Rhamnan sulphate from green algae Monostroma nitidum improves constipation with gut microbiome alteration in double-blind placebo-controlled trial

Rhamnan sulphate (RS), a sulphated polysaccharide from Monostroma nitidum, possesses several biological properties that help in treating diseases such as viral infection, thrombosis, and obesity. In the present study, we first administered RS (0.25 mg/g food volume) orally to high-fat diet-treated mice for 4 weeks. RS increased the faecal volume and calorie excretion with decreased plasma lipids, which was in accordance with the results of our previous zebrafish study. Notably, as the excretion amount by RS increased in the mice, we hypothesised that RS could decrease the chance of constipation in mice and also in human subjects because RS is considered as a dietary fibre. We administrated RS (100 mg/day) to subjects with low defaecation frequencies (3–5 times/week) for 2 weeks in double-blind placebo-controlled manner. As a result, RS administration significantly increased the frequency of dejection without any side effects, although no effect was observed on the body weight and blood lipids. Moreover, we performed 16s rRNA-seq analysis of the gut microbiota in these subjects. Metagenomics profiling using PICRUSt revealed functional alternation of the KEGG pathways, which could be involved in the therapeutic effect of RS for constipation.


Introduction
Seaweeds contain high levels of iodine, iron, vitamin C (which aids iron absorption), anti-oxidants, soluble and insoluble bre, vitamin K, vitamin B-12, and various nutrients that promote human health. Of these, green algae belong to Monostroma genus, and are commercially cultivated in East Asia and South America for edible purposes, as popular sushi wraps. Rhamnan sulphate (RS) is a sulphate polysaccharide comprising L-rhamnose and sulphated L-rhamnose found in green algae, and was puri ed as the main constituent from the cell walls of Monostroma lattisimum and Monostrom nitudum in 1998 1 , as an activator for anti-thrombin 2 . For the following 20 years, several biological activities of RS have been identi ed such as its anti-coagulant [3][4][5][6] and anti-viral effects 7-10 and others 11 . Of these, we discovered lipid-lowering properties of RS to improve hepatic steatosis, using a diet-induced obesity model of zebra sh in 2015. 12 In the present study, to validate the anti-obesity properties of RS in mammals, we administered RS to high-fat diet-induced obese mice and found that RS increased the excretion amount and calories with lipid-lowering effects. Furthermore, we performed a clinical trial on subjects with constipation tendency to determine whether RS can be used as a therapeutic molecule.

RS improved constipation in subjects with low defaecation frequency
From the mouse experiment, we hypothesised that RS has therapeutic properties to improve constipation, thereby subsequently decreasing blood lipids and body weight. Thus, we performed a clinical trial with chronic constipation. The present study was performed from 28th February to 23rd April 2020. Seventythree volunteers were initially screened, as illustrated in Figure S2. The nal 38 healthy volunteers (participants), who had relatively low defaecation frequencies (3-5 times a week), were randomly allocated to the groups to receive RS or placebo. The baseline characteristics of the participants are summarized in Table 1. No signi cant difference was observed between RS and the placebo group for any baseline characteristics (p ≥ 0.05). In contrast to the mouse experiment, RS did not decrease the body weight ( Fig. 2A), plasma TG (Fig. 2B) and TCHO (Fig. 2C); however, blood glucose was signi cantly (p < 0.01) decreased by RS administration (90.7 ± 8.3 mg/dL at 0 week vs. 86.2 ± 6.0 mg/dL at 2 weeks in the RS group; Fig. 2D), which was in accordance with that of the results with mouse experiment (Fig. 1D).
RS signi cantly (p < 0.05) increased the excretion frequency (4.1 ± 1.5 time/week at 0 week vs. 5.6 ± 1.9 time/week at 2 weeks in RS group; Fig. 2E) with increase in the excretion frequency from 0 to 2 weeks (0.3 ± 1.7 time/week in placebo group vs. 1.5 ± 1.6 time/week in RS group at 2 weeks; Fig. 2F). Moreover, the excretion days per week were also increased by RS administration (3.8 ± 1.0 days/week at 0 week vs. 4.9 ± 1.2 days/week at 2 weeks in the RS group; Fig. S3). Presumably, these results are similar to those observed in our mouse study ( Fig. 1E and 1F). No important harms or unintended effects was observed during the study.

