Novel composition for increasing NAD+ in blood

Abstract

[Problem] To provide a composition for increasing NAD+ in blood. [Solution] The present invention is achieved by a composition for increasing NAD+, the composition containing rhamnan sulfate. The composition is preferably a composition for ameliorating dementia.

Description

Novel composition for increasing blood NAD+

[0001] The present invention relates to novel compositions for increasing blood NAD+, particularly those containing rhamnan sulfate.

[0002] Seaweed is rich in essential minerals, vitamins, dietary fiber, polyunsaturated fatty acids, and polysaccharides, and is known to be beneficial for maintaining health. Seaweed consumption has been associated with various beneficial effects on human health, including antioxidant and anti-inflammatory effects, immune support, vascular health, anti-obesity, and improved gut microbiota. Various functional substances have been discovered in seaweed, and their efficacy has been confirmed in various animals, including humans. Among these functional substances, polysaccharides have been extensively studied for their functional properties and physiological activities. For example, fucoidan is known to have anti-cancer properties. Fucoidan and carrageenan also possess anti-obesity properties due to a decrease in the Bacteroides / Firmicutes ratio, a marker related to gut microbiota, and an increase in specific gut bacteria in obese individuals.

[0003] Ulva crustifolia (Ulva crustifolia), commonly known as Ulva crustifolia (Ulva crustifolia), is a type of green algae belonging to the Chlorophyta phylum. Ulva crustifolia is used in many dishes, including tempura, miso soup, and tsukudani (simmered food in soy sauce and soy sauce). Its mild flavor and high nutritional value, including essential vitamins, minerals, and dietary fiber, are well-known. Ulva crustifolia contains rhamnan sulfate (RS), a type of sulfated polysaccharide. RS has attracted attention for its anticoagulant, antiviral, and anti-inflammatory properties. Furthermore, its inhibitory effects on hepatic steatosis and lipid reduction have been demonstrated in zebrafish models, and human clinical trials have shown that RS improves constipation by altering the intestinal microbiota (Non-Patent Document 1). The present inventors have demonstrated that RS administration improves constipation through alterations in the intestinal microbiota (Non-Patent Document 1).

[0004] NAD+ (nicotinamide adenine dinucleotide) is an essential component (coenzyme) in many biological oxidation-reduction reactions and plays a central role in oxidative phosphorylation. The following health benefits are expected from NAD+ intake: (1) Improved energy metabolism: It plays an important role in cellular energy production and may contribute to improving overall energy levels; (2) Slowing the aging process: It is involved in the cellular aging process and may have rejuvenating and longevity-related effects; (3) Supporting DNA repair and cellular health: It helps repair DNA damage and contributes to maintaining cellular health; (4) Neuroprotective effects: It may protect neurons and help maintain and improve cognitive function; (5) Supporting cardiovascular health: It may contribute to reducing the risk of heart disease; and (6) Blood sugar control: It may help manage blood sugar levels by improving insulin sensitivity and streamlining energy metabolism. Focusing on these health benefits, various supplements containing NMN, a precursor to NAD+, are commercially available.

[0005] Currently, numerous issues and concerns have been raised regarding the use of NAD+ supplements. Due to a lack of data regarding long-term safety and side effects, supplements should be used with caution. Particular attention should be paid to potential interactions with existing health conditions and other medications. The quality and purity of NAD+ supplements on the market vary, and some products may not contain the advertised ingredients or the recommended amounts. Furthermore, there are no clear guidelines regarding the appropriate dosage of NAD+ supplements, raising concerns about the health risks of overdosing. Many NAD+ supplements are expensive, placing a significant financial burden on some consumers.

[0006] JP 2023-159833 A JP 2021-191257 A JP 2019-189563 A

[0007] Shimada, Y.; Terasawa, M.; Okazaki, F.; Nakayama, H.; Zang, L.; Nishiura, K.; Matsuda, K.; Nishimura, N. Rhamnan sulphate from green algaeMonostroma nitidum improves constipation with gut microbiome alteration in double-blind placebo-controlled trial. Sci. Rep. 2021, 11, 13384.

[0008] Although RS has been known to have functions such as improving brain function (Patent Document 1), relieving constipation (Patent Document 2), and antiviral activity (Patent Document 3), the history of research on RS is not long, and there is still room for further research into the physiological effects of RS. The present invention has been made in view of the above circumstances, and its object is to provide a novel composition containing rhamnan sulfate, particularly a novel composition that increases blood NAD+.

[0009] Thus, the composition for increasing NAD+ according to the present invention is characterized by containing rhamnan sulfate. Furthermore, the composition for improving insulin resistance is characterized by containing rhamnan sulfate. In this case, a composition for improving dementia containing the composition can be provided. The composition of the present invention can be provided as a pharmaceutical or quasi-drug. It can also be provided as a food or beverage. The daily dosage of rhamnan sulfate is preferably 10 mg to 1000 mg, more preferably 30 mg to 500 mg, and even more preferably 50 mg to 300 mg. The composition of the present invention can be provided by combining an amount of rhamnan sulfate effective for improving insulin resistance or increasing NAD+ with a pharmaceutically acceptable carrier or additive. This composition can also be provided as a pharmaceutical or quasi-drug. The pharmaceutical composition can be used internally or externally. This pharmaceutical composition can be used in the form of an oral preparation, an injection such as an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, and / or an intraperitoneal injection, a transmucosal application preparation, a transdermal application preparation, or the like. In particular, rhamnan sulfate is effective when administered orally or transdermally, and is therefore preferably used as an oral agent, a transmucosal agent, or a transdermal agent.

