Online LC-ESI-MS/MS comparative analysis of N/O-glycopatterns in human colostrum from different ethnic groups in Northwest China.

Human milk oligosaccharides, including free oligosaccharides and glycoconjugates, exert a key role in neonatal health and development. Changes in free oligosaccharides of milk from different ethnic groups have been documented. In this study, human milk was collected from Han, Hui, and Tibetan populations in northwest China, and differences in N/O-glycome among these three ethnic groups were systematically compared using online high-performance liquid chromatography-tandem mass spectrometry. Among the 63 detected N-glycans, 35 showed significant differences between the three ethnic groups (p < 0.05). Among the 70 detected O-glycans, four neutral O-glycans and six acidic O-glycans exhibited significant differences among the three ethnic groups (p < 0.05), with six acidic O-glycans reported for the first time. Overall, the extent of milk N/O-glycosylation was higher in the Han population than in the Hui or Tibetan groups. This trend was particularly pronounced for the main sialylated N/O-glycans. Except for sulfated O-glycans, which were higher in the milk from Tibetan mothers, the other types of N/O-glycans were present in similar proportions across all ethnic groups. Understanding the composition of N/O-glycans in human milk can help research on the structure-function relationship of glycans.

Improved Glycoqueuing Strategy Reveals Novel α2,3-Linked Di-/Tri-Sialylated Oligosaccharide Isomers in Human Milk.

Sialylated human milk oligosaccharides (SHMOs) possess unique biological activities. Qualitative and quantitative analyses of SHMOs at different lactation stages are limited by interference from neutral oligosaccharides, glycan structural complexity, and low detection sensitivity. Herein, our previously developed glycoqueuing strategy was improved and applied to enable an isomer-specific quantitative comparison of SHMOs between colostrum milk (CM) and mature milk (MM). A total of 49 putative structures were determined, including 1 α2,6-linked and 13 α2,3-linked isomers separated from seven newly discovered SHMO compositions. The content of most oligosaccharides was more than 50% lower in MM than in CM, and α2,3-sialylation was observed in 43.74% of SHMOs from CM and 22.95% of SHMOs from MM. Finally, the fucosylation level of the SHMOs increased from 16.45 to 22.28% with prolonged lactation. These findings provide the basis for further studies on the structure-activity relationship of SHMOs and a blueprint to improve infant formula.

Impact of Lycium barbarum arabinogalactan on the fecal metabolome in a DSS-induced chronic colitis mouse model.

Ulcerative colitis (UC) is often accompanied by the dysbiosis of gut microbiota and metabolism. Our previous study indicated that arabinogalactan from Lycium barbarum (LBP-3) could markedly attenuate the symptoms of chronic UC in mice by modulating the structure of gut microbiota. This study explored the impact of LBP-3 on the fecal metabolomic profiling of the same cohort of mice by HPLC-TripleTOF/MS. Untargeted metabolomic analyses indicated that supplementation with LBP-3 markedly reversed 18 of the 48 differential metabolites (mainly belonging to amino acids and organic acids) disturbed by DSS. Targeted metabolomics revealed that the lower levels of tryptophan, lysine, diiodothyronine, kynurenine, and betaine and higher levels of phenylalanine, leucine, glutamine, isoleucine, homoserine, (S)-2-hydroxyglutarate, 2-isopropylmalic acid, ascorbic acid, gluconic acid, and taurine, which were caused by DSS induction, were reversed by LBP-3 treatment. In addition, pathway analysis showed that the pentose phosphate pathway, phenylalanine metabolism, ascorbate and aldarate metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis were strongly affected by LBP-3. More importantly, the above amino acids, organic acids, and metabolic pathways changed by LBP-3 were correlated with the abundance of gut microbiota such as Turicibacter, Lactobacillus, Parasutterella, Odoribacter, Veillonella, Faecalibacterium, and Ruminococcaceae. This study advances our understanding of the interaction between the microbiome and metabolomics in DSS-induced chronic colitis after LBP-3 treatment.

Qualitative and quantitative mass spectrometry comparison of characteristic galactosyl lactose isomers from goat milk at different lactation stages.