Metabolic functional pathways in RS administrated subjects
To understand the RS-induced functional alterations in the gut microbiota of participants, bacterial metagenomes were predicted by PICRUSt using 16S rRNA sequencing, as previously reported 13 . Predicted proteins in each bacterium were classi ed into KEGG ortholog (KO) entities, which resulted in the identi cation of 6188 entities across all samples. Of these, 588 KOs and 553 KOs were up-regulated (> 2.0) and down-regulated (0.5<) in the RS group, whereas 593 KOs and 1425 KOs were up-regulated and down-regulated in the placebo group. The up-regulated 414 KOs and the down-regulated 130 KOs were selective in RS groups (Fig. 4A). Thereafter, we mapped the differentially expressed KOs to the KEGG Mapper in order to identify the altered pathways in the participants. As illustrated in Fig. 4B, we identi ed 199 and 214 pathways in the RS and placebo groups, respectively. Furthermore, 175 pathways were common in both groups and 24 pathways were speci c to RS administration. Because the RS-selective pathways (Table S4) contained few KOs (maximum 3 KOs in lysin biosynthesis [map00300]), we further performed different analyses. We calculated the ratio of KO counts in each KEGG pathway in the common 175 pathways and found that 56 pathways were altered in the RS group compared to placebo (> 2 or < 0.5; Table 3). After evaluating these 56 pathways manually (represented as images in

RS improved constipation
Although we identi ed the therapeutic effects of RS in an HFD-induced obesity model with increased defaecation, we evaluated these effects only in constipated human subjects, and not in obese populations, as it was di cult to nd obese people with constipation. Even with this limitation, we con rmed that RS increased the excretion amount both in mouse and human for the rst time. In general, intake of seaweeds is bene cial for gut health and improves constipation, as they contain bres and polysaccharides. In particular, sulphated polysaccharides from marine seaweeds affect the human microbiome 14 and improve loperamide-induced constipation in rats 15 . Since RS is categorised in sulphated polysaccharides, our results seem reasonable for ameliorating constipation with alteration of gut microbiota.
Although RS signi cantly improved constipation-related phenotypes between 0 and 2-week administration, no signi cant difference was found between the RS and placebo groups at 2 weeks ( Fig. 2E and 2F). As Mancabelli et al. previously reported that constipated people increased the diversity of gut microbiota 16 , we separated the participants into two groups: those who possessed greater diversity in gut microbiota than the average diversity of whole participants (high diversity subjects) and smaller ones (less diverse subjects). Moreover, as illustrated in Figure S4, RS signi cantly (p < 0.05) improved constipation between placebo and RS group on excretion day per week (3.7 ± 1.6 time/week vs. 5.1 ± 1.1 time/week at 2 weeks), increased the excretion frequency (0.0 ± 2.1 time/week vs. 2.0 ± 1.7 time/week at 2 weeks), and increased the excretion days (− 0.2 ± 1.8 time/week vs. 1.4 ± 1.3 time/week at 2 weeks) in high diversity group, which are not detected in low diversity group. For the Bristol scale (faeces property), the placebo group increased (p < 0.01) whereas RS did not, even in the high diversity group (data not shown). An increase in the Bristol scale indicates softening of faeces. Since RS retains water as a soluble bre in faeces, this result seems inconsistent. We hypothesised that RS-induced defaecation occurred at a faster pace than water retention by RS, and that no increase was observed in the faecal water content as well as in the Bristol scale.