[0010] The dosage form of the pharmaceutical composition can be appropriately determined, and examples thereof include solid preparations such as tablets, granules, capsules, powders, and dusts; liquid preparations such as solutions and suspensions; and semi-solid preparations such as ointments and gels. As for food and beverage products, rhamnan sulfate can be incorporated into various foods as a food ingredient to produce food compositions. Examples of food compositions include general foods, as well as foods for specified health uses, foods with nutrient functions, foods with functional claims, foods for hospital patients, and supplements. It can also be used as a food additive. Examples of food compositions include beverages (soft drinks, alcoholic beverages, carbonated beverages, dairy beverages, fruit juice beverages, tea, coffee, energy drinks, concentrated beverages, etc.), powdered beverages (powdered juice, powdered soup, etc.), confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chocolate, etc.), bread, cereal, seasonings, etc.

[0011] Foods for specified health uses, foods with nutrient functions, and foods with functional claims can also be provided in the form of capsules, lozenges, syrups, granules, powders, etc. Foods for specified health uses are foods containing functional health ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. In the present invention, foods can be sold with specific uses such as improving insulin resistance, increasing NAD+, or improving dementia. Foods with nutrient functions are foods used to supplement nutrients (vitamins and minerals) and display the function of the nutritional ingredients. To be sold as a food with nutrient functions, the amount of nutrients contained in the recommended daily intake must be within specified upper and lower limits, and not only nutritional function claims but also warning labels must be included. Foods with functional claims are foods that display scientifically based functionality at the responsibility of the business operator. Information on safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale. NAD+ refers to the oxidized form of nicotinamide adenine dinucleotide.

[0012] Examples of aerobic respiration reactions involving NAD+ include (1) the reaction in the Embden-Meyerhof pathway that produces 1,3-diphosphoglycerate and NADH from glyceraldehyde triphosphate and NAD+ (EC1.2.1.12), and the reaction in the citric acid cycle that produces acetyl-CoA, NADH, and CO2 from pyruvate, SH-CoA, and NAD+ (EC1.8.1.4). (2) the reaction in the citric acid cycle that produces α-ketoglutarate, NADH, and CO2 from isocitrate and NAD+. (EC 1.1.1.41), the reaction from α-ketoglutarate, NAD+, and SH-CoA to produce succinyl-CoA, NADH, and CO2 (EC 1.2.4.2), the reaction from malate and NAD+ to produce oxaloacetate and NADH (EC 1.1.1.37), and (3) the reaction during beta-oxidation that produces acetyl-CoA, FADH2, NADH, and H+ from palmitoyl-CoA, CoA, FAD, NAD+, and HO. Therefore, increasing NAD+ can speed up the above reactions.

[0013] In addition, pathways for oxidizing NADH to NAD+ include (1) the oxidation pathway during aerobic respiration, which produces NAD+, H+, and e- from NADH; and (2) the oxidation pathway during anaerobic respiration, which produces lactate and NAD+ from pyruvate and NADH (EC 1.1.1.27), ethanol and NAD+ from acetaldehyde and NADH (EC 1.1.1.1), and glycerol 3-phosphate and NAD+ from dihydroxyacetone phosphate and NADH (EC 1.1.1.8). The present invention, due to its NAD+-increasing effect, may activate one of these pathways. Compounds that increase NAD+ in the brain have been shown to suppress neuroinflammation and thereby reduce neurological damage (https: / / www.kanazawa-u.ac.jp / rd / 92323). Additionally, multiple NAD+-dependent enzymes are known to be involved in synaptic plasticity and neuronal stress resistance (https: / / www.cell.com / cell-metabolism / fulltext / S1550-4131(19)30502-9). Therefore, the present composition may be able to reduce brain inflammation by increasing blood NAD+. The present invention contains rhamnan sulfate as an active ingredient and is used as a food for specified health uses, a food with nutrient functions, or a food with functional claims for healthy individuals, frail individuals, frail individuals with unclear illnesses but who are not considered healthy, and the elderly.

[0014] The present invention provides a composition containing rhamnan sulfate for increasing NAD+. Ingesting rhamnan sulfate increases blood NAD+, and health benefits equivalent to those of NAD+ supplements can be expected. Data on the long-term safety and side effects of rhamnan sulfate are available, establishing its safety. Rhamnan sulfate is manufactured in a standardized manner, allowing it to be supplied with consistent quality and purity. Overdose testing has demonstrated that the intake amount poses no health risks, making it safe. Furthermore, because it is a seaweed extract, it is relatively inexpensive. In this way, the problems associated with NAD+ supplements can be resolved by ingesting rhamnan sulfate and increasing NAD+.

