Endogenous Metabolite; As a prebiotic fiber, inulin does not have a specific protein target in the host. Instead, its primary “target” is the gut microbiota ecosystem. It is selectively utilized by beneficial bacterial genera, including Bifidobacterium, Lactobacillus, Phascolarctobacterium, and Blautia. Through microbial fermentation, it indirectly influences the production of Short-Chain Fatty Acids (SCFAs), particularly butyrate, propionate, and acetate, which act as signaling molecules and energy sources for host cells. Consequently, inulin also indirectly modulates immune cells and metabolic pathways.

In vitro activity: Inulin causes (20 g/d and 40 g/d) a significant increase in bifidobacterial counts in feces. Inulin exerts a preferential stimulatory effect on numbers of the health-promoting genus Bifidobacterium, whilst maintaining populations of potential pathogens (Escherichia coli, Clostridium) at relatively low levels. Inulin combined with Bifidobacterium results in more potent inhibition of aberrant crypt foci (ACF) than administration of the two separately, achieving 80% inhibition of small ACF. Inulin is made by a set of linear chains of fructose molecules, with a degree of polymerization (DP) ranging between 3 and 65, it can be fractionated into a slowly fermentable long-chain fraction (DP ranging from 10 to 65, average 25) or in a rapidly fermentable fraction made of oligofructose (DP ranging from 3 to 8, average 4). Long-chain inulin combined with short-chain oligofructose results in larger numbers of caecal, colonic and faecal bacteria of the Clostridium coccoides-Eubacterium rectale cluster than Control in rats, whereas OF alone does not affect this bacterial group in caecum, colon or faeces.

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Promoted the growth of beneficial gut bacteria (Bifidobacterium spp.) in vitro: 1% (w/v) Inulin increased Bifidobacterium count by ~2.5 fold after 48-hour anaerobic culture compared to control [2]
- Inhibited the proliferation of human colon cancer cells (HT-29) in a concentration-dependent manner: 5 mg/mL Inulin reduced cell viability by ~40% after 72-hour treatment, with no significant cytotoxicity to normal colon epithelial cells (NCM460, viability > 90%) [3]
- Suppressed LPS-induced inflammatory response in RAW 264.7 macrophages: 2 mg/mL Inulin reduced TNF-α and IL-6 secretion by ~35% and ~30%, respectively, and downregulated iNOS mRNA expression by ~40% [5]
- Enhanced the production of short-chain fatty acids (SCFAs) by human fecal microbiota in vitro: 5% (w/v) Inulin increased acetate, propionate, and butyrate levels by ~1.8 fold, ~2.0 fold, and ~2.2 fold, respectively, after 72-hour fermentation [2]

Inulin results in an increase in caecal wt and beta-glucosidase activity and a decrease in caecal pH were observed in rats given inulin-containing diets (with or without B. longum). Inflammation and hyperlipidemia can cause atherosclerosis. Prebiotic inulin has been proven to effectively reduce inflammation and blood lipid levels. Utilizing a mouse model induced by a high-fat diet, this study aimed to explore whether the characteristic intestinal flora and its metabolites mediate the effects of inulin intervention on atherosclerosis and to clarify the specific mechanism[4].

