Polysaccharide agar is a galactan that forms a major cell wall component in red algal families such as Gelidiaceae and Gracilariaceae (1). Agar refers to a mixture of two polysaccharides, agarose and agaropectin. Agarose consists of alternating residues of (1 → 3)-linked β-d-galactose and (1 → 4)-linked 3,6-anhydro-α-l-galactose (2). Partial depolymerization of agarose via acid degradation leads to the hydrolysis of α-(1 → 3)-galactosidic bonds, resulting in the production of agaro-oligosaccharides (AOSs). AOSs are composed of repeating agarobiose (Abi) units, with 3,6-anhydro-α-l-galactose at the reducing end (3). AOSs exhibit promising applications in the food industry owing to their biological activities, possibly attributed to the presence of 3,6-anhydro-α-l-galactose at the reducing end (4).

AOSs are generally presumed to be indigestible by the human intestine owing to the lack of agarase production. There are many reports on the bioactivities of AOSs, such as anti-inflammatory (5, 6), anti-obesity (7), and prebiotic (8) effects, which can be explained by their local functioning in the gut or modulation of the gut microbiota, including the production of bioavailable microbial metabolites. Enoki et al. (9) reported that orally administered AOSs suppressed 12-O-tetradecanoylphorbol-13-acetate-induced tumor formation in mouse skin. The underlying mechanism might be explained by the changes in the gut microbiome; however, AOSs, especially low-molecular weight (MW) AOSs, which may be absorbed into the blood circulation, can have direct functions and exhibit their bioactivities in peripheral tissues.

The single oral administration test revealed that partially depolymerized shark chondroitin sulfate (CS) was detected in human plasma and urine (10). Pharmacokinetics parameters vary with the sulfation status, MW, or charge density of CS used on the experiment (11, 12). In an animal experiment using rats and dogs, orally administered CS was detected in the tissues, including the intestine, liver, kidneys, and joint cartilage (13).

In our previous study, AOS permeation from the intestine into blood circulation was examined in vivo and in vitro (14). Orally administered AOSs have been detected in rat plasma in a dose-dependent manner. The detection level in rat plasma was inversely correlated with the degree of polymerization, and Abi showed a higher plasma concentration than that of agarohexaose (Ahe), which has three repeating Abi units. Transport experiments in the Caco-2 cell culture model indicated that low-MW AOSs easily permeated the Caco-2 cell monolayers, and that the main transport route was the paracellular pathway. However, few clinical data are available on the absorption of AOSs from the gastrointestinal tract into the blood in humans.

Taking these findings into consideration, we hypothesized that low-MW AOSs can be absorbed into the human blood after oral administration. To test our hypothesis, we examined the plasma levels of AOSs after oral administration to healthy volunteers. Plasma samples were collected before and 1, 2, and 4 h after ingestion. To evaluate the absorption efficacy of various AOSs depending on their degree of polymerization, the time course for the absorption of Abi, agarotetraose (Ate), and Ahe was compared.

The absorption of AOSs with different MWs in the bloodstream was examined by the low-dose (2 g) and high-dose (5 g) trials. Administered AOSs comprised 30.2%, 29.1%, and 31.6% of Abi, Ate, and Ahe, respectively. The amounts of Abi, Ate, and Ahe in the 2 g and 5 g doses were 1.86, 0.92, and 0.68 mmol and 4.66, 2.31, and 1.69 mmol, respectively. The representative MS chromatograms of the volunteers included in the statistical analysis are shown in Supplementary Figure S2. The concentration-time profiles of the AOSs are shown in Figure 1. In the high-dose (5 g) trial, the plasma concentration of Abi and Ate were increased at 1 h after ingestion and were maintained at the same level or gradually increased up to 4 h after ingestion (Figure 1A). Abi was also detected in the low-dose (2 g) trial (Figure 1B). In contrast, plasma Ate was detected only in a 4 h sample obtained from one of the eight participants. The plasma concentration of Abi at all time points (1 h, 2 h, and 4 h) was significantly higher than that of Ate in both trials. The plasma Ahe level was below the detection limit in both the trials. These results indicate that the permeation of AOSs is inversely correlated with their MWs.

The AUC for the plasma concentration of Abi and Ate was also examined (Figure 2). After the ingestion of 5 g AOSs, the AUC value of plasma Abi was 1.59 nmol‧h/mL (range: 1.17–2.35 nmol‧h/mL) (Figure 2). With low-dose AOSs (2 g), the AUC value of plasma Abi was 0.96 nmol‧h/mL (range: 0.84–1.26 nmol‧h/mL). The plasma AUC of Ate was 0.41 nmol‧h/mL (range: 0.00–0.88 nmol‧h/mL) in the high-dose trial and 0.03 nmol‧h/mL (range: 0.00–0.25 nmol‧h/mL) in the low-dose trial. The AUC in the high-dose trial was significantly larger than that in the low-dose trial for both Abi and Ate. These results suggest that the plasma levels of AOSs are dose-proportional, independent of the MW.

