that may negatively alter the immune system ever, no data regarding the impact of AA supplementation [17]. A high level of physical demand during such events on healthy marathon runners performing strenuous phys- induces a wide range of metabolic changes and causes ical activity is available. micro-injuries in the muscles and other tissues. This Our hypothesis is that glycan supplementations before physical demand increases the migration of white blood strenuous physical activity enhances immune function cells to the sites of injury, and induces acute phase in- and balances pro-inflammatory and anti-inflammatory flammatory reactions [5, 16, 28]. The local response to proteins. Therefore, the aim of this study is to determine tissue injury involves the production of a large number the impact of AA in the levels of sIgA, pro-inflammatory of acute phase proteins and cytokines related to innate chemokines and anti-inflammatory proteins before and immunity such as secretory Immunoglobulin A (sIgA) after running a marathon in non-elite marathoners. or inflammatory chemokines [9, 30]. Secretory Immuno- globulin A (sIgA) is a first line of defense against exter- Methods nal agents, serving as a noninvasive biomarker of Participants mucosal immunity [10]. Several studies have shown This study is a part of the SUMMIT project (Health in significant sIgA changes following strenuous exercise Ultra-Marathons and their Limits), whose objective is to [3, 19, 24]. Other important immune factors present evaluate the behavior of certain clinical parameters in in mucosal secretion also change after prolonged running different races and was approved by an institutional re- [6, 20, 24]. These changes include: pro-inflammatory che- view board (IIBSP-SUMMIT-2016-02). In this study, 41 mokines, such as Gro-alpha (neutrophil-activating protein male non-elite runners of the Barcelona Marathon 2016 3 and melanoma growth stimulating activity), Gro-beta participated and they gave signed informed consent. (macrophage inflammatory protein, secreted by mono- All runners were weighed on race morning within 2 h cytes and macrophages and chemotactic for polymorpho- of race start in racing attire with running shoes and im- nuclear leukocytes and hematopoietic stem cells) and mediately after completing the race using the same scale MCP-1 (to recruit monocytes, memory T cells, and den- Jata 565 model. Later the body mass index (BMI) was dritic cells to the sites of inflammation produced by either calculated using the formula BMI = weight (kg) / height tissue injury or infection); anti-inflammatory proteins such (m2). The mean age was 39.3 ± 9.2 years old. as Angiogenin (associated with altered normality through angiogenesis and through activating gene expression that Study groups suppresses apoptosis), Adipokine ACRP30 (cell signaling Twenty participants (48%) received AA supplementation protein secreted by adipose tissue involved in regulating prior to the race (AA group), while 21 participants (52%) glucose levels as well as fatty acid breakdown) and Siglec5 did not receive the AA supplement prior to the race (Sialic acid-binding Ig-like lectin 5). (non-AA group). AA was administrated at dose of 8 g/ Glycans (a generic term for any sugar or assembly of day (with Arabinolactan, Aloe Vera extract, rice starch, sugars, in free form or attached to another molecule) are ghatti gum, gum Tragacanth, Glucosamine HCl, Wak- directly involved in physiology [31]. Glycans are involved ame algae extract) for 15 days before the marathon. The in inflammation and immune system activation [11]. A 41 athletes were selected for being homogeneous in age, standardized polysaccharide-based multi-ingredient sup- weekly training hours, years of training, weight and plement including glycans (Advanced Ambrotose© com- height (Table 1), and were randomly assigned into the plex powder (AA)) may produce a significant overall two study groups. shift towards an increased sialylation of serum glycopro- teins. Sialylation changes can have a key role in many as- Saliva and