Ginsenoside Rh4 alleviates gastrointestinal mucositis and enhances chemotherapy efficacy through modulating gut microbiota.

BACKGROUND: Gastrointestinal mucositis stands as one of the most severe side effects of irinotecan (CPT-11). however, only palliative treatment is available at present. Therefore, there is an urgent need for adjunctive medications to alleviate the side effects of CPT-11. PURPOSE: In this study, our objective was to explore whether ginsenoside Rh4 could serve as a modulator of the gut microbiota and an adjunctive agent for chemotherapy, thereby alleviating the side effects of CPT-11 and augmenting its anti-tumor efficacy. STUDY DESIGN: A CPT-11-induced gastrointestinal mucositis model was used to investigate whether ginsenoside Rh4 alleviated CPT-11-induced gastrointestinal mucositis and enhanced the anti-tumor activity of CPT-11. METHODS: In this study, we utilized CT26 cells to establish a xenograft tumor model, employing transcriptomics, genomics, and metabolomics techniques to investigate the impact of ginsenoside Rh4 on CPT-11-induced gastrointestinal mucositis and the effect on the anti-tumor activity of CPT-11. Furthermore, we explored the pivotal role of gut microbiota and their metabolites through fecal microbiota transplantation (FMT) experiments and supplementation of the key differential metabolite, hyodeoxycholic acid (HDCA). RESULTS: The results showed that ginsenoside Rh4 repaired the impairment of intestinal barrier function and restored intestinal mucosal homeostasis in a gut microbiota-dependent manner. Ginsenoside Rh4 treatment modulated gut microbiota diversity and upregulated the abundance of beneficial bacteria, especially Lactobacillus_reuteri and Akkermansia_muciniphila, which further regulated bile acid biosynthesis, significantly promoted the production of the beneficial secondary bile acid hyodeoxycholic acid (HDCA), thereby alleviating CPT-11-induced gut microbiota dysbiosis. Subsequently, ginsenoside Rh4 further alleviated gastrointestinal mucositis through the TGR5-TLR4-NF-κB signaling pathway. On the other hand, ginsenoside Rh4 combination therapy could further reduce the weight and volume of colon tumors, promote tumor cell apoptosis, and enhance the anti-tumor activity of CPT-11 by inhibiting the PI3K-Akt signaling pathway, thus exerting a synergistic anti-tumor effect. CONCLUSION: In summary, our findings confirm that ginsenoside Rh4 can alleviate CPT-11-induced gastrointestinal mucositis and enhance the anti-tumor activity of CPT-11 by modulating gut microbiota and its related metabolites. Our study validates the potential of ginsenoside Rh4 as a modulator of the gut microbiota and an adjunctive agent for chemotherapy, offering new therapeutic strategies for addressing chemotherapy side effects and improving chemotherapy efficacy.

Expression of Multicopy Tandem Recombinant Ginseng Hexapeptide in Bacillus subtilis and the Evaluation of Antiaging Activity.

Ginseng oligopeptides are naturally occurring small-molecule peptides extracted from ginseng that exhibit positive effects on health and longevity. However, the current industrial production of ginseng oligopeptides primarily relies on plant extraction and chemical synthesis. In this study, we proposed a novel genetic engineering approach to produce active ginseng peptides through multicopy tandem insertion (5 and 15 times). The recombinant ginseng peptides were successfully produced from engineered Bacillus subtilis with an increasing yield from 356.55 to 2900 mg/L as the repeats multiple. Additionally, an oxidative stress-induced aging model caused by H2O2 was established to evaluate whether the recombinant ginseng peptides, without enzymatic hydrolysis into individual peptides, also have positive effects on antiaging. The results demonstrated that all two kinds of recombinant ginseng peptides could also delay cellular aging through various mechanisms, such as inhibiting cell cycle arrest, suppressing the expression of pro-inflammatory factors, and enhancing cellular antioxidant capacity.