RS effects on gut microbiota in various subjects
Surprisingly, the compositions of gut microbiota and KOs in the placebo group were altered more than those in the RS group (Table 2 and Fig. 4). Because our trial was conducted during the rst wave of the coronavirus disease 2019 (COVID-19) crisis (from February to April 2020), many participants underwent lifestyle changes such as work from home, consumed home-made meals, and avoided alcohol consumption and eating out. In this situation, we identi ed several gut bacteria that are involved in the therapeutic effect of RS. Although Clostridiales and Negativicutes were neighbouring in the Firmicutes phylum, Clostridiales decreased (Fig. 3C) and Negativicutes increased (Fig. 3D) in the RS group. Clostridia produce medium-length fatty acids that increase water absorption, and subsequently dry up faeces, causing constipation 17 . Thus, one of the therapeutic mechanisms of RS against constipation is the decrease in the Clostridiales class. In particular, prebiotic supplementation in constipation patients reduced the composition of Clostridiales 18 . RS selectively increased Negativicutes (Fig. 3D) and Acidaminococcales (Fig. 3E). Their biological effects on constipation remain unclear; however, several studies reported that the increase in these bacteria is positively related to improved constipation.
Negativicutes were increased by Psyllium Husk (derived from seeds of Plantago ovata) administration 19 and Bi dobacterium-based probiotic treatment 20 during constipation improvement. Acidaminococcus may be one of the main factors in curing constipation during faecal microbial transplantation in clinical application 21 and administration of partially hydrolyzed guar gum (water-soluble bres) in children 22 . Moreover, Veillonellales, a pro-in ammatory and lactate-fermenting bacterium increased in the irritable bowel syndrome patients 23 , in both RS and placebo groups (Fig. 3F), thereby revealing stress from the new lifestyle due to COVID-19 crisis.
Predictive functional analysis of gut microbiota in subjects Combination analysis using PICRUSt and KEGG Mapper was a powerful tool to predict functional alterations in gut microbiota 24 . KOs predicted by PICRUSt revealed that the number of down-regulated KOs in the placebo group was larger than that in the RS group (Fig. 4A). We mapped these KOs to the KEGG Mapper in order to predict functional pathways differentially expressed between the RS and placebo groups (Table 3 and supplementary materials). Of these, several KOs in the pathway "metabolism of xenobiotics by cytochrome p450" were up-regulated in the RS group, whereas they were down-regulated in the placebo group (Fig. 4C). Reactions catalysed by cytochrome p450 usually turn xenobiotics, such as polysaccharides RS, to excretable metabolites 25 . This result was in accordance with that obtained in the present study. As illustrated in Fig. 4D, in "cationic anti-microbial peptide (CAMPs) resistance" pathway, several KOs were up-regulated in RS group, whereas they were down-regulated in the placebo. CAMPs are critical frontline contributors to host defence against invasive bacterial infections.
For successful survival and colonisation of the host, bacteria have a series of mechanisms that interfere with CAMP activity 26 . This result suggests that RS induced CAMP resistance, which promotes the proliferation of pathogenic bacteria; however, Assoni et al. reported that CAMP resistance mediates the recovery of prominent gut commensals during in ammation 27 . In general, constipation is accompanied by in ammation in the gut mucosa, implying that RS would improve not only constipation but also mucosal damage by altering the composition of gut microbiota. Nicotinamide metabolism was downregulated in the placebo group but up-regulated in the RS group (Fig. 4E). Nicotinamide, known as vitamin B3, is essential for life as it is part of the coenzyme NADH/NAD+, which is crucial for biological redox reactions. Moreover, it ameliorates experimental colitis in mice by improving host defence and enhancing bacterial clearance in Citrobacter rodentium-induced colitis 28 and Staphylococcus aureus infection in mice 29,30 . Furthermore, NAD replenishment ameliorates constipation in aged mice 31 , suggesting that one of the therapeutic mechanisms of RS is the increase in bacteria related to nicotinamide synthesis and/or secretion. In particular, our functional analysis based on PICRUSt and KEGG Mapper is just a prediction; further studies are necessary to demonstrate our speculation.