[0015] These graphs show (A) food intake, (B) blood triglycerol levels, and (C) blood total cholesterol levels in mice fed rhamnan sulfate. In the figures, "*" indicates a significant difference (p<0.05, "**" indicates a significant difference (p<0.01), "***" indicates a significant difference (p<0.001), and "****" indicates a significant difference (p<0.0001). Furthermore, when p<0.05 is greater, a significance level (p) is indicated (the same applies to all figures). These graphs show (A) fasting blood glucose (FBG), (B) fasting blood insulin (FBI), and (C) homeostasis model assessment index for insulin resistance (HOMA-IR) in mice fed rhamnan sulfate. These graphs show (A) NAD+ and (B) NAD+ / NADH ratio measurements. These graphs show (A) Observed, (B) Chao1 index, and (C) Firmicutes / Bacteroidetes ratio (F / B ratio) in mice fed rhamnan sulfate.

[0023] Figure 1 is a diagram showing the citric acid cycle.

[0024] Figure 2 is a diagram showing the glycolysis / gluconeogenesis pathway.

[0025] Figure 3 is a diagram showing the nicotinic acid and nicotinamide metabolic pathways.

[0026] Figure 4 is a graph showing the results of measuring the amounts of seven types of short-chain fatty acids.

[0027] Figure 5 is a heat map of the intestinal microbiota.

[0016] Graphs showing (A) body weight change and (B) weight gain rate when mice were fed a normal diet (ND), a normal diet containing rhamnan sulfate (RS) (NS+RS), a high-fat diet (HFD), and a high-fat diet containing RS (HFD+RS). (A) Time series changes in fasting blood glucose levels, (B) fasting blood glucose levels during the final week of the study. (A) Fasting blood insulin and (B) Homeostasis Model Assessment Index for Insulin Resistance (HOMA-IR). (A) Blood triglycerides (TG), (B) blood total cholesterol (TCHO), and (B) food intake. (A) Liver weight, (B) visceral fat weight (eWAT weight), and (C) visceral fat (VAT). (A) NAD+ and (B) NAD+ / NADH ratios were measured. (A) Subcutaneous fat volume, (B) Body fat, (C) Representative photographs of subcutaneous fat and body fat in each group examined by 3D-microCT.

[0017] 23 is a flowchart showing the tracking of test participants in this embodiment in Test 3.

[0049] FIG. 24 is a bar graph showing the results of scores where significant differences were observed between the test group and the placebo group. (A) shows a bar graph showing the mean ± standard deviation of the actual data, and (B) shows a bar graph showing the mean ± standard deviation of the difference data (the same applies to Figures 19 to 23).

[0050] FIG. 24 is a graph showing the results of standardized score data for NCI. (B) shows the results of standardized score data for reaction time. (C) shows the results of standardized score data for overall attention. (D) shows the results of standardized score data for cognitive flexibility. (E) shows the results of standardized score data for executive function. (F) shows the results of standardized score data for logical thinking. (G) shows the results of measuring NAD+ and (B) the NAD+ / NADH ratio in skeletal muscle. (G) shows the results of measuring NAD+ and (B) the NAD+ / NADH ratio in the large intestine.

[0018] Next, embodiments of the present invention will be described with reference to the accompanying drawings. However, the technical scope of the present invention is not limited to these embodiments, and various embodiments can be implemented without departing from the spirit of the invention. <Test Method 1> <Preparation of Rhamnan Sulfate> Any naturally occurring rhamnan sulfate (RS) can be used. In this embodiment, rhamnan sulfate obtained by hot water extraction from Single-stranded algae was used. Dried seaweed was washed with water, extracted with hot water, and the resulting hot water extract was filtered to obtain an extract containing RS as the main component. This was used as the sample in this embodiment. The molecular weight of RS was determined by gel permeation chromatography (GPC) using a Shodex RI-71 refractive index detector (Showa Denko K.K.) and a Shodex SB-806M HQ column (8.0 x 300 mm). Rhamnan sulfate can also be prepared by methods other than those described above. The raw material is not limited to Single-stranded algae, but can also be Ulva pertusa (Ulva radiata) or Ulva ribbon (Ulva ribbon). In the following animal tests, RS with a purity of 95% or higher was used.

[0019] Animals: BALB / c mice were purchased from SLC Japan and housed at the Mie University Laboratory Animal Research Institute under a 12-hour light / dark cycle. Five-week-old male mice were divided into two groups of six and fed either a CE-7 standard diet (ND: CLEA Japan) or a CE-7 diet supplemented with RS (250 mg / kg body weight) for 12 weeks. Body weight and food intake were measured weekly during the feeding experiment. For blood chemistry tests, mice were fasted for 14 hours before blood collection. All animal experiments were approved by the Ethics Committee of Mie University and conducted in accordance with the "Act on the Welfare and Management of Animals" (Ministry of the Environment) and in accordance with international guidelines (Animal Experimentation Committee approval number: 28-4-3). After euthanasia with carbon dioxide gas, the cecum was removed and its contents were used for 16s rRNA sequencing.