Ginsenoside Rg5 inhibits lipid accumulation and hepatocyte apoptosis via the Notch1 signaling pathway in NASH mice.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a prevalent chronic liver disease that lacks an FDA-approved treatment medicine. Despite the known antitumor and hypoglycemic properties of Ginsenoside Rg5, its effects and underlying mechanisms in the context of NASH remain largely unexplored. PURPOSE: This study aims to investigate the effect of Rg5 on NASH mice induced by a high-fat diet and CCl4. STUDY DESIGN: In vivo experiments, a mouse NASH model was established by a HFHC diet plus intraperitoneal injection of low-dose CCl4. In vitro experiments, a cellular steatosis model was established using free fatty acids (FFA) induced HepG2 cells. In addition, a fibrogenesis model was established using HSC-LX2 cells. METHODS: The effects of Ginsenoside Rg5 on lipid accumulation and oxidative damage were analyzed by ELISA kit, H&E staining, Oil Red O staining, flow cytometry and Western blot. The effects of Ginsenoside Rg5 on liver fibrosis were analyzed by Masson staining, Sirus Red staining, immunohistochemistry and Western blot. The effect of Ginsenoside Rg5 on Notch1 signaling pathway in liver was studied by protein Oil Red staining, protein immunoblotting and immunofluorescence. RESULTS: In terms of lipid accumulation, Rg5 has the ability to regulate key proteins related to lipogenesis, thereby inhibiting hepatic lipid accumulation and oxidative stress. Additionally, Rg5 can reduce the occurrence of hepatocyte apoptosis by regulating the p53 protein. Moreover, after Rg5 intervention, the presence of fibrotic proteins (α-SMA, Collagen 1, TGF-ß) in the liver is significantly suppressed, thus inhibiting liver fibrosis. Lastly, Rg5 leads to a decrease in the expression levels of Notch1 and its ligand Jagged-1 in the liver. CONCLUSION: In summary, the regulatory effects of Rg5 on the Notch1 signaling pathway play a crucial role in modulating hepatic lipid metabolism and preventing hepatocyte apoptosis, thereby impeding the progression of NASH. These findings highlight the potential of Rg5 as a promising natural product for interventions targeting NASH.

Residue Effect-Guided Design: Engineering of S. Solfataricus ß-Glycosidase to Enhance Its Thermostability and Bioproduction of Ginsenoside Compound K.

ß-Glycosidase from Sulfolobus solfataricus (SS-BGL) is a highly effective biocatalyst for the synthesis of compound K (CK) from glycosylated protopanaxadiol ginsenosides. In order to improve the thermal stability of SS-BGL, molecular dynamics simulations were used to determine the residue-level binding energetics of ginsenoside Rd in the SS-BGL-Rd docked complex and to identify the top ten critical contributors. Target sites for mutations were determined using dynamic cross-correlation mapping of residues via the Ohm server to identify networks of distal residues that interact with the key binding residues. Target mutations were determined rationally based on site characteristics. Single mutants and then recombination of top hits led to the two most promising variants SS-BGL-Q96E/N97D/N302D and SS-BGL-Q96E/N97D/N128D/N302D with 2.5-fold and 3.3-fold increased half-lives at 95 °C, respectively. The enzyme activities relative to those of wild-type for ginsenoside conversion were 161 and 116%, respectively..

Ginsenoside Rh4 Alleviates Amyloid ß Plaque and Tau Hyperphosphorylation by Regulating Neuroinflammation and the Glycogen Synthase Kinase 3ß Signaling Pathway.

Alzheimer's disease (AD) is a primary neurodegenerative disease. It can be caused by aging and brain trauma and severely affects the abilities of cognition and memory of patients. Therefore, it seriously threatens the mental and physical health of humans worldwide. As a traditional Chinese medicine, ginsenosides have been proven to have a variety of pharmacological activities. Ginsenoside Rh4 (Rh4) is one of the rare ginsenosides with higher pharmacological activity than ordinary ginsenosides, but its effect on alleviating AD and its molecular mechanism have not been studied. Here, we investigated the anti-AD effects of Rh4 and its potential mechanisms using an AD mouse model induced by a combination of AlCl3·6H2O and d-galactose. The results showed that Rh4 could significantly improve the ability of cognizance and reduce neuronal damage in mice. Concurrently, Rh4 attenuates amyloid ß accumulation, increases the density of dendritic spines, and logically inhibits synaptic structural damage as a result of neuronal excessive apoptosis and autophagy. Rh4 can not only inhibit the inflammatory response caused by the overactivation of microglia and astrocytes, reduce the levels of pro-inflammatory factors, increase the level of antioxidant enzymes in serum, and significantly improve the activity of antioxidant enzyme SOD1 in the hippocampus but also inhibit the hyperphosphorylation of tau protein in the hippocampus of mice by regulating the Wnt2b/GSK-3ß/SMAD4 signaling pathway. Together, this study provides a theoretical basis for Rh4 in the treatment of AD and reveals that Rh4 is a potential drug for the treatment of AD.