blood collection pects of the immune response [2]. It is therefore not Saliva samples were collected 48 h before the marathon surprising that one of the physiological effects of these (d0 samples) and two days after the end of the race (d2 polysaccharides is the immunomodulation through the samples). Saliva was collected into clean, sterile tubes, glycosylation of proteins [15]. AA is a saccharide supple- maintained at 4 °C before centrifugation at 9500×g 10 ment containing a standardized combination of plant min at 4 °C. Supernatant was aspirated and 3 different polysaccharides (a source of mannose, galactose, fucose, aliquots were frozen at − 20 °C, until evaluated. Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 3 of 8 Table 1 Participant characteristics, anthropometric and training data before the marathon Baseline characteristics Total cohort Non-AA group AA group p-value Number of runners 41 21 20 Age (years) 39.3 ± 9.2 39.3 ± 12.8 39.2 ± 4.9 0.717 Weight (Kg) 77.0 ± 11.0 74.0 ± 12.0 78.5 ± 12.0 0.208 Height (cm) 178.0 ± 8.0 178.0 ± 7.0 179.5 ± 8.0 0.142 BMI (kg/m2) 24.1 ± 3.4 23.8 ± 3.0 24.2 ± 3.7 0.696 Training (years) 8.0 ± 8.5 7.0 ± 6.5 9.0 ± 12.3 0.537 Training (hours/week) 7.0 ± 5.3 6.0 ± 4.5 7.0 ± 4.9 0.106 Loss weight (%) 3.1 ± 1.8 3.1 ± 2.9 3.1 ± 1.7 0.276 Race time 3.5 ± 0.5 3.4 ± 0.6 3.6 ± 0.5 0.265 AA group: Participants received Advanced Ambrotose Complex Powder (AA) supplementation prior the race Non-AA group: Participants did not received any AA supplement prior the race BMI: body mass index Values are reported as mean ± SD for the quantitative and in percentage for the categorical. ANOVA test was used for parametric variables; Kruskal Wallis test was used for non parametric variables; Chi-Square Pearson for categorical variables Three 10 mL blood samples were obtained from the (Pressage® ST2 assay, Critical Diagnostics, San Diego, antecubital vein 48 h before the marathon (d0 samples), at CA, USA). completion (d1 samples), and 48 h after the race (d2 sam- ples). After the marathon, samples (d1) and weights were Statistical analysis obtained within the 10 min interval after completing the The Kolmogorov-Smirnov test was applied to test the nor- race and before drinking any fluid or emptying the bladder. mal distribution of the data. All variables, with a normal distribution, were reported as mean ± standard deviation Saliva analysis (SD). The rest of the variables were reported as median Saliva samples were thawed and total saliva protein was (interquartile rank) (IQR). T-test and paired t-test were re- quantified using Qubit protein Assay Kit (molecular spectively used for the comparison of independent and re- probes Life Technologies). Salivary IgA (Human IgA, lated variables with normal distribution. Wilcoxon test Platinum ELISA, ebioscience, Affymetrix, Santa Clare, was used for the comparison of related variables with CA), Lactoferrin, Lysozyme (AssayPro, St, Charles, MO, non-normally distributed data. Pearson’s and Spearman’s USA), Gro-alpha/CXCL1, Gro-beta/CXCL2 and MCP-1 coefficients were respectively used to correlate changes (Elabscience, Houston, Texas) were measured by ELISA between normal and between non-normal distributed var- according to manufacturer’s instructions. Limits of detec- iables. ANOVA and Kruskal-Wallis test were respectively tion were for IgA: 1.6 ng/ml; lactoferrin: 0.625 ng/ml; lyso- used for comparative analyses of multiple normal and zyme: 0.0781 ng/ml; Gro-alpha/CXCL1: 15.63 pg/ml, non-normal distributed data. Chi-square tests were used Gro-beta/CXCL2: 15.63 pg/ml, MCP-1: 15.63 pg/ml, for the comparison of frequencies. P values less than 0.05 Angiogenin: 1.64 pg/ml, ARCP30: 24.69 pg/ml and Siglec were considered significant. 5: 6.86 pg/ml. The data was expressed as concentration of each protein relative to total saliva protein concentration. Results Study participants Blood analysis Forty-one male non-elite marathon runners completed The complete blood counts were performed on the Uni- the 42.195 km of the 2016 Barcelona Marathon. Mean cel DxH800 automated hematology analyzer (Beckman finishing time was 3 h and 28 min ± 0. 