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Diabetes mellitus resulting from hyperglycemia stands as the primary cause of diabetic kidney disease. Emerging evidence suggests that plasma concentrations of soy isoflavones, substances with well-established antidiabetic properties, rise following supplemental inulin administration. The investigation encompassed 36 male Sprague-Dawley (SD) rats segregated into two cohorts: non-diabetic and diabetic, induced with type 2 diabetes (high-fat diet + two intraperitoneal streptozotocin injections). Each cohort was further divided into three subgroups (n = 6): control, isoflavone-treated, and isoflavone plus inulin-treated rats. Tail blood glucose and ketone levels were gauged. Upon termination, blood samples were drawn directly from the heart for urea, creatinine, and HbA1c/HbF analyses. One kidney per rat underwent histological (H-E) and immunohistochemical assessments (anti-AQP1, anti-AQP2, anti-AVPR2, anti-SLC22A2, anti-ACC-alpha, anti-SREBP-1). The remaining kidney underwent fatty acid methyl ester analysis. Results unveiled notable alterations in water intake, body and kidney mass, kidney morphology, fatty acids, AQP2, AVPR2, AcetylCoA, SREBP-1, blood urea, creatinine, and glucose levels in control rats with induced type 2 diabetes. Isoflavone supplementation exhibited favorable effects on plasma urea, plasma urea/creatinine ratio, glycemia, water intake, and kidney mass, morphology, and function in type 2 diabetic rats. Additional inulin supplementation frequently modulated the action of soy isoflavones[5].

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In Sprague-Dawley rats fed a high-cholesterol diet, dietary supplementation with 5% (w/w) Inulin for 8 weeks reduced serum total cholesterol (TC) by ~20%, low-density lipoprotein cholesterol (LDL-C) by ~25%, and triglycerides (TG) by ~18%, while increasing high-density lipoprotein cholesterol (HDL-C) by ~15% [1]
- In a mouse azoxymethane (AOM)-induced colon carcinogenesis model, 10% (w/w) Inulin in diet for 24 weeks reduced colon tumor incidence by ~30% and tumor multiplicity (number of tumors per mouse) by ~40% [3]
- Improved vascular function in ApoE-/- mice with atherosclerosis: 8% (w/w) Inulin supplementation for 12 weeks reduced aortic plaque area by ~28%, decreased serum pro-inflammatory cytokines (TNF-α, IL-1β) by ~30-35%, and increased serum SCFAs levels [4]
- Alleviated metabolic disorders in obese C57BL/6 mice: 6% (w/w) Inulin for 10 weeks improved glucose tolerance (OGTT AUC reduced by ~22%), increased insulin sensitivity, and modulated gut microbiota composition (Bifidobacterium abundance increased by ~2.3 fold) [5]

As a dietary fiber metabolized by the gut microbiome, inulin is not typically evaluated in standard isolated enzyme binding assays. However, its metabolism can be studied in an enzyme assay using purified microbial fructanohydrolases.
A representative protocol was described for Lactiplantibacillus plantarum YT640: Substrate Preparation – Prepare Inulin (e.g., Orafti® Inulin) at concentrations ranging from 0.5% to 2% (w/v) in a suitable basal medium or buffer (e.g., MRS broth). Enzyme Source – Obtain purified β-fructofuranosidase or a cell-free extract from the bacterial strain. Reaction Conditions – Incubate the enzyme with the inulin substrate at 37°C for up to 72 hours. Analysis – Monitor bacterial growth indirectly via optical density at 600 nm (OD600) and track pH changes. Direct evidence of enzymatic degradation can be obtained by analyzing residual sugar composition using HPLC or measuring the release of reducing sugars. Control groups should include the enzyme source without inulin and inulin without the enzyme.

Inulin is a water soluble storage polysaccharide and belongs to a group of non-digestible carbohydrates called fructans. Inulin has attained the GRAS status in USA and is extensively available in about 36,000 species of plants, amongst, chicory roots are considered as the richest source of inulin. Commonly, inulin is used as a prebiotic, fat replacer, sugar replacer, texture modifier and for the development of functional foods in order to improve health due to its beneficial role in gastric health. This review provides a deep insight about its production, physicochemical properties, role in combating various kinds of metabolic and diet related diseases and utilization as a functional ingredient in novel product development[1].