Ginsenoside Rh4 delays skeletal muscle aging through SIRT1 pathway.

BACKGROUND: The aging of skeletal muscle is the leading cause of physical disability in older adults, currently effective treatment methods are lacking. Ginsenoside Rh4, an active component extracted from ginseng, possesses beneficial anti-inflammatory and anti-oxidative effects. PURPOSE: The aim of this study was to elucidate the antioxidant effect of ginsenoside Rh4 on aging skeletal muscle and its molecular mechanism of anti-aging of skeletal muscle. STUDY DESIGN: In this study, we employed a D-galactose-induced model of skeletal muscle aging to investigate whether ginsenoside Rh4 can delay the process of skeletal muscle senescence. METHODS: The effects of ginsenoside Rh4 on oxidative damage and inflammation in aging skeletal muscle were analyzed using immunofluorescence, immunohistochemistry, ELISA kits, H&E staining, flow cytometry, and protein immunoblotting. The changes of ginsenoside Rh4 on mitochondrial morphology were observed by transmission electron microscopy, and ELISA kits and protein immunoblotting analyzed the effects of ginsenoside Rh4 on mitochondrial homeostasis in skeletal muscle cells. The influence of ginsenoside Rh4 on the SIRT1 signaling pathway in aging skeletal muscle were investigated by protein immunoblotting, immunofluorescence, and ß-galactosidase staining. RESULTS: Our results showed that Rh4 improved the morphology of muscle fibers and produced an anti-inflammatory response. Furthermore, in vitro experiments indicated that ginsenosides reduced the production of senescent cells, while Rh4 effectively alleviated oxidative damage in skeletal muscle and restored mitochondrial balance. Transcriptome analysis and molecular docking showed that Rh4 improved mitochondrial homeostasis and delayed skeletal muscle aging by regulating the PGC-1α-TFAM and HIF-1α-c-Myc pathways via targeting SIRT1. CONCLUSION: Ginsenoside Rh4 improves oxidative stress and inflammation in skeletal muscle by activating SIRT1, deacetylating Nrf2, regulating PGC-1α-TFAM and HIF-1α-c-Myc pathways, and enhancing mitochondrial homeostasis, thus achieving the effect of delaying skeletal muscle aging.

Grazing-incidence diffraction reveals cellulose and pectin organization in hydrated plant primary cell wall.

The primary cell wall is highly hydrated in its native state, yet many structural studies have been conducted on dried samples. Here, we use grazing-incidence wide-angle X-ray scattering (GIWAXS) with a humidity chamber, which enhances scattering and the signal-to-noise ratio while keeping outer onion epidermal peels hydrated, to examine cell wall properties. GIWAXS of hydrated and dried onion reveals that the cellulose ([Formula: see text]) lattice spacing decreases slightly upon drying, while the (200) lattice parameters are unchanged. Additionally, the ([Formula: see text]) diffraction intensity increases relative to (200). Density functional theory models of hydrated and dry cellulose microfibrils corroborate changes in crystalline properties upon drying. GIWAXS also reveals a peak that we attribute to pectin chain aggregation. We speculate that dehydration perturbs the hydrogen bonding network within cellulose crystals and collapses the pectin network without affecting the lateral distribution of pectin chain aggregates.

Mussel-inspired adhesive bilayer hydrogels for bacteria-infected wound healing via NIR-enhanced nanozyme therapy.

Preventing bacterial infection in situ and accelerating skin generation simultaneously are essentially important for wound healing. Herein, a mussel-inspired Ag nanozyme-based bilayer hydrogel is constructed to address the above concerns. The bilayer hydrogel is composed of a layer with large pores absorbing the wound exudate and allowing oxygen exchange and a layer with small pores keeping the wound moist and preventing bacterial invasion. Benefitting from the polydopamine (PDA) coating-reduced Ag nanoparticles (AgNPs), the hydrogel exhibits high near infrared (NIR) absorption at 808 nm to generate hyperthermia and NIR-enhanced peroxidase (POD-like) activity to produce hydroxyl radicals (•OH), which endows the hydrogel with excellent antibacterial properties when combined with the released Ag+. In addition, the hydrogel presents adhesiveness due to the catechol group on a PDA molecule. The in vivo test results demonstrate that the bilayer hydrogel can accelerate infected skin generation by facilitating collagen deposition, decreasing tumor necrosis factor-α secretion, and promoting vascular endothelial growth factor expression.