41 h. Median Coulter, Miami, FL, USA). N-terminal pro-B-type natri- (interquartile range) age was 39 ± 9 years old, with a uretic peptide (NT-proBNP) was quantified in whole weight of 77 ± 11 kg, height of 178 ± 8 cm and Body EDTA blood using the AQT90 FLEX immunoassay Mass Index (BMI) of 24.1 ± 3.4. Median training years (Radiometer Medical, Copenhagen, Denmark). Serum were 8 ± 8, with a median of 7 ± 5 h per week. creatine kinase (CK) and C-reactive protein (CRP) were No significant differences in age, weight, height, and determined using the AU-5800 Chemistry Analyzer training were found between runners who received AA (Beckman Coulter). Troponin T was measured from supplementation and those who did not (Table 1). serum, using a High Sensitive Troponin-T assay in a Cobas e601 platform (Roche Diagnostics, Barcelona, Plasma and blood measurements Spain). ST2 was measured from serum samples using a C-reactive protein increased significantly 48 h after the high-sensitivity sandwich monoclonal immunoassay marathon (d2), although no significant differences Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 4 of 8 between AA and non-AA groups were found. Similarly, supplementation (13.2 ± 4.6 vs 16.1 ± 4.4 × 109/L, p = 0.03) all evaluated cardiac (Hs-TnT, St2 and NTproBNP) and (Table 2). muscle damage (CK) biomarkers increased after the race, but no significant differences between participants in either group were found (Table 2). Salivary IgA Hemoglobin, hematocrit, erythrocytes and platelets No differences in salivary IgA levels normalized to total counts did not change after the race (d1), with no differ- salivary protein before (d0) and 48 h after the race (d2) ences between groups. However, a marked increase in were found (0.47 ± 0.44 vs 0.37 ± 0.29, p = 0.6). How- leukocyte counts per L was found in both groups after the ever, when participants with and without AA supple- race (d1) and participants who received AA supplementa- mentation were compared, a decrease of sIgA 48 h after tion for 15 days prior to the race experienced a lower in- the race (d2) was observed in the non-AA group (0.55 crease compared to those without the AA ± 0.37 vs 0.31 ± 0.35, p = 0.01), while no differences Table 2 Plasma and blood measurements before, after and 48 h after the marathon in 41 runners Biochemical values Total cohort Non-AA group AA group p-value CRP_d0 (mg/dL) 0.85 ± 1.18 0.60 ± 1.28 1.00 ± 1.23 0.683 CRP_d1 (mg/dL) 0.55 ± 0.85 0.45 ± 2.91 0.95 ± 2.48 0.606 CRP_d2 (mg/dL) 7.6 ± 4.65 6.80 ± 5.00 8.95 ± 4.25 0.359 CK_d0 (U/L) 170.5 ± 123.8 188.5 ± 172.3 136.0 ± 125.3 0.633 CK_d1 (U/L) 566.0 ± 282.3 566.0 ± 251.5 567.5 ± 396.3 0.958 CK_d2 (U/L) 764.5 ± 540.8 788.5 ± 787.8 650.0 ± 513.8 0.368 Hs-TnT_d0 (ng/L) 3.1 ± 2.7 2.9 ± 2.0 3.4 ± 5.0 0.407 Hs-TnT_d1 (ng/L) 49.6 ± 68.1 48.9 ± 68.8 49.9 ± 68.8 0.449 Hs-TnT_d2 (ng/L) 4.8 ± 6.3 4.9 ± 7.2 4.6 ± 3.7 0.334 St2_d0 (ng/mL) 34.0 ± 14.8 34.7 ± 16.2 28.6 ± 16.0 0.394 St2_d1 (ng/mL) 54.9 ± 30.3 59.0 ± 40.5 43.3 ± 30.4 0.256 St2_d2 (ng/mL) 32.9 ± 20.5 37.7 ± 19.3 29.4 ± 17.1 0.059 NTproBNP_d0 (ng/L) 70.0 ± 0 70.0 ± 0 70.0 ± 0 0.336 NTproBNP_d1 (ng/L) 94.5 ± 70.5 100.5 ± 50.3 85.5 ± 221.3 0.392 NTproBNP_d2 (ng/L) 70.0 ± 0 70.0 ± 0 70.0 ± 0.5 0.845 Hemoglobin_d0 (g/L) 14.8 ± 1.3 14.8 ± 1.1 14.7 ± 1.6 0.350 Hemoglobin_d1 (g/L) 14.7 ± 1.1 14.8 ± 1.0 14.5 ± 0.3 0.173 Hemoglobin_d2 (g/L) 14.3 ± 0.7 14.4 ± 0.7 14.2 ± 0.9 0.170 Hematocrit_d0 43.7 ± 3.0 43.7 ± 3.1 43.8 ± 3.1 0.327 Hematocrit_d1 44.0 ± 4.2 44.3 ± 3.2 43.3 ± 3.4 0.132 Hematocrit_d2 42.7 ± 2.2 42.8 ± 1.8 42.2 ± 3.7 0.114 Erythrocytes_d0 (10Exp12/L) 4.9 ± 0.6 4.9 ± 0.4 5.1 ± 0.7 0.903 Erythrocytes_d1 (10Exp12/L) 5.0 ± 0.5 5.0 ± 0.4 5.0 ± 0.8 0.586 Erythrocytes_d2 (10Exp12/L) 4.8 ± 0.4 4.8 ± 0.3 4.9 ± 0.5 0.532 Platelets_d0 (10Exp9/L) 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.063 Platelets_d1 (10Exp9/L) 0.2 ± 0.0 0.2 ± 0.1 0.2 ± 0.0 0.852 Platelets_d2 (10Exp9/L) 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.555 9 Leukocyte_d0 (10 /L) 6.5 ± 1.9 6.4 ± 1.3 6.5 ± 3.2 0.860 Leukocyte_d1 (109/L) 12.2 ± 5.3 16.1 ± 4.4 13.2 ± 4.6 0.032 9 Leukocyte_d2 (10 /L) 6.6 ± 1.3 6.6 ± 1.4 6.6 ± 1.8 0.217 AA group: Participants received Advanced Ambrotose Complex Powder (AA) supplementation prior the race Non-AA group: Participants did not received any AA supplement prior the race CK creatine kinase, CRP C-reactive protein, HGB hemoglobin, Hs-TnT high-sensitivity troponin T, NT-proBNP N-terminal pro-B-type natriuretic peptide Values are reported as mean ± SD for the quantitative and in percentage for the categorical. ANOVA test was used for parametric variables; Kruskal Wallis test was used for non parametric variables; Chi-Square Pearson for categorical variables Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 5 of 8 before (d0) and 48 h after the race (d2) in the AA group These systemic correlations were not observed in the were found (0.25 ± 0.33 vs 0.24 ± 0.32, p = 0.5) (Fig. 1). non-AA group (Fig. 4). Salivary pro inflammatory proteins Discussion A decrease of pro-inflammatory salivary proteins such In our study, we demonstrated significant changes in sal- as Gro-alfa, Gro-beta and MCP-1 after the race was ivary biomarkers of immune function in healthy, observed in all participants, although differences were non-elite athletes before and after a strenuous exercise not statistically significant (data not shown). Neverthe- like an asphalt marathon, after consuming a less, the decline in Gro-alpha (Fig. 2a) and Gro-beta polysaccharide-based multi-ingredient supplement, AA, (Fig. 2b) was statistically significant in those runners who for 15 days prior to exercise. Specifically, a decrease in did not receive the AA supplement (0.38 ± 0.20 vs salivary sIgA, Gro-alfa and Gro-beta were observed after 0.24 ± 0.18, p = 0.02 and 0.47 ± 0.26 vs 0.32 ± 0.26, p = the marathon in those runners who did not receive AA 0.03), while differences were not observed in the AA supplement prior the race, while those runners who re- group. Furthermore, basal levels of Gro-alpha and ceived the AA supplement showed a decrease in a saliv- Gro-beta were significantly lower (p = 0.03) in the AA ary Angiogenin. Gro-alpha and Gro-beta salivary levels group at baseline before the marathon (d0) compared were lower before the race in the AA group and corre- to the non-AA group (Fig. 2). lated with the counts of blood leukocytes and platelets. These findings suggest that AA supplementation pro- Salivary anti-inflammatory proteins duces changes in salivary immunity that may have a No statistically significant differences of salivary positive effect on immunity before and after a marathon. anti-inflammatory proteins such as ACRP, angiotensin, sIgA is a key component of innate immunity and pro- and Siglec 5 were observed before (d0) and 48 h after vides a first line of defense against pathogens at mucosal the marathon (d2) (data not shown). However, in those surfaces [8]. Numerous studies have assessed the saliva runners of the AA group, a significant decrease of sIgA response to prolonged strenuous exercise to ex- Angiogenin 48 h after the race (d2) was observed (0.18 plore whether immunity may be temporarily compro- ± 0.08 vs 0.14 ± 0.07, p = 0.04), while differences were mised after the exercise. Most of the studies described a not observed in the non-AA group (0.3 ± 0.13 vs 0.17 ± post-exercise decrease in sIgA, including Nordic skiers 0.10, p = 0.7) (Fig. 3). after a 50 km race [29] or trained cyclists cycling for more than 2 h on a stationary ergometer [32]. In mara- Systemic correlations thon runners, a significant decrease of sIgA following a After the race, those runners who received the AA supple- marathon race has been described [19], which was inde- ment (AA group) showed positive correlations be- pendent of gender, age or carbohydrate ingestion. In our tween salivary Gro-alpha levels with leukocyte counts per study, runners with AA supplementation did not experi- L (r = 0.38, p = 0.02) and a tendency between Gro-beta sal- ence a significant decrease in sIgA