Conclusion
RS promoted defaecation in mice and human subjects without any side effects and improved gut microbiota. Therefore, co-administration of RS with other prebiotics and probiotics may enhance this effect in future studies. In conclusion, with other health-promoting properties of RS (lipid-lowering, antiviral and anti-thrombotic), it is a powerful constituent in M. nitidum, and can be used as a therapeutic or preventive supplement due to its anti-constipation properties.

Preparation of rhamnan sulphate (RS)
For mouse experiments, we used puri ed RS (> 95% purity) as previously described 7 . For the clinical study, Rhamnox® (Konan Chemical Manufacturing, Mie, Japan) was used for RS. The preparation of Rhamnox is described as follows according to a previous study 9 , with certain modi cations. Dried M. nitidum (700 g) was washed with H 2 O, by adding up to 18 L of H 2 O, and then extracted for 6 h at 100°C.
To prevent foaming, 2-5 g of citrate acid was added after 40 min of heating. Thereafter, celite (540 g) was added to the extract, and then centrifuged to remove the algal fronds. Next, celite (90 g) was readded to the extract and ltered with qualitative lter paper grade 2 (Pellicon Cassette System P2B010V01; Millipore, Billerica, MA, USA). Hot water extract obtained in this manner was fractionated via ultra ltration (Millipore; cutoff MW 10,000 University. Six-month-old male mice were assigned to three groups with six mice, housed individually, and were fed either the CE-7 normal diet (ND; CLEA Japan, Tokyo, Japan), high-fat diet (Test Diet 58Y1; TestDiet, Richmond, IN, USA) or a high-fat diet (HFD) supplemented with RS (250 mg/g BW) for 4 weeks to induce obesity. The compositions of ND and HFD are described in Table S1. During the feeding experiment, body weight, food intake, and faecal weight were measured once per week. Mice were subjected to fasting for 14 h before blood sampling to assess the fasting blood glucose levels. Blood glucose was measured using a hand-held glucometer (Glutest Neo Super; Sanwa Kagaku, Nagoya, Japan). The plasma levels of triacylglycerol (TG), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TCHO) were measured using Wako L-type TG, Wako L-type LDL-C, and Wako L-type TCHO (Wako Pure Chemicals, Osaka, Japan) assay kits according to the manufacturer's protocol. The mice were euthanized with CO 2 gas, and then the organ samples were collected and subsequently dissected for analysis.

Caloric analysis of mouse stools
The stool samples were collected daily and stored at − 20°C. The nutritional composition (fat, protein, moisture, ash, carbohydrate and energy) was determined as described elsewhere 33 with certain modi cations. The fat, protein, moisture, and ash contents were evaluated by the Folch method, Kjeldahl method, atmospheric heating drying method and by direct ashing method, respectively; moreover, the carbohydrate content was assessed by subtracting the fat, protein, moisture and ash contents from the total amount. Energy content was calculated using modi ed Atwater factors (4, 9 and 4 kcal/g for protein, fat, and carbohydrate, respectively).

Design of clinical study
This was a randomised, double-blind, placebo-controlled, and parallel-group study carried out in a single clinical centre (Chiyoda Paramedical Care Clinic; CPCC, Tokyo, Japan) in Japan. Mie University Graduate School of Medicine and Konan Chemical Manufacturing together prepared the study protocol. All study procedures were undertaken by a clinical research organisation (CPCC) on consignment from Konan Chemical.