Adult stem cell transplantation combined with conventional therapy for the treatment of end-stage liver disease: a systematic review and meta-analysis.

End-stage liver disease (ESLD) is characterized by the deterioration of liver function and a subsequent high mortality rate. Studies have investigated the use of adult stem cells to treat ESLD. Here, a systematic review and meta-analysis was conducted to determine the efficacy of a combination therapy with adult stem cell transplantation and traditional medicine for treating ESLD. Four databases-including PubMed, Web of Science, Embase, and Cochrane Library-were investigated for studies published before January 31, 2021. The main outcome indicators were liver function index, model for end-stage liver disease (MELD) scores, and Child‒Turcotte‒Pugh (CTP) scores. Altogether, 1604 articles were retrieved, of which eight met the eligibility criteria; these studies included data for 579 patients with ESLD. Combination of adult stem cell transplantation with conventional medicine significantly improved its efficacy with respect to liver function index, CTP and MELD scores, but this effect gradually decreased over time. Moreover, a single injection of stem cells was more effective than two injections with respect to MELD and CTP scores and total bilirubin (TBIL) and albumin (ALB) levels, with no significant difference in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. With respect to the TBIL levels, patients receiving mononuclear cells (MNCs) experienced a significantly greater therapeutic effect-starting from twenty-four weeks after the treatment-whereas with respect to ALB levels, CD34+ autologous peripheral blood stem cells (CD34+ APBSCs) and MNCs had similar therapeutic effects. Severe complications associated with adult stem cell treatment were not observed. Although the benefits of combination therapy with respect to improving liver function were slightly better than those of the traditional treatment alone, they gradually decreased over time.Systematic review registration: PROSPERO registration number: CRD42021238576.

Protective effect of ginsenoside Rg5 against kidney injury via inhibition of NLRP3 inflammasome activation and the MAPK signaling pathway in high-fat diet/streptozotocin-induced diabetic mice.

Diabetic nephropathy (DN) is a common and serious complication of diabetes and causes kidney failure. Ginsenoside Rg5 (Rg5) is an important monomer in the main protopanaxadiol component of black ginseng. Rg5 has exhibited some beneficial biological effects, such as anti-cancer, neuroprotection, and anti-depression, but the effect of Rg5 on DN and its potential mechanism remains unclear. The aim of this study is to investigate the effect of Rg5 on kidney injury of C57BL/6 diabetic mice induced by high-fat diet and streptozotocin. After treatment with different concentration of Rg5 (30 and 60 mg kg-1·d-1) for 6 consecutive weeks, the fasting blood glucose, insulin levels, serum creatinine, serum urea, and serum UA in Rg5-treated DN mice were significantly reduced, while the renal histopathology was remarkably improved, compared with untreated DN mice. Moreover, ROS production, oxidative stress markers (MDA, SOD, and GSH-PX), Nox4 and TXNIP expressions of kidney in DN mice were significantly reduced after Rg5 treatment. Additionally, the expression levels of the NLRP3 inflammasome (NLRP3, ASC, and Caspase-1) and the inflammatory cytokines IL-1ß and IL-18 were significantly inhibited, and the expression of NF-kB and the phosphorylation of p38 MAPK were also decreased with Rg5 treatment compared with no treatment in DN mice. Together, our results indicate that Rg5 attenuated renal injury in diabetic mice by inhibiting oxidative stress and NLRP3 inflammasome activation to reduce inflammatory responses, indicating that Rg5 is a potential compound to prevent or control diabetic renal injury.

The effect of human-like collagen calcium complex on osteoporosis mice.