after the marathon, ivary levels and blood platelet counts (r = 0.27, p = 0.06). which was observed in runners without the AA. These findings suggested that AA supplement enhanced immun- ity by avoiding the sIgA decrease after the race. Different studies have postulated that glycosylation plays an import- ant role in the biosynthesis and biological activity of the proteins involved in antigen recognition, such as sIgA [23]. Further studies are needed to explore this pathway, which may be crucial in order to better understand the ef- fect of strenuous exercise on mucosal immunity. Supplementation with glycans can also result in salivary glycol-modifications of proteins. These modifications can regulate the synthesis and/or degradation of pro- and anti-inflammatory molecules participating in the immune response of runners to strenuous effort. Several inflamma- tory chemokines have demonstrated a potential role in the regulation of immune response during exercise [27]. In our study, we demonstrated that Gro-alfa and Gro-beta, two pro-inflammatory chemokines, involved in the attrac- Fig. 1 Salivary IgA levels before (d0) and 48 h after the marathon tion of neutrophils to the site of inflammation, decreased (d2) between groups in saliva after the race in those runners who did not Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 6 of 8 Fig. 2 Salivary pro inflammatory proteins, gro alpha (a) and gro beta (b) before (d0) and 48 h after the marathon (d2) between groups receive AA. It has been described that moderate exercise The systemic effect of glycans has been described in suppressed Gro-alfa [4] and, in experimental models, exer- several studies [15]. Interestingly, it is postulated that cise down-regulates multiple inflammatory cytokines [26], glycans may regulate leukocyte activity [21]. In our including Gro-alfa and Gro-beta, that results in a systemic study, runners who received the AA supplement experi- anti-inflammatory effect [1, 13]. These findings were not enced a lower increase in total blood leukocyte counts observed in runners who received the AA supplement, after the race compared to those runners without the suggesting that they had better immunoregulation. Run- AA supplement. Furthermore, a correlation between sal- ners with AA supplementation have also showed a de- ivary levels of Gro-alpha with blood leukocyte counts crease in salivary Angiogenin levels, which was not and salivary Gro-beta levels with blood platelet counts detected in runners without AA. It suggests that salivary were observed in the AA group. All in all, these findings pro-inflammatory proteins can be buffered with suggested a relationship between salivary and systemic anti-inflammatory ones, especially those produced by run- immunity, especially in those runners who received the ners supplemented with AA. In addition, it is important to AA supplement prior to the race. The association be- note that after 15 days on AA, the baseline levels of tween high dietary protein during high-intensity training Gro-alfa, Gro-beta and Angiogenin were different in run- and reduction in respiratory symptoms in elite cyclists ners with and without AA. Interpretations of all these sal- by restoring impairments in leukocyte trafficking has ivary inflammatory changes are complex and require also been reported [35] The influence of ingredients further more detailed studies. other than glycans on the immune system has been re- ported. For example, brown seaweed [33], Aloe vera [12] and glucosamine [14] have antioxidant activity and anti-inflammatory effects. Arabinogalactan decreases the incidence of infectious episodes by improving serum- antigen specific IgG and IgE response to Streptococcus pneumonia [7]. Starch supplementation promotes the growth of commensal bacteria that may improve bowel health [34]. However, more work is needed to clarify the mechanism that may explain these important results. Our study has limitations. First, we only have studied men, and we cannot exclude the effect of glycans in sal- ivary immunity based on