Galactooligosaccharides are composed mainly of galactosyl lactose, which is important for infant growth and as a functional food additive. Although galactosyl lactose is abundant in goat milk, its complex structure has hindered the separation and analysis of its isomers. In this study, 5 isomers of goat milk galactosyl lactose were separated by HPLC: ß6'-galactosyl lactose (ß6'-GL), α6'-galactosyl lactose (α6'-GL), ß4'-galactosyl lactose (ß4'-GL), α3'-galactosyl lactose (α3'-GL), and ß3'-galactosyl lactose (ß3'-GL). This composition differs from that of commercial galactooligosaccharide products, which comprise mainly ß-configuration oligosaccharides. The isomers were then qualitatively and quantitatively compared at different lactation stages using online HPLC-mass spectrometry. Relative quantitative analysis showed that the total content of the 5 galactosyl lactose isomers was highest in transitional goat milk. Specifically, ß3'-GL was the main isomer in colostrum and α3'-GL was the main isomer in transitional and mature milk. ß6'-Galactosyl lactose and ß4'-GL tended to increase and then decrease during lactation. Moreover, α3'-GL content was 2 times higher than in colostrum and 10 times higher in transitional milk than in mature milk; in contrast, for ß3'-GL, the values were 5 and 2 times higher, respectively. Absolute quantitative analysis revealed that ß3'-GL was the most abundant isomers in colostrum (32.3 mg/L), and α3'-GL was the most abundant in transitional milk (88.1 mg/L) and mature milk (36.3 mg/L). These findings provide an important quantitative basis for understanding the relationship between structure and function of galactosyl lactose in goat milk, as well as its exploitation as a functional food.

Pregnancy-Related Diseases and Delivery Mode can Affect the Content of Human Milk Oligosaccharides: A Preliminary Study.

Human milk oligosaccharides (HMOs) are the second most abundant carbohydrates in colostrum. In this study, we performed a quantitative analysis of 13 oligosaccharides in 99 colostrum samples obtained from mothers living in Northwest China. The analysis combined liquid chromatography-mass spectrometry (LC-MS) with 2-amino-N-(2-aminoethyl)benzamide (AEAB) labeling and nonsecretors accounted for 17%. Compared with healthy secretor mothers, those with gestational diabetes mellitus presented lower levels of sialylated oligosaccharides, especially 3'-sialyllactose. Colostrum from mothers with pregnancy-induced hypertension had higher levels of fucosylated oligosaccharides, but the difference was not significant, and hypothyroidism appeared to have no effect on HMOs. Most HMOs (especially 6'-sialyllactose) were more abundant in colostrum from mothers who underwent vaginal delivery than a C-section. These findings show that the concentration of total or individual HMOs is affected by multiple factors. These findings provide a reference for evaluating variations in HMO expression among different populations and potential guidance for providing personalized clinical nutrition.

Ferroptosis as a novel form of regulated cell death: Implications in the pathogenesis, oncometabolism and treatment of human cancer.

The treatment of cancer mainly involves surgical excision supplemented by radiotherapy and chemotherapy. Chemotherapy drugs act by interfering with tumor growth and inducing the death of cancer cells. Anti-tumor drugs were developed to induce apoptosis, but some patient's show apoptosis escape and chemotherapy resistance. Therefore, other forms of cell death that can overcome the resistance of tumor cells are important in the context of cancer treatment. Ferroptosis is a newly discovered iron-dependent, non-apoptotic type of cell death that is highly negatively correlated with cancer development. Ferroptosis is mainly caused by the abnormal increase in iron-dependent lipid reactive oxygen species and the imbalance of redox homeostasis. This review summarizes the progression and regulatory mechanism of ferroptosis in cancer and discusses its possible clinical applications in cancer diagnosis and treatment.

Alteration of the goat milk glycoproteins N/O-glycome at different lactation stages.