The objective of this study was to assess the effect of a modified human-like collagen calcium complex on osteoporosis mice. BHK (Baby Hamster Kidney) cells were used to compare the cytotoxicity of different calcium reagents with the MTT test. Six-week-old male mice (n = 80) were randomly divided into eight groups: a blank group (blank), control group (control), human-like collagen calcium group (HLC-Ca), thiolated human-like collagen calcium group (SH-HLC-Ca), phosphorylated human-like collagen calcium group (Pi-HLC-Ca), gluconate group (Glc-Ca), calcium carbonate group (CaCO3) and D-cal group (B). A systematic analysis of the results available in vivo after 3 months of treatment was presented. The effects of several Ca supplements on osteoporosis mice were investigated by detecting serum calcium, alkaline phosphate activity (ALP), bone hydroxyproline (BHP) and bone mineral density (BMD). The results proved that the BMD and BHP of osteoporosis mice were significantly increased in the Pi-HLC-Ca group, while serum calcium and ALP were decreased. Therefore, Pi-HLC-Ca is likely a good calcium supplement for clinical applications. In this review, the advantage of Pi-HLC-Ca in preventing and delaying osteoporosis is highlighted. In addition to the current progress, further investigations are necessary to reveal the relative influences of collagen and calcium proportions on the long-term clinical effects of osteoporosis.

Microencapsulation of Phosphorylated Human-Like Collagen-Calcium Chelates for Controlled Delivery and Improved Bioavailability.

The bioavailability of Phosphorylated Human-like Collagen-calcium chelates (PHLC-Ca) as calcium supplement is influenced by the extremely low pH and proteolytic enzymes in the gastrointestinal tract. This study addresses these issues by microencapsulation technology using alginate (ALG) and chitosan (CS) as wall materials. The different ratio of ALG to PHLC-Ca on microcapsules encapsulation efficiency (EE) and loading capacity (LC) was evaluated and 1:1/2 was selected as the optimal proportion. The microcapsules were micron-sized and spherical in shape. PHLC-Ca was successfully entrapped into the matrix of ALG through forming intermolecular hydrogen bonding or other interactions. The confocal laser scanning microscopy (CLSM) indicated that CS was coated on ALG microspheres. The MTT assay exhibited that CS/ALG-(PHLC-Ca) microcapsules extracts were safe to L929. The animal experiment showed that CS/ALG-(PHLC-Ca) microcapsules was superior to treating osteoporosis than PHLC-Ca. These results illustrated that the bioavailability of PHLC-Ca was improved by microencapsulated.

Anticancer effects of ginsenoside Rk3 on non-small cell lung cancer cells: in vitro and in vivo.

Ginsenoside Rk3 (Rk3) is present in the roots of processed Panax notoginseng herbs and it exerts anti-platelet aggregation, pro-immunogenic and cardioprotective effects. However, little is known regarding the anticancer activities of this compound, especially in lung cancer. This study was designed to investigate the anticancer effects of Rk3 on non-small cell lung cancer (NSCLC) cells and in an H460 xenograft tumor model. Our results showed that Rk3 reduced cell viability, inhibited both cell proliferation and colony formation, and induced G1 phase cell cycle arrest by downregulating the expression of cyclin D1 and CDK4 and upregulating the expression of P21. Rk3 also induced apoptosis in a concentration-dependent manner in H460 and A549 cells by Annexin V/PI staining, TUNEL assay and JC-1 staining, resulting in a change in the nuclear morphology. Moreover, Rk3 induced the activation of caspase-8, -9, and -3, promoted changes in mitochondrial membrane potential, decreased the expression of Bcl-2, increased the expression of Bax, and caused the release of cytochrome c, which indicated that the apoptosis-inducing effects of Rk3 were triggered via death receptor-mediated mitochondria-dependent pathways. Furthermore, Rk3 significantly inhibited the growth of H460 xenograft tumors without an obvious effect on the body weight of the treated mice. Histological analysis indicated that Rk3 inhibited tumor growth by altering the proliferation and morphology of tumor cells. In addition, we confirmed that Rk3 inhibited angiogenesis via CD34 staining and chick embryo chorioallantoic membrane (CAM) assay in vivo. Taken together, our findings revealed not only the anticancer effect of Rk3 on NSCLC cells but also a new promising therapeutic agent for human NSCLC.

Higher iron bioavailability of a human-like collagen iron complex.