[0020] Blood chemistry analysis: Blood glucose levels were measured using a handheld Lab Glucometer (Foracare Japan). Blood triglycerides (TG) and total cholesterol (TCHO) were measured using Wako L-type TG and Wako L-type TCHO (Fujifilm Wako Pure Chemical Industries, Ltd.). Insulin concentrations were measured using a mouse insulin ELISA kit (Mercodia AB) according to the manufacturer's protocol. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by dividing the product of fasting insulin (μU / mL) and glucose (mmol / L) by 22.5. Analysis of SCFAs in Mouse Feces. Fecal organic acid concentrations were measured using a JASCO HPLC system equipped with a UV-4070 detector (JASCO), a guard column (KC-G 6B, Resonac), and two separation columns (Shodex Ionpack KC-811, Resonac). Organic acids were separated by post-column separation at 60°C. The HPLC mobile phase used was 3 mM perchloric acid at a flow rate of 1.0 mL / min. For the post-column reaction, a 125 mg / L BTB solution containing 5.3 g / L NaHPO was used at a flow rate of 1.2 mL / min. Fecal samples were stored at -80°C until analysis. Fecal samples (100–120 mg) were suspended in 400 μL of PBS(-) and homogenized. After centrifugation at 15,000 rpm and 4°C for 10 minutes, 200 μL of the supernatant was collected, and 200 μL of 5% perchloric acid was added to remove proteins. This was then centrifuged at 15,000 rpm and 4°C for 10 minutes. Next, 200 μL of the supernatant was collected and added to 800 μL of mobile phase. The mixed supernatant was filtered through a membrane filter (0.45 μm), and 50 μL of the solution was analyzed by HPLC.

[0021] Genomic DNA Extraction: Fecal samples from mouse cecums were freeze-dried using a VD-250R freeze dryer (Tytec) and homogenized using a Multi-Beads Shocker (Yasui Kikai). After homogenization, lysis solution F (Nippon Gene) was added, heated at 65°C for 10 minutes, centrifuged (12,000 × g, 2 minutes), and the supernatant was collected. Genomic DNA was then isolated using the Lab-Aid 824s DNA Extraction Kit (Zesan) according to the manufacturer's protocol. 16S rRNA Sequencing: Illumina MiSeq paired-end sequencing of the hypervariable V3-V4 region of 16S rRNA was performed at Bioengineering Lab (Kanagawa, Japan). DNA content was determined using a Synergy LX (Biotech) and a QuantiFluor dsDNA System (Promega). A two-step tail PCR method was used for 16S metagenomic sequencing library preparation (Illumina) according to the protocol. Indexed libraries were analyzed on a fragment analyzer using the dsDNA 915 Reagent Kit (Advanced Analytical Technologies). The prepared libraries were used for paired-end sequencing using a MiSeq (Illumina) equipped with MiSeq v3 reagents and 2 × 300 bp reads.

[0022] Bacterial composition analysis in the 16S rRNA dataset: Paired-end reads of the 16S rRNA gene were assembled using the DADA2 method in QIIME2 (ver. 2022.8). Qualitatively processed reads were processed for operational taxonomic units (OTUs) (100% identity) using de novo OTU picking and taxonomic assignment using the Feature Classification plugin against the EzBioCloud 16S database (https: / / www.ezbiocloud.net / ). Microbiome analysis: Alpha diversity analysis using the Observed index and Chao1 index was performed in QIME2 with default parameters. PICRUST2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, version 2.3.0b) was used to predict functional gene products of the fecal microbiota based on classifications obtained from the METACYC pathway database. GraphPad Prism version 9.5.1 (GraphPad Software) was used to calculate Spearman correlations between the top 50 bacteria and phenotypic changes, and the circlize (version 0.4.15) and ComplexHeatmap (version 2.16.0) packages in R were used for visualization. Statistical analysis: All results are presented as mean ± standard deviation (SD). Data were analyzed by Student's t-test using GraphPad Prism version 8 (GraphPad Software). A significance level of 5% (p < 0.05) was considered significant.

[0023] <Test Results 1> <Effect of RS on Insulin Resistance in Mice> Figure 1 shows the results of measurements of food intake, blood triglycerides (TG), and blood total cholesterol (TCHO) in mice fed a rhamnan sulfate-containing diet. The RS group significantly (p<0.0001) increased food intake compared with the control group (Figure 1(A)). Regarding blood lipids, triglycerides (TG) did not change with RD feeding (Figure 1(B)), but total cholesterol (TCHO) significantly (p<0.05) decreased with RS (Figure 1(C)). Figure 2 shows the results of measurements of fasting blood glucose (FBG), fasting blood insulin (FBI), and homeostasis model assessment index for insulin resistance (HOMA-IR). RS significantly (p<0.05) decreased FBG compared with the control group (Figure 2(A)). Furthermore, RS tended to decrease FBI (Figure 2(B)) and significantly (p<0.0001) reduced HOMA-IR (Figure 2(C)). Thus, RS demonstrated hypoglycemic properties that improved insulin resistance. Figure 3 shows the results of measurements of NAD+ (Figure 3(A)) and the NAD+ / NADH ratio (Figure 3(B)). Although no statistically significant differences were observed, RS administration tended to increase NAD+. Because the fluctuations in NAD+ and NADH are biochemically very drastic, significant differences require considerable technical care. Therefore, a difference of this magnitude is sufficient to physiologically indicate an increase in NAD+.