Goat milk oligosaccharides represent an unexplored multi-functional ingredient for the dairy industry. Here, we qualitatively and quantitatively compared the N/O-glycome at different lactation stages via online hydrophilic interaction chromatography-tandem mass spectrometry. Complex N-glycans and high mannose N-glycans constituted 82.1% and 17.9% of the glycan pool, respectively. N-glycans with isomers containing non-bisected antenna complex structures accounted for 30.8%. N-glycans modified with Neu5Ac, Neu5Gc and fucosylated were 3.7%, 5.3% and 35.3%. The triantennary trifucosylated complex N-glycan (H5N5F3) was reported for the first time. A comparison between colostrum and mature milk revealed a 1.20-fold decrease in total N-glycans and 1.66-fold decrease in fucosylation with ongoing lactation, echoing the trend in human milk. Similarly, Neu5Ac- and Neu5Gc-modified sialylation decreased by 1.69 and 3.62 times, respectively. In the O-glycome, 46.2% of structures were O-linked core 1, 23.1% were O-linked core 2, 7.7% were O-linked core 3 and core 4. As lactation progressed, overall O-glycans content decreased by 1.26-fold. Unlike human milk, Neu5Ac- and Neu5Gc-modified sialylation increased by 4.4 and 2 times, respectively. These findings will facilitate research on the structure-function relationship of goat milk oligosaccharides and the development of formula food targeting different age groups.

A preliminary study on isomer-specific quantification of sialylated N-glycans released from whey glycoproteins in human colostrum and mature milk using a glycoqueuing strategy.

Sialylated N-glycans are an integral component of whey proteins in human milk and play an irreplaceable role in infant growth and development. Currently, there are few studies on quantitative comparison of sialylated N-glycans in milk obtained at different lactation stages. Here, a preliminary isomer-specific quantification of whey sialylated N-glycans of human colostrum milk (CM) and mature milk (MM) was performed by using our recently developed glycoqueuing strategy. Such a preliminary comparison revealed that the whey sialylated N-glycan content was 86.4% lower in MM than in CM. Twenty-three α2,6-linked sialylated N-glycan isomers were detected with no α2,3-linked isomer observed. For the first time, three mono-sialylated and four bi-sialylated glycan isomers were reported. With the prolongation of lactation, the relative abundance of mono-sialylated glycans increased, whilst the relative abundance of bi-sialylated glycans decreased significantly. These findings contribute to the understanding of the structure-function relationship of sialylated N-glycans in the human whey fraction.

Online LC-UV-ESI-MS/MS Comparative Analysis of Changes in Goat Colostrum N/O-Glycopatterns at Different Parities.

Goat milk oligosaccharides are complex carbohydrates with a variety of biological functions. Free oligosaccharides from goat milk show more similarity to human milk than cow milk. At present, changes in goat milk glycoconjugates at different parities remain poorly studied. Herein, we qualitatively and quantitatively compared the goat milk glycoprotein N/O-glycome at different parities using a stable isotope labeling followed by electrospray ionization mass spectrometry and online hydrophilic interaction chromatography. N-Glycans were mainly fucosylated and nonfucosylated nonsialylated, and both fucosylation and sialylation gradually increased with parity, amounting (at the third parity) to 1.25 times and 3.3 times those of the first parity, respectively. O-Glycans were mostly nonfucosylated and nonsialylated, and sialylation increased with increasing parity, and Neu5Ac-sialylated was up to 9 times higher in the third parity than in the first parity, whereas Neu5Gc-sialylated was 5.5 times higher. This study provides a reference for exploring an alternative milk source closest to human milk and for the development of humanized formula milk.

Arabinogalactan derived from Lycium barbarum fruit inhibits cancer cell growth via cell cycle arrest and apoptosis.

Previous studies have shown that crude polysaccharides from the Lycium barbarum fruit could inhibit cancer cell growth, but the major effective constituents are yet to be identified. In this study, we compared the effects of L. barbarum fruit polysaccharide fractions on the growth of hepatoma cells (SMMC-7721 and HepG2), cervical cancer cells (HeLa), gastric carcinoma cells (SGC-7901), and human breast cancer cells (MCF-7). LBGP-I-3 showed stronger inhibitory effects on MCF-7 cells (cell viability of 48.96%) than SMMC-7721 (cell viability of 78.91%) and HeLa cells (cell viability of 55.94%), and had no effect on HepG2 and SGC-7901 cells. In addition, LBGP-I-3 had no inhibitory effect on normal liver cells (L02, cell viability of 115.58%). Investigation of the underlying mechanism suggested that LBGP-I-3 inhibited the growth of cancer cells by cell cycle arrest and apoptosis. LBGP-I-3 arrested the cell cycle at the G0/G1 phase, altered mitochondrial function, activated oxidative stress, and regulated the MAPK signaling pathway to induce apoptosis. Thus, LBGP-I-3 may be a potential functional food ingredient for the prevention of cancer without toxicity to normal cells in vitro. These results could help further elucidate the structure-activity relationship of L. barbarum fruit polysaccharides and functional food development.