sex. Second, due to the small sample size of runners included, the results should be validated in further studies before generalizing them. Third, runners received the AA supplement for 15 days prior the race, and the effect of taking this supplement for a longer time may be different. Fourth, the influence Fig. 3 Salivary anti-inflammatory protein Angiogenin before (d0) of ingredients included in the AA supplements which and 48 h after the marathon (d2) between groups are not glycans such as glucosamine and Aloe Vera on Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 7 of 8 Fig. 4 Systemic correlations 48 h after the marathon (d2) between: salivary Gro-alpha levels with leukocyte counts in non-AA group (a) and AA group (b) and Gro-beta salivary levels and blood platelet counts in non-AA group (c) and AA group (d) the immune system cannot be ruled out, and further Funding studies in non-elite marathon runners should be per- This study was funded by an unconditional product grant from Mannatech Inc. (Coppell, TX). The researchers in this study independently collected, formed to better clarify this important point. analyzed, and interpreted the results from this study and have no financial interests in the results of this study. Conclusion Availability of data and materials The datasets used and/or analyzed during the current study are available In conclusion, our study documented significant changes from the corresponding author on reasonable request. in salivary and systemic immunological biomarkers in marathon runners after consuming a polysaccharide-based Authors’ contributions multi-ingredient supplement AA, before and after the race. All authors participated at the conception and design of the study. ER and LN were involved at data collection. All authors participated at data Further research using randomized, double-blind, interpretation and analysis. ER, EC, OS and SV participated at manuscript placebo-controlled design will extend knowledge of the drafting. All authors read and approved the final manuscript. potential benefits nutritional dietary supplementation with polysaccharides may have in marathon runners. Ethics approval and consent to participate As discussed in the Participants section of the manuscript, this study was approved by an institutional review board. Protocol number: IIBSP-SUMMIT- Abbreviations 2016-02. All participants provided written consent prior to their participation AA: Standardized dietary plant-derived polydisperse polysaccharide in the study. Advanced Ambrotose© complex powder; BMI: Body mass index; CK: Serum creatine kinase; CRP: C-reactive protein; HGB: Hemoglobin; Hs-TnT: High- Consent for publication sensitivity troponin T; NT-proBNP: N-terminal pro-B-type natriuretic peptide; Not applicable. sIgA: Salivary secretory Immunoglobulin A Competing interests The study was started prior to the corresponding author, Emma Roca joining Acknowledgements the Global Scientific Advisory Board (GSAB)* at Mannatech, Inc. as related The authors would like to thank the Barcelona marathon organization for research to her PhD studies. The study was approved by the supervising their cooperation, UPC students for their help in data collection, Hospital Faculty/Professor and the University (Universitat Politècnica de Catalunya Universitari Germans Trias i Pujol and Hospital de la Santa Creu i Sant Pau for (UPC), Research Centre for Biomedical Engineering (CREB-UPC) and IRB data analysis, and finally the marathon runners for their participation. without input from Mannatech, Inc. Roca et al. Journal of the International Society of Sports Nutrition (2019) 16:14 Page 8 of 8 Publisher’s Note 16. Kim HJ, Lee YH, Kim CK. Biomarkers of muscle and cartilage damage and Springer Nature remains neutral with regard to jurisdictional claims in inflammation during a 200 km run. Eur J Appl Physiol. 2007;99(4):443–7. published maps and institutional affiliations. 17. Kłapcińska B, Waśkiewicz Z, Chrapusta SJ, Sadowska-Krępa E, Czuba M, Langfort J. 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