[0024] <Effect of RS on Microbiota Changes> At level 6 (genus), of the 166 bacteria detected, the number of significantly altered bacteria decreased by 9 (5.4%) and increased by 8 (4.8%) (p<0.05; Table 1). At level 7 (species), of the 403 bacteria detected, the number of significantly altered bacteria decreased by 27 (6.7%) and increased by 22 (5.5%) (p<0.05; Table 2). RS significantly (p<0.05) decreased the α diversity of the Observed and Chao1 indices (Figures 4(A) and 4(B)). The Firmicutes / Bacteroidetes ratio (F / B ratio), a marker related to gut microbiota dysbiosis in obese individuals, was significantly (p<0.01) decreased by RS feeding (Figure 4(C)).

[0025]

[0026]

[0027] <Function Predictions from Altered Gut Microbiota> To confirm the effects of RS on the metabolic functions of the gut microbiota, bacterial metagenomes were predicted using PICRUSt2. Each predicted bacterial protein was classified into KEGG ortholog (KO) entities, and 4,851 entities were identified across all samples. Of these, 510 KOs were significantly upregulated (p<0.05) and 64 KOs were significantly downregulated (p<0.05) in the RS-treated group. To identify altered pathways, differentially expressed KOs were mapped using KEGG Mapper. As a result, 199 pathways were identified, and the ratio of the number of KOs involved in RS for each pathway to the total number of KOs for that pathway was calculated. Pathways with an occurrence rate of 5% or higher were extracted (Table 3). Pathway entries were obtained from the KEGG TATHWAY Database (https: / / www.genome.jp / kegg / pathway.html). The citric acid cycle (TCA cycle) is shown in Figure 5, the glycolysis / gluconeogenesis pathway in Figure 6, and the nicotinic acid and nicotinamide metabolic pathways in Figure 7.

[0028]

[0029] Correlations between RS-Induced Gut Microbiota, Body Weight Change, Blood Chemistry, and Short-Chain Fatty Acids. The fecal contents of seven short-chain fatty acids (SCFAs) were measured in the control and RS-fed groups. As shown in Figure 8, acetate and propionate levels were significantly increased by RS (p<0.0001 and p<0.05, respectively). Next, the relationship between body weight change, blood TG, blood TCHO, FBG, FBI, HOMA-IR, and fecal SCFA levels and the top 50 most abundant genera in all samples was analyzed using Spearman correlation coefficients. In Figure 9, bacteria marked with stars were highly correlated with each phenotype (blood chemistry and fecal SCFAs) (*, p<0.05, **, p<0.01, ***, p<0.001). In particular, bacteria located in the lower right corner of the heatmap showed a high correlation with glycemic control and fecal acetate levels.

[0030] <Test Method 2> NSY / Hos male mice (n = 24) were used. These mice were randomly divided into a group receiving a normal diet (ND) without RS or a group receiving a normal diet (ND_RS) containing 0.25% RS in addition to ND. They were then further divided into four groups (n = 6 per group): the ND control group (ND Control), the RS-treated ND group (ND RS), the high-fat diet control group (HFD Control), and the high-fat diet RS group (HFD RS). The RS dose was 250 mg / kg body weight / day for 10 weeks. Mice were purchased at 6 weeks of age and raised to 12 weeks of age, then housed three per cage (with feces collected individually in small cages). During the RS diet administration, body weight, food intake, and fasting blood glucose levels were measured weekly. Ten weeks after the start of the study, blood samples were collected and analyzed for FBG, TG, TCHO, and blood NAD / NADH levels using a Biovision kit (https: / / www.biovision.com / documentation / datasheets / K337.pdf). After the study, animals were observed using 3D-microCT. Intestinal (cecal) feces were collected for analysis of gut microbiota and organic acids. Liver weight, visceral fat weight (eWAT weight), and visceral adipose tissue volume (VAT volume) were also measured. Livers were then collected and fixed in RNAlater, T-PER, and PFA. Analysis was performed using qPCR, RNA-seq, WB, histology, and fluorescent immunohistochemistry (FIHC).

[0031] <Test Results 2> Figure 10 shows the weight change (A) and weight gain rate (B) for the four groups. The HFD group showed greater weight gain than the ND group. In contrast, weight gain was suppressed in the RS-fed group. In particular, weight gain was significantly suppressed in the HFD+RS group compared to the HFD group. As shown in Figure 11, fasting blood glucose levels increased significantly in the HFD group compared to the ND group from week 6. In contrast, the HFD+RS group showed a significant suppression of fasting blood glucose levels, similar to the ND group. Similarly, as shown in Figure 12, fasting blood insulin levels increased significantly in the HFD group compared to the ND group, whereas the HFD+RS group showed a significantly suppressed increase in fasting blood insulin levels compared to the HFD group. As shown in Figure 13, the HFD group showed significantly increased TG and TCHO compared to the ND group. In contrast, the HFD+RS group showed significantly decreased TG and TCHO compared to the HFD group. Furthermore, feeding RS did not affect food intake. As shown in Figure 14, liver weight, eWAT weight, and VAT volume were significantly increased in the HFD group compared with the ND group. In contrast, liver weight was significantly decreased in the HFD+RS group compared with the HFD group. Furthermore, as shown in Figure 15, NAD+ and the NAD+ / NADH ratio were significantly increased in the RS-fed groups (ND+RS and HFD+RS) compared with the ND and HFD groups. As shown in Figure 16, subcutaneous fat and body fat were significantly increased in the HFD group compared with the ND group. In contrast, subcutaneous fat volume was significantly decreased in the HFD+RS group compared with the HFD group. Thus, RS administration for more than 10 weeks reduced fasting blood glucose and plasma TCHO, as well as improved insulin resistance. Functional analysis of the gut microbiota revealed that glycolysis, the TCA cycle pathway, and the nicotinic acid and nicotinamide metabolic pathways were correspondingly activated, and RS administration was found to increase NAD+ and the NAD+ / NADH ratio.