Mass Spectrometry Analysis of Changes in Human Milk N/O-Glycopatterns at Different Lactation Stages.

Human milk oligosaccharides are complex carbohydrates with multibiofunctional health benefits to newborns. Human milk free oligosaccharides (HMOs) are well characterized. However, changes in the N/O-glycome during lactation are poorly reported. Herein, we qualitatively and quantitatively investigated N/O-glycome profiles and their alteration in human milk at different lactation stages. N-Glycans were mainly fucosylated and nonsialylated, nonfucosylated throughout lactation. O-Glycans mainly consisted of sialylated and nonsialylated, nonfucosylated in colostrum and transitional milk, and fucosylated and nonfucosylated, nonsialylated in mature milk. Fucosylated and sialylated N-glycans gradually decreased and increased, respectively, as lactation progressed; O-glycans showed the reverse. Interestingly, changes in HMO abundance decreased during lactation, complementing HMG N/O-glycome changes. In conclusion, temporal HMG glycosylation changes provide the groundwork for developing infant formula that is closer to breast milk at different lactation stages.

Preparation, structural characterization and bioactivity of 4-O-Methylglucuronoxylan from Artemisia sphaerocephala Krasch.

We obtained four soluble acid xylan fractions AGP-III-A, AGP-III-B, AGP-III-C and AGP-III-D from the insoluble Artemisia sphaerocephala Krasch gum (ASKG) polysaccharide by weak alkali treatment combined with H2O2-Vc oxidative degradation. Activity studies showed that the degradation components could reduce the cell viability of several cancer cell lines in a concentration-dependent manner, especially 4-O-Methylglucuronoxylan AGP-III-C with specific molecular weight and branching degree significantly reduced cancer cells viability and induced HepG2 apoptosis, also caused mitochondrial membrane dysfunction upregulated ROS levels, and induced G0/G1 arrest in HepG2 cells by cell cycle assay. Further, AGP-III-C mediates apoptosis in HepG2 cells by upregulating MAPK phosphorylation. The structure of AGP-III-C was characterized by uronic acid reduction, permethylation with GC-MS, and 2D-NMR analysis. The structure of AGP-III-C had a linear (1→4)-linked ß-Xylf residue backbone with one branched 4-O-Me-α-GlcAp attached to the main chain by a (1→2)-glycosidic bond at every two ß-(1→4)-Xylf units.

Lycium barbarum polysaccharides extend the mean lifespan of Drosophila melanogaster.

The fruits of Lycium barbarum are considered medicinal foods with high nutritional value and bioactivity. In this study, we aimed to evaluate the effect of a crude L. barbarum polysaccharide (LBP) and two derived fractions, LBP-1 and LBP-2, on the lifespan of Drosophila melanogaster (fruit fly). The average lifespan of fruit flies was extended by supplementing their diet with either of the three LBP preparations. In vivo analysis of antioxidant activities detected increased superoxide dismutase (SOD) and catalase (CAT) activities and decreased malondialdehyde (MDA) levels. Dietary LBP supplements significantly reduced the mortality rate of fruit flies induced by paraquat and hydrogen peroxide. Importantly, the strongest anti-aging activity was exhibited by the LBP-2 fraction, containing arabinogalactan with a molecular weight of 9 × 104 Da. Further studies showed that the anti-aging activity of LBP was, at least in part, mediated by an age-related signaling pathway (MAPK, TOR, S6K) and the expression of longevity genes (Hep, MTH, and Rpn11).

Physicochemical properties, antioxidant activity and immunological effects in vitro of polysaccharides from Schisandra sphenanthera and Schisandra chinensis.