Iron deficiency remains a public health problem around the world due to low iron intake and/or bioavailability. FeSO4, ferrous succinate, and ferrous glycinate chelate are rich in iron but have poor bioavailability. To solve the problem of iron deficiency, following previous research studies, a thiolated human-like collagen-ironcomplex supplement with a high iron content was prepared in an anaerobic workstation. In addition, cell viability tests were evaluated after conducting an MTT assay, and a quantitative analysis of the thiolated human-like collagen-iron digesta samples was performed using the SDS-PAGE method coupled with gel filtration chromatography. The iron bioavailability was assessed using Caco-2 cell monolayers and iron-deficiency anemia mice models. The results showed that (1) one mole of thiolated human-like collagen-iron possessed approximately 35.34 moles of iron; (2) thiolated human-like collagen-iron did not exhibit cytotoxity and (3) thiolated human-like collagen- iron digesta samples had higher bioavailability than other iron supplements, including FeSO4, ferrous succinate, ferrous glycine chelate and thiolated human-like collagen-Fe iron. Finally, the iron bioavailability was significantly enhanced by vitamin C. These results indicated that thiolated human-like collagen-iron is a promising iron supplement for use in the future.

Formation mechanism and biological activity of novel thiolated human-like collagen iron complex.

To develop an iron supplement that is effectively absorbed and utilized, thiolated human-like collagen was created to improve the iron binding capacity of human-like collagen. A thiolated human-like collagen-iron complex was prepared in a phosphate buffer, and one mole of thiolated human-like collagen-iron possessed approximately 28.83 moles of iron. The characteristics of thiolated human-like collagen-iron were investigated by ultraviolet-visible absorption spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and differential scanning calorimetry. The results showed that the thiolated human-like collagen-iron complex retained the secondary structure of human-like collagen and had greater thermodynamic stability than human-like collagen, although interactions between iron ions and human-like collagen occurred during the formation of the complex. In addition, to evaluate the bioavailability of thiolated human-like collagen-iron, an in vitro Caco-2 cell model and an in vivo iron deficiency anemia mouse model were employed. The data demonstrated that the thiolated human-like collagen-iron complex exhibited greater bioavailability and was more easily utilized than FeSO4, ferric ammonium citrate, or ferrous glycinate. These results indicated that the thiolated human-like collagen-iron complex is a potential iron supplement in the biomedical field.

A novel thiolated human-like collage zinc complex as a promising zinc supplement: physicochemical characteristics and biocompatibility.

To improve zinc binding ability to human-like collagen (HLC) and stability of metal complex, HLC was thiolated by mercaptosuccinylation reaction with S-acetylmercaptosuccinic anhydride (S-AMSA) at pH8.0. One mole of thiolated HLC-Zn (SHLC-Zn) complex possessed 24.3mol zinc ions when pH was 8.0 and zinc concentration was 15 mM. The physicochemical properties and biocompatibility of thiolated HLC-Zn (SHLC-Zn) complex were investigated by UV-vis, CD, electrophoresis analysis, differential scanning calorimetry (DSC) and cell viability assay, respectively. The results showed that SHLC-Zn complex(1) exhibited higher zinc ions than that of native HLC and still maintained the secondary structure of HLC though interaction occurred between SHLC and zinc ions, (2) increased the apparent molecular weight when compared with native HLC, (3) exhibited greater thermal stability than native HLC, and (4) presented toxicity free for BHK cells. This study suggests that the SHLC-Zn complex is a potential nutrition as well as zinc supplement in the medical application.

Formation and characterization of iron-binding phosphorylated human-like collagen as a potential iron supplement.

Iron incorporated into food can induce precipitation and unwanted interaction with other components in food. Iron-binding proteins represent a possibility to avoid these problems and other side effects, as the iron is protected. However, there are several technical problems associated with protein-iron complex formation. In this paper, the iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared under physiological conditions through phosphorylated modification. One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. Spectroscopy analysis, differential scanning calorimetry (DSC) and equilibrium dialysis techniques were employed to investigate the characteristics of the Fe-G6P-HLC. The binding sites (nb) and apparent association constant (Kapp) between iron and phosphorylated HLC were measured at nb=23.7 and log Kapp=4.57, respectively. The amount of iron (Fe(2+) sulfate) binding to phosphorylated HLC was found to be a function of pH and phosphate content. In addition, the solubility and thermal stability of HLC were not significantly affected. The results should facilitate the utilization of HLC as a bioactive iron supplement in the food and medical industry and provide an important theoretical evidence for the application of HLC chelates.