[0032] <Study Method 3> 1. Subjects (1) Eligibility Criteria The enrollment criteria for subjects were healthy Japanese people (both males and females) between the ages of 40 and 65. Exclusion criteria included those currently undergoing treatment for or with a history of malignant tumors, heart failure, or myocardial infarction; those with a pacemaker or implantable cardioverter defibrillator; those undergoing treatment for arrhythmia, liver damage, kidney damage, cerebrovascular disease, rheumatism, diabetes, dyslipidemia, hypertension, or other chronic diseases; those regularly consuming foods for specified health uses, foods with functional claims, or other foods or beverages thought to have functional properties; those regularly consuming seaweed or products containing seaweed-derived ingredients; those regularly using medicines (including herbal medicines) or supplements; those with allergies (to medicines or foods related to the test food); those who are pregnant, breastfeeding, or intend to become pregnant during the study period; those who have participated in other clinical trials in the 28 days prior to obtaining consent or plan to participate during the study period; smokers; and those deemed inappropriate by the principal investigator for this study.

[0033] (2) Management of subjects When subjects visited the hospital, their physical condition was ascertained through interviews. During the study period, they recorded daily in a diary designated by the contract clinical trial organization whether they had taken the test food and whether they had their period (women only). The diaries were submitted by mail every week, and if there was a diary that had not been submitted at the time of the final examination, they brought it with them and submitted it at the time of the visit. The diary from the final examination was submitted at the time of the visit. If it could not be submitted at the time of the visit, it was filled out on the spot. Subjects were asked to strictly adhere to the following points while participating in the study. (a) consume the test food according to the prescribed usage and dosage, (b) consume the test food so that the intake rate is 80% or more, (c) avoid overeating and drinking from the date of obtaining the consent form for the study until the final test (the test 8 weeks after intake) and do not change your previous lifestyle habits, (d) refrain from drinking alcohol or engaging in excessive exercise from the day before each test until the end of the test on the day, (e) refrain from eating or drinking for 6 hours before blood sampling (this also includes the intake of test food. However, only water was permitted; functional water and tea were not permitted), (f) if any changes in your physical condition occur during the test period, immediately contact the contracted clinical trial organization and ask for instructions on how to proceed, and (g) avoid, as much as possible, consuming foods for specified health uses, foods with functional claims, or other foods / drinks that are thought to have functional properties during the test period.

[0034] (3) Dropout / Discontinuation Policy Subjects who met any of the following criteria were considered to have dropped out of the study. Safety assessments were conducted on an individual basis. (a) The study was discontinued for the subject's own convenience; (b) The subject was found not to have followed the instructions of the investigator or study organizer; (c) The subject significantly deviated from the protocol; or (d) The investigator otherwise deemed it appropriate to drop the subject. Furthermore, subjects who met any of the following criteria were considered to have been discontinued: (a) A serious adverse event occurred, and the investigator determined that the subject's participation in the study should be discontinued; (b) The investigator determined that objective symptoms made it difficult for the subject to continue the study; or (c) The investigator otherwise determined that it was difficult to continue the study. The study was also discontinued if an unexpected serious adverse event occurred, or if a serious violation or non-compliance with ethical guidelines or the protocol occurred.

[0035] 2. Test Food The test food group used a seaweed-derived extract (containing rhamnan sulfate), and the placebo group used a placebo containing no seaweed-derived extract. 3. Test Period The test food or placebo was taken for 8 weeks. Cognitive function was checked twice, before and after intake. 4. Dosage The dosage was as follows: Test food group: 1 capsule per day Placebo group: 1 capsule per day However, the daily dose was to be taken on the same day, and if a dose was missed, it was to be taken as soon as remembered. The test food was stored at room temperature, avoiding direct sunlight, high temperatures and humidity. The test food was manufactured by Konan Chemical Co., Ltd. The test foods were indistinguishable in appearance, shape, color, odor, and taste. The composition of the test food (per capsule) was as follows: the test food was a capsule containing 120 mg of rhamnox (containing approximately 90 mg of rhamnan sulfate) and a 60 mg cellulose capsule, and the placebo was a capsule containing 120 mg of dextrin and a 60 mg cellulose capsule.

[0036] 5. Measurement Items The following cognitive function tests were measured. (1) Ten tests (verbal memory test, visual memory test, finger tapping test, SDC test, Stroop test, attention shifting test, sustained processing test, facial expression recognition test, logical reasoning test, and four-part sustained processing test) were conducted using Cognitrax. (2) Standardized scores for overall memory and standardized scores for other cognitive domains (Neurocognitive Index (NCI), verbal memory, visual memory, cognitive function speed, reaction time, overall attention, cognitive flexibility, processing speed, executive function, social cognition, logical reasoning, working memory, sustained attention, simple attention, and motor speed) were used. (3) Evaluation methods included screening and pre-intake tests and an 8-week post-intake test. (4) Physical measurements included height, weight, body fat percentage, and temperature. BMI was calculated by dividing weight (kg) by the square of height (m). These were conducted at screening and pre-intake tests and an 8-week post-intake test. However, height was measured only during the information session.