Schisandra sphenanthera and Schisandra chinensis are widely consumed either as food or for medicinal purposes. Nevertheless, no detailed comparative assessments of their physicochemical properties and biological activity have been reported. In this paper, using hot-water extraction, alcohol precipitation, and deproteinization, we obtained polysaccharidic extracts from Schisandra sphenanthera and Schisandra chinensis (denoted as SSP and SCP, respectively) and investigated their antioxidant and immunological activities. The extracts were different from each other with regard to sugar, protein, and uronic acid contents. Both extracts were mainly composed of arabinose, glucose, and galactose, but their contents varied greatly; SSP had more galacturonic acid. Compared with SCP, SSP had stronger free radical scavenging ability, protective effects on biomolecules, cellular antioxidant activity, owing to its higher protein (35.35 ±â€¯1.73%) and uronic acid (12.81 ±â€¯1.15%) contents. With respect to cell viability, neutral red phagocytosis, NO production, and acid phosphatase activity, SCP had stronger effects than SSP; this was largely due to its high levels of mannose, galactose, arabinose, and glucose. These results provide evidence to support the use Schisandra-derived polysaccharides for several purposes, including clinical, agricultural, and industrial applications.

Reductive chemical release of N-glycans as 1-amino-alditols and subsequent 9-fluorenylmethyloxycarbonyl labeling for MS and LC/MS analysis.

Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS. This new method employs aqueous ammonia solution containing NaBH3CN as the reaction medium to release glycans from glycoproteins as 1-amino-alditol forms. The released glycans are conveniently labeled with 9-fluorenylmethyloxycarbonyl (Fmoc) and analyzed by ESI-MS and online LC/MS. Using the method, the neutral and acidic N-glycans were successfully released without peeling degradation of the core α-1,3-fucosylated structure or detectable de-N-acetylation, revealing its general applicability to various types of N-glycans. The Fmoc-derivatized N-glycans derived from chicken ovalbumin, Fagopyrum esculentum Moench Pollen and FBS were successfully analyzed by online LC/MS to distinguish isomers. The 1-amino-alditols were also permethylated to form quaternary ammonium cations at the reducing end, which enhance the MS sensitivity and are compatible with sequential multi-stage mass spectrometry (MSn) fragmentation for glycan sequencing. The Fmoc-labeled N-glycans were further permethylated to produce methylated carbamates for determination of branches and linkages by sequential MSn fragmentation. SIGNIFICANCE OF THE STUDY: N-Glycosylation represents one of the most common post-translational modification forms and plays pivotal roles in the structural and functional regulation of proteins in various biological activities, relating closely to human health and diseases. As a type of informational molecule, the N-glycans of glycoproteins participate directly in the molecular interactions between glycan epitopes and their corresponding protein receptors. Detailed structural and functional characterization of different types of N-glycans is essential for understanding the functional mechanisms of many biological activities and the pathologies of many diseases. Here we describe a simple, versatile method to indistinguishably release all types of N-glycans as functionalized forms without remarkable side reactions, enabling convenient, rapid analysis and preparation of released N-glycans from various complex biological samples. It is very valuable for studies on the complicated structure-function relationship of N-glycans, as well as for the search of N-glycan biomarkers of some major diseases and N-glycan related targets of some drugs.

Physicochemical properties and biological activities of polysaccharides from Lycium barbarum prepared by fractional precipitation.

Traditional separation and purification process of Lycium barbarum polysaccharides (LBP) includes water extraction, alcohol precipitation, deproteinization and ion-exchange column chromatography, which is complicated and time-consuming. In our study, retentate LBP-I and dialysate LBP-O were obtained from LBP by water extraction, alcohol precipitation and deproteinization. LBP-I was separated by fractional precipitation and three fractions (LBP-I-1, LBP-I-2 and LBP-I-3) were obtained. The three fractions were further purified by gel permeation chromatography to LBGP-I-1, LBGP-I-2 and LBGP-I-3 with yields of 0.05%, 0.03%, and 0.19%, respectively, which are higher than yields by traditional method. The physicochemical properties, biological activities of LBGP-I-1, LBGP-I-2 and LBGP-I-3 were investigated. The results indicated that LBGP-I-1 (3.19 × 104 Da) consists of arabinose (21.95%), glucose (51.22%) and galactose (17.07%); LBGP-I-2 (2.92 × 104 Da) mainly consists of arabinose (19.35%), glucose (32.26%) and galactose (35.48%); LBGP-I-3 (9.12 × 104 Da) mainly consists of arabinose (48.15%) and galactose (44.44%). LBGP-I-1 and LBGP-I-2 were different from the components purified by traditional method. LBGP-I-3 could most significantly enhance macrophages NO, phagocytic capacity, and acid phosphatase. LBP-O exhibits the strongest anti-oxidant activities in vitro. These results provided a reference for applications of Lycium barbarum polysaccharides which would benefit the development of industry and agriculture.