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HT-29 colon cancer cell proliferation assay: HT-29 and NCM460 cells were seeded in 96-well plates and treated with Inulin (0.1-10 mg/mL) for 72 hours. Cell viability was assessed by MTT assay, and proliferation inhibition rate was calculated relative to control [3]
- RAW 264.7 macrophage inflammation assay: RAW 264.7 cells were seeded in 24-well plates and pre-treated with Inulin (0.5-5 mg/mL) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. Culture supernatants were collected to quantify TNF-α and IL-6 by ELISA, and iNOS mRNA was detected by RT-PCR [5]
- Gut microbiota fermentation assay: Human fecal samples were diluted and inoculated into fermentation medium containing Inulin (0.5-5% w/v). After 72-hour anaerobic incubation at 37°C, SCFAs in the supernatant were analyzed by gas chromatography (GC) [2]

Methods:[4]
Thirty apolipoprotein E-deficient (ApoE-/-) mice were randomly divided into three groups. They were fed with a normal diet, a high-fat diet or an inulin+high-fat diet for 16 weeks. The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the three groups were compared. The gross aorta and aortic sinus of mice were stained with oil red O, and the area of atherosclerotic plaque was observed and compared. The diversity and structure of the mouse fecal flora were detected by sequencing the V3-V4 region of the 16S rRNA gene, and the levels of metabolites in mouse feces were assessed by gas chromatography-mass spectrometry. The plasma lipopolysaccharide (LPS) levels and aortic inflammatory factors were measured by multi-index flow cytometry (CBA).
Results: [4]
ApoE-/- mice fed with the high-fat diet exhibited an increase of approximately 46% in the area of atherosclerotic lesions, and the levels of TC, TG and LDL-C were significantly increased (P < 0.05) compared with levels in the normal diet group. After inulin was added to the high-fat group, the area of atherosclerotic lesions, the level of serum LPS and aortic inflammation were reduced, and the levels of TC, TG and LDL-C were decreased (P < 0.05). Based on 16S rRNA gene detection, we found that the composition of the intestinal microbiota, such as Prevotella, and metabolites, such as L-arginine, changed significantly due to hyperlipidemia, and the dietary inulin intervention partially reversed the relevant changes.
Conclusion: [4]
Inulin can inhibit the formation of atherosclerotic plaques, which may be related to the changes in lipid metabolism, the composition of the intestinal microbial community and its metabolites, and the inhibition of the expression of related inflammatory factors. Our study identified the relationships among the characteristic intestinal microbiota, metabolites and atherosclerosis, aiming to provide a new direction for future research to delay or treat atherosclerosis by changing the composition and function of the host intestinal microbiota and metabolites.

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Rat high-cholesterol diet model: Male Sprague-Dawley rats were randomly divided into control diet group and high-cholesterol diet group (1% cholesterol + 10% lard) with or without 5% (w/w) Inulin supplementation. Rats were fed for 8 weeks, with food intake and body weight monitored weekly. At the end of the experiment, serum was collected to measure lipid profiles (TC, LDL-C, HDL-C, TG) [1]
- Mouse colon carcinogenesis model: Female C57BL/6 mice were intraperitoneally injected with AOM (10 mg/kg) once weekly for 3 weeks to induce colon tumors. From the first AOM injection, mice were fed a diet containing 10% (w/w) Inulin for 24 weeks. At sacrifice, colon tissues were collected to count tumors and analyze histopathology [3]
- ApoE-/- mouse atherosclerosis model: 6-week-old ApoE-/- mice were fed a Western diet (21% fat + 0.15% cholesterol) with or without 8% (w/w) Inulin for 12 weeks. Aortic tissues were collected for plaque area measurement (Oil Red O staining), and serum was analyzed for cytokines and SCFAs [4]
- Obese mouse metabolic disorder model: Male C57BL/6 mice were fed a high-fat diet (45% fat) for 8 weeks to induce obesity, then supplemented with 6% (w/w) Inulin for another 10 weeks. OGTT was performed to assess glucose tolerance, and gut microbiota composition was analyzed by 16S rRNA sequencing [5]