[0037] (5) In addition, as part of the physical examination, blood pressure (systolic blood pressure, diastolic blood pressure) and pulse rate were measured. In addition, urine tests (protein, glucose, urobilinogen, bilirubin, ketone bodies, pH, occult blood) and peripheral blood tests (white blood cell count, red blood cell count, hemoglobin, hematocrit value, platelet count, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), white blood cell picture (neutrophil rate, lymphocyte rate, monocyte rate, eosinophil rate, basophil rate, neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), AST (GOT), ALT (GPT), γ-GT (γ-GTP), ALP, LD (LDH), LAP, total bilirubin, direct bilirubin, indirect bilirubin, cholinesterase (ChE), total protein, urea nitrogen, creatinine, uric acid, CK, calcium, serum amylase, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides (TG: Neutral lipids), glycoalbumin, serum iron (Fe), sodium (Na), potassium (K), chloride (Cl), inorganic phosphorus (IP), glucose, hemoglobin A1c (HbA1c: NGSP), and nonspecific IgE were measured.

[0038] (6) Subject Recruitment, Allocation, and Double-Blinding The number of subjects to be screened was 15, the target number 10, and the number of subjects to be tested 12. Study participants were recruited through a monitor recruitment website operated by the contract clinical trial organization. The 12 subjects were divided into two groups of six and assigned to either the test food group or the placebo group. All subjects were assigned on the same day after the target number of registered subjects set in the protocol was reached. Random numbers were generated on a computer, and an allocation list was created using a completely random method with specified variables as factors. Study participants were enrolled based on the allocation list. The allocation ratio was 1:1. The study was conducted using a double-blind method. Blinding was achieved by using indistinguishable test foods. The test food shipping staff at the contract clinical trial organization confirmed the indistinguishability of the test foods and entered and confirmed the screening test data, then communicated the identification number to the person responsible for allocation.

[0039] (7) Data collection and management Physical measurement and physical examination data were entered into paper medical records by staff at the study institution. Questionnaires were completed by the study participants themselves on questionnaire forms, which were then collected by staff at the study institution. Paper-based measurement results (medical records and questionnaire forms) were obtained by the monitoring staff. Urine test and peripheral blood test results were obtained as ".txt" data (electronic data) from m-Line (http: / / www.medience.co.jp / mline / ), operated by LSI Medience Corporation. Data management was led by the contracted clinical trial organization. The ".csv" data collected by the monitoring staff and the electronic data obtained by m-Line were imported into Access and saved at the contracted clinical trial organization. Paper-based measurement results were entered into Microsoft Access by one staff member. The entered data was checked for errors by two staff members.

[0040] The medical institutions conducting the trial, institutions with ethical review committees, contract clinical trial organizations, and the sponsor agreed to keep documents that should be kept at their respective institutions for two years after the end of the trial. When storing documents, they were to be managed appropriately to prevent the leakage, mixing, theft, or loss of personal information. Furthermore, once the storage period had expired, they were to be anonymized and discarded. Biological samples (blood and urine) collected from the trial participants were to be used for no purpose other than measurement, and after measurement they were to be discarded as medical waste while remaining anonymized. The storage and disposal of the samples was outsourced to LSI Medience Corporation.

[0041] (8) Statistical methods The full registered population set consisted of cases who provided informed consent and were enrolled in the study. The full analysis set (FAS) was a group obtained by excluding cases in the full registered population set that met one or more of the following conditions: (a) cases who did not receive the allocated intervention, (b) cases who did not meet the conditions for the target population (cases with a confirmed diagnosis of some disease or cases that met clearly defined, objectively assessable important inclusion / exclusion criteria), (c) cases who never received the intervention after allocation, and (d) cases with no post-allocation data. The per protocol analysis set (PPS) was a group obtained by excluding cases in the FAS that met one or more of the following conditions: (a) Cases in which the intake rate of the test food was less than 80%, (b) cases in which actions that significantly undermined the reliability of the test results, such as missing diary records, were found to have met the exclusion criteria after enrollment in the study, (d) cases in which violations of compliance rules were found during the study period, (e) cases in which patients consumed food or medicines during the study period that are expected to significantly affect the test results, (f) cases in which patients engaged in activities that were significantly different from their lifestyle at the time of enrollment in the study, and (g) cases in which there were other clear reasons that made it appropriate to exclude the patient.

[0042] The safety analysis population (SAF) was a subset of the entire enrollment population, excluding cases who met one or more of the following criteria: (a) cases who did not receive the assigned intervention, (b) cases who never received the intervention after allocation, and (c) cases for which safety endpoints were never measured after allocation. Demographic data for study participants was collected from the entire enrollment population set or other analysis datasets. Gender was compared between groups using the chi-squared test, and other parameters were compared using the Student's t-test. For statistical analysis of efficacy endpoints, the PPS was used as the analysis dataset for the primary outcome. Means and standard deviations were presented, and intergroup comparisons were performed using ANCOVA with baseline as a covariate. If other tests were necessary from multiple perspectives, such as unpaired t-tests, they were also used to evaluate efficacy. The PPS was then used as the analysis dataset for secondary outcomes. Means and standard deviations were presented, and actual values ​​were compared between groups using ANCOVA with baseline as a covariate. The amount of change from the screening test and the amount of change from the pre-intake test were compared between groups using unpaired t-tests.If other tests were necessary from a multifaceted perspective other than the above analytical methods, they were adopted and their effectiveness was evaluated.