The haematopoietic effect of Panax japonicus on blood deficiency model mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Panax japonicus C. A. Mey. var. major (Burk.) C. Y. Wu et K. M. Feng (PJ) is a commonly used traditional Chinese medicine to promote hematopoietic effects, promote blood circulation and supporting healthy energy. Aim of the study is to investigate the haematopoietic effects of PJ and determine the mechanism of its haematopoietic activity. MATERIALS AND METHODS: The crude extract from PJ (PJE) was separated into two fractions: polysaccharides (PJPS) and low-molecular-weight compounds (PJSM). PJPS, and PJSM were incubated with mice spleen cells, and their haematopoietic activities were evaluated by determining the haematopoietic growth factor levels (HGFs) in vitro. The in vivo experiments used anaemia model mice that were given hypodermic injections of N-acetyl phenylhydrazine (APH) and intraperitoneal injections of cyclophosphamide (CTX). RESULTS: Both PJPS and PJSM were significantly involved in the haematopoietic effect of PJE. The administration of PJPS and PJSM could accelerate the recovery of the white blood cell (WBC), red blood cell (RBC), and haemoglobin (HGB) levels in the blood deficiency model mice. Haematopoietic activity may result from stimulating the secretion of interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO), GM colony-stimulating factor (CSF), and M-CSF and by the resistance of spleen cells to apoptosis. CONCLUSIONS: The study results support the potential use of PJPS and PJSM for the treatment of anaemia.

Lycium barbarum polysaccharide LBPF4-OL may be a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer.

Recognition of the utility of the traditional Chinese medicine Lycium barbarum L. has been gradually increasing in Europe and the Americas. Many immunoregulation and antitumor effects of L. barbarum polysaccharides (LBP) have been reported, but its molecular mechanism is not yet clear. In this study, we reported that the activity of the polysaccharide LBPF4-OL, which was purified from LBP, is closely associated with the TLR4-MAPK signaling pathway. We found that LBPF4-OL can significantly induce TNF-α and IL-1ß production in peritoneal macrophages isolated from wild-type (C3H/HeN) but not TLR4-deficient mice (C3H/HeJ). We also determined that the proliferation of LBPF4-OL-stimulated lymphocytes from C3H/HeJ mice is significantly weaker than that of lymphocytes from C3H/HeN mice. Furthermore, through a bio-layer interferometry assay, we found that LPS but not LBPF4-OL can directly associate with the TLR4/MD2 molecular complex. Flow cytometry analysis indicated that LBPF4-OL markedly upregulates TLR4/MD2 expression in both peritoneal macrophages and Raw264.7 cells. As its mechanism of action, LBPF4-OL increases the phosphorylation of p38-MAPK and inhibits the phosphorylation of JNK and ERK1/2, as was observed through Western blot analysis. These data suggest that the L. barbarum polysaccharide LBPF4-OL is a new Toll-like receptor 4/MD2-MAPK signaling pathway activator and inducer.

Characterization of an immunologically active pectin from the fruits of Lycium ruthenicum.

An immunologically active pectin, named LRGP5, was firstly isolated from the fruits of Lycium ruthenicum Murr. It contained rhamnose, arabinose, xylose, galactose and galacturonic acid in the molar ratio of 1.0:2.2:0.5:1.2:4.7. Its molecular weight was estimated to be approximately 1.37 × 10(5)Da by high performance gel permeation chromatography. The structure was elucidated using methylation analysis, partial acid hydrolysis, NMR and ESI-MS analysis. Results showed that LRGP5 consisted of a (1 → 4)-linked galacturonic acid backbone occasionally interrupted by (1 → 2)-linked rhamnose. The side chains were attached to position 4 of the rhamnosyl units, including (1 → 3)-linked arabinose, (1 → 3)-linked galactose, (1 → 3,6)-linked galactose, (1 → 4)-linked galacturonic acid, (1 → 2)-linked rhamnose and (1 → 2,4)-linked rhamnose, and the termini were arabinose and rhamnose. Immunological assay results demonstrated that LRGP5 could significantly promote macrophage proliferation and enhance the secretion of nitrogen monoxide in vitro.