Poor absorption in the small intestine: Inulin is not hydrolyzed by human digestive enzymes (amylase, lactase) and reaches the colon intact [1, 2]
- Metabolized by the colonic microbiota: fermented in the colon into short-chain fatty acids (acetic acid, propionic acid, butyric acid), about 95% of which are absorbed into the blood [2, 5]
- No significant systemic distribution: unfermented inulin is excreted in feces, accounting for about 5-10% of the administered dose [1]
- The plasma half-life of inulin-derived short-chain fatty acids is about 1.5 hours, and they are mainly metabolized in the liver and peripheral tissues [5]

Acute toxicity: LD50 > 20 g/kg (oral administration to rats); no deaths or acute adverse reactions were observed at doses up to 20 g/kg [1]
- Subchronic toxicity: Daily dietary supplementation of rats with 10% (w/w) inulin for 6 months did not cause significant changes in body weight, liver and kidney function (ALT, AST, creatinine), or hematological parameters [1, 3]
- Gastrointestinal tolerance: Mild and transient abdominal distension, flatulence, or diarrhea have been reported at human doses > 20 g/day; no adverse reactions were observed at doses ≤ 15 g/day [4, 5]
- No genotoxicity or carcinogenicity: Ames test and animal carcinogenicity model results were negative [3]

[1]. J Nutr.1998 Jan;128(1):11-9;
[2]. J Appl Bacteriol.1993 Oct;75(4):373-80;
[3]. Carcinogenesis.1998 Feb;19(2):281-5.
[4]. Coron Artery Dis. 2024 May 17. doi: 10.1097/MCA.0000000000001377.
[5]. Int J Mol Sci. 2024 May 16;25(10):5418. doi: 10.3390/ijms25105418.

Inulin is a fructan polysaccharide naturally found in plants such as chicory, garlic, and onions, and is classified as a soluble dietary fiber and prebiotic [1, 2, 5]. Its core mechanism of action is as a prebiotic, selectively stimulating the growth and activity of beneficial gut bacteria (Bifidobacteria, Lactobacilli), thereby increasing the production of short-chain fatty acids (SCFAs). SCFAs regulate gut homeostasis, lipid metabolism, inflammation, and cell proliferation [2, 4, 5]. Potential therapeutic applications include metabolic disorders (dyslipidemia, type 2 diabetes, obesity), colorectal cancer prevention, and atherosclerosis management [1, 3, 4, 5]. It has been recognized as clinically safe (GRAS) by regulatory agencies and is widely used in foods and dietary supplements [1, 5]. Unlike other dietary fibers, its unique fermentation properties allow it to preferentially promote the growth of beneficial bacteria without stimulating the proliferation of pathogenic bacteria [2].

C6NH10N+2O5N+1

490.411

285.101

9005-80-5

254762074

White to off-white solid powder

1,35 g/cm3

563.5±60.0 °C at 760 mmHg

176-181ºC

294.6±32.9 °C

0.0±1.5 mmHg at 25°C

1.665

Plant/Helianthus tuberosus

1.91

C1(O)[C@](CO)(OC[C@]2(O[C@H]3OC(CO)[C@@H](O)C(O)C3O)C(O)C(O)[C@@H](CO)O2)O[C@H](CO)C1O

UMGSZTYVVMHARA-RYKCJHNISA-N

InChI=1S/C17H30O16/c18-1-5-7(21)12(26)16(3-20,31-5)29-4-17(13(27)8(22)6(2-19)32-17)33-15-11(25)9(23)10(24)14(28)30-15/h5-15,18-28H,1-4H2/t5-,6-,7-,8-,9+,10+,11-,12+,13+,14+,15-,16-,17+/m1/s1

(2S,3S,4S,5R,6R)-6-(((2S,3S,4S,5R)-2-((((2R,3S,4S,5R)-3,4-dihydroxy-2,5-bis(hydroxymethyl)tetrahydrofuran-2-yl)oxy)methyl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)oxy)tetrahydro-2H-pyran-2,3,4,5-tetraol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: Water soluble

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Solubility in Formulation 4: 27.5 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)