[0043] For statistical analysis of safety endpoints, the SAF was used in the analysis dataset as the primary safety endpoint. Occurring side effects and adverse events were tabulated for each participant. The incidence rates of side effects and adverse events were tabulated by group, and the 95% confidence intervals for the incidence rates and differences between groups were calculated. The incidence rates of side effects and adverse events in each group were compared using chi-square tests and other methods. The SAF was used in the analysis dataset as a secondary safety endpoint. The proportion of cases in which urine and peripheral blood test values ​​within the reference range at screening and pre-intervention testing fell outside the reference range after intervention was calculated, and inter-group comparisons were performed at each time point using chi-square tests. If other tests were necessary from multiple perspectives, they were also used to evaluate safety. For other safety endpoints, a data list was created for each participant. Additionally, the principal investigator or subinvestigator reviewed the safety endpoints on an individual basis to confirm that no medically significant changes occurred due to the intake of the test food. In addition, when other tests or stratified analyses were deemed necessary from a multifaceted perspective, such as at case conferences, appropriate other tests or stratified analyses were performed. All statistical analyses were performed using two-sided tests, with a significance level of 5%. SPSS Statistics version 23 or higher was used as the software, and other statistical software was used as necessary. This study focused on the primary outcome, and multiplicity that occurs in secondary outcomes set using multiple hypotheses was not taken into consideration.

[0044] <Study Results 3> The following abbreviations and full names were used: ITT: Intention to treat; PPS: Per protocol set; SAF: Safety analysis population; VISIT 1: Screening and pre-intake test (baseline); VISIT 2: 8-week post-intake test; Change: Change from screening and pre-intake test to 8-week post-intake test; Mean: Mean; SD: Standard deviation; SE: Standard error; 95% CI- and 95% CI+: 95% confidence interval. Figure 17 shows a flowchart of the follow-up of study participants. This study targeted healthy Japanese men and women aged 40 to 65 years. Of the 23 participants who agreed to participate in the study, 12 who met the eligibility criteria were enrolled in the study, with six participants assigned to the test food group and six to the placebo group. All participants completed the study, and no participants violated the compliance requirements. The analysis datasets were the PPS and SAF, which were determined at the time of study planning, and consisted of 12 participants each. Participant demographics are shown in Table 4.

[0045] The mean and SD of the cognitive function tests, the intergroup differences and their SE, 95% CI-, 95% CI+, and statistical analysis results are shown in Tables 5 and 6.

[0046]

[0047]

[0048] As shown in the table above, no significant differences were observed between the test food group and the placebo group in the standardized scores for overall memory, verbal memory, visual memory, cognitive speed, social cognition, working memory, sustained attention, simple attention, motor speed, and processing speed. In contrast, significant differences (P<0.05) were observed between the test food group and the placebo group in the standardized scores for NCI, reaction time, overall attention, cognitive flexibility, executive function, and reasoning. Specifically, the differences in VISIT 2 were P=0.004, P=0.049, P=0.043, P=0.019, and P=0.042, respectively. A trend toward improvement was also observed in reasoning (P=0.070).

[0049] Figures 18 to 23 show (A) bar graphs showing the mean ± standard deviation of the actual data and (B) bar graphs showing the mean ± standard deviation of the difference data for the NCI standardized scores, reaction time standardized score, overall attention standardized score, cognitive flexibility standardized score, executive function standardized score, and logical thinking standardized score. No significant differences were observed in the incidence of side effects, adverse events, or secondary safety endpoints between the subject groups. Additionally, individual anthropometric and physical examination data confirmed that no medically significant changes occurred with the intake of the test food.

[0050] <Test Method 4> Next, a similar test was conducted to replicate the results of "Test Method 2" above. The test method followed "Test Method 2." <Test Results 4> Figures 24 and 25 show the NAD+ and NAD+ / NADH ratios in skeletal muscle and colon. As shown in the figures, in the colon, the RS-administered group (HFD + RS) showed significantly increased NAD+ and NAD+ / NADH ratios compared to the HFD group. Furthermore, in skeletal muscle, the RS-administered group (HFD + RS) also showed a tendency for increased NAD+ and NAD+ / NADH ratios compared to the HFD group, although no significant difference was observed. Thus, the composition of this test was found to safely improve brain function. Thus, this embodiment provides novel compositions containing rhamnan sulfate, particularly compositions for increasing NAD+ and improving insulin resistance.

Claims

1. A composition for increasing blood NAD+ containing ramnansulfate.

2. A composition for improving insulin resistance containing ramnansulfate.

3. A composition for improving dementia containing the composition according to claim 1.

4. A pharmaceutical or quasi-drug containing the composition according to any one of claims 1 to 3.

5. A food or drink containing the composition according to any one of claims 1 to 3.

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