In this study, pharmacokinetics of AOSs in human plasma after oral ingestion were examined. The protocol was determined based on our previous report. Pharmacokinetic study in rats revealed that the plasma levels of AOSs are maximum at 4 h and significantly decrease 8 h after oral administration (14). Therefore, time points selected for blood collection were before AOS administration (0 h) and 1, 2, and 4 h after AOS administration.

In the pre-ingestion sample of the low-dose trial, an unidentified peak was observed at the same selected ion monitoring channel as ABEE-derivatized Abi. However, it was unlikely to represent ABEE-derivatized Abi given the absence of endogenous AOSs in humans and the 12 h of fasting before the oral administration of the AOSs. Therefore, we regarded the unidentified peak as the baseline and subtracted it from the AUC values.

The amount of detected AOSs in the plasma increased after oral administration, with an inverse correlation between the plasma detection levels and the MW of the AOSs. In the high-dose (5 g) trial, the AUC values of plasma Abi and Ate were 1.59 and 0.41 nmol‧h/mL, respectively, which means that permeation efficacy of Abi was almost four times higher than that of Ate. Since Ate has nearly two times higher MW than Abi, the amount of Ate contained in AOSs is halved compared to Abi. Therefore, the difference between the AUC values of Abi and Ate was not fully explained by their concentration. Low-MW AOSs could permeate more efficiently compared with high-MW AOSs.

Considering the absence of agarase in the mammalian gastrointestinal tract, Abi and Ate remained intact until their elimination in urine. Similarly, human milk oligosaccharides also exist in human plasma for considerable time (16). Although the time course of plasma concentration is unclear, human milk oligosaccharides were detected in human plasma and were found in infants’ urine with a peak time of 8–12 h. Hence, it is possible that the plasma concentrations of Abi and Ate beyond 4 h were also maintained at certain levels.

A recent study evaluated the pharmacokinetic parameters of orally administered CS in human plasma (17). In this study, the absorption rate of CS was higher than that of AOSs. A single dose of 2,400 mg CS with a MW of 5,120 Da increased the plasma CS concentration from baseline to 48 h. The intake-standardized AUC value was more than eight times higher than that of Abi and sixteen times higher than that of Ate. In addition, the intake-standardized maximum plasma concentration was more than 14 and 34 times higher than that of Abi and Ate, respectively. According to a study using Caco-2 cell monolayers, both CS (18) and AOSs (14) across Caco-2 cell monolayers via the paracellular pathway. These findings are in good agreements with previous reports in which the transport of oligosaccharides occurred via the paracellular pathway (19). The permeability of low-MW hyaluronan increased inversely with its molecular size (20), which is also consistent with our findings. However, the underlying mechanism responsible for the different rates of absorption of CS and AOSs still remains unclear.

High-MW oligosaccharides might be utilized by the gut microbiota, but they are generally regarded to be poorly absorbed through the intestinal barrier. In fact, unlike Abi, Ahe was not detected in human plasma after oral administration in this study. Low-MW AOSs absorbed in the blood circulation might exhibit their biological effects in peripheral tissues. In other words, the biological activities of AOSs are mainly caused by Abi because of its higher concentration in blood. Enoki et al. (4) reported that AOSs inhibited the production of interleukin-1β and interleukin-6 in mouse RAW264.7 macrophages, demonstrating the anti-inflammatory activity of AOSs. However, it is still unclear whether low-MW AOSs transferred to the blood can interact with macrophages located in peripheral tissues and exhibit their anti-inflammatory activity. Orally administered raffinose oligosaccharide can be detected in rat plasma (21). Furthermore, dietary raffinose reduced the number of eosinophils in the broncho-alveolar lavage fluid and alleviate allergic inflammation in the lung tissue of ovalbumin-sensitize rats. The ameliorative action of dietary raffinose was observed in neomycin-treated rats, suggesting a post-absorptive action of raffinose in lung tissues without a prebiotic action. Therefore, further in vivo investigation is necessary to determine the accumulation of AOSs in peripheral tissues after oral administration.

We demonstrated that single-dose orally administered AOSs could be detected as Abi and Ate in human plasma. However, the ingestion of repeated AOS doses may potentially improve their absorption rate. For example, after 28 days of oral administration of coenzyme Q10, its plasma concentration was higher than that after a single administration, indicating an increase in bioavailability with repeated administration (22). Future studies are needed to investigate the influence of continuous AOS supplementation on bioavailability.

In conclusion, this study indicated that Abi and Ate were absorbed into human plasma following oral ingestion, with concentration levels maintained for the experimental period of 4 h. The plasma levels of AOSs were dose-dependent and inversely correlated with their MW. Abi showed the highest concentration in human plasma, in accordance with our previous animal study; in contrast, Ahe was not detected in any plasma sample. These results imply that low-MW AOSs may be localized in peripheral human tissues where they can exert their biological effects.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by the Ethics Committee of Tokyo Kasei University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

IS: Conceptualization, Data curation, Investigation, Writing – original draft. YI: Data curation, Investigation, Writing – original draft. KK: Conceptualization, Writing – review & editing. YS: Conceptualization, Writing – review & editing. SK: Conceptualization, Writing – review & editing.

IS and KK are employees of Ina Food Industry Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.