Subjects
Seventy-three healthy Japanese male and female volunteers (20-65 years) were selected from the total volunteers registered in the CPCC. The study details were disclosed to the subjects before enrolment, and the investigators obtained their written informed consent. Thereafter, the subjects underwent various tests (lifestyle questionnaire, medical interview, physiological, biochemical, and haematological tests). Each of these tests was performed at the CPCC. Subjects who did not meet the exclusion criteria, but met the inclusion criteria, were enrolled in the 2-week screening. Selected subjects recorded their defaecation status (number of defaecations, date of defaecation, con rmation of how many defaecations were collected on the day of defaecation, Bristol scale (faecal properties), amount of defaecation, colour of stool, smell of stool, refreshing feeling at the time of defaecation and stomach condition (abdominal pain, swelling, gurgling, bloating, atulence, nausea) in a diary. Health adults aged 20-65 years who had relatively low defaecation frequencies (3-5 times/week) were selected for this study. There were 15 exclusion criteria, as described in Table S2. Eventually, 38 subjects were selected as the study subjects.

Randomization
Subjects were randomly allocated into the RS or placebo group to balance the sex, age, faecal frequency and BSS. Allocation was operated by a researcher of the CPCC who was not involved in taking measurements and analysis.

Blinding
In total, 100 mg of RS (Rhamnox®) was lled in a cellulose white capsule (Matsuya, Osaka, Japan). The placebo involved an empty capsule, identical in appearance and avour, and was then provided to CPCC with a coded name. Correspondence of the coded name and the true name of the product was disclosed to CPCC after completion of data analysis.

Study protocol
After a 3-week screening, 19 and 19 participants were allocated to the RS or placebo group, respectively. Each participant ingested 1 capsule/day for 2 weeks. After the end of the trial, no participant experienced adverse events. Participants recorded their life survey and defaecation questionnaire in diary every day during the study period. The test schedule is illustrated in Figure S1.

Data collection
The primary outcomes were defaecation frequency, defaecation date and Bristol Scale. The secondary outcomes were gut microbiota and faecal condition (faecal odour, colour, volume and feeling after defaecation). Faecal frequency and condition were assessed by recording the defaecation times and faecal condition every day in a diary. Analysis exclusion criteria are described in Table S3; however, no subject was excluded from the analysis.

Faecal sample collection and DNA extraction
Faecal samples were collected using a guanidine thiocyanate solution (Faeces Collection kit; Techno Suruga Lab, Shizuoka, Japan). After vigorous mixing, the samples were stored at 4°C for a maximum of 7 days until DNA extraction. After homogenisation with lysis solution F (Nippon Gene, Tokyo, Japan), the genomic DNA was heated at 65°C for 10 min and puri ed from the supernatants using the MPure Bacterial DNA Extraction Kit (MP Biomedicals, Solon, OH, USA) with MPure-12 system (MP Biomedicals).
The puri ed DNA was quanti ed using Synergy LX (BioTek Instruments, Winooski, VT, USA) and

Analysis of bacterial composition in 16S rRNA datasets
The paired-end reads of the 16S rRNA gene were assembled using QIIME2 (ver. 2020.2), with the default parameter values, were applied for sequence de-noising, primer sequence trimming and chimaera checking using the DADA2 method 34,35 . Quality ltered reads were assigned to operational taxonomic units (OTUs) (100% identity) using de novo OTU picking and taxonomic assignment using the feature-

Safety assessment
The principal investigator assessed the safety of RS based on the results of participant communication, tests (physiological, biochemical, haematological) and urinalyses. The daily diary content was also referred to for safety assessment.
Sample size Although RS was tested on subjects with constipation, no testing was performed on these healthy subjects; hence, we were unable to estimate the minimum number of subjects. Thereafter, we set the minimum number of subjects to 19 for statistical analysis.

Statistical analysis
All results were represented as means with standard deviations (SD). Data were analysed using Student's t-test or one-way ANOVA with the Dennett multiple comparison procedure, depending on the number of comparisons, using GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA) or IBM SPSS software (IBM, Armonk, NY, USA).  Tables 2 and 3