Characterization, prebiotic and immune-enhancing activities of rhamnogalacturonan-I-rich polysaccharide fraction from molokhia leaves

https://doi.org/10.1016/j.ijbiomac.2021.02.019Get rights and content

Abstract

Plant-derived polysaccharides possess potential health benefits that improve intestinal health and the immune system. Molokhia leaves have a large amount of mucilage polysaccharide; in the present study, crude polysaccharide extract was prepared from molokhia leaves. The molecular weight of molokhia leaf polysaccharide fraction (MPF) was estimated to be 51.2 × 103 Da. Polysaccharide was methylated and the structure of MPF was mainly composed of rhamnogalacturonan-I structure with side chains, such as galactans and linear glucan (starch), as shown by GC–MS analysis. To study the biofunctional effects of MPF, its prebiotic and intestinal immune-enhancing activities were assayed in vitro. MPF exhibited good prebiotic activity, as shown by its high prebiotic scores, and increased contents of total short-chain fatty acids on five probiotic strains. In addition, MPF showed immune-enhancing activity on Peyer's patches, as revealed by the high bone marrow cell proliferating activity and production of immunoglobulin A and cytokines. These results demonstrate that MPF may be a potential beneficial prebiotic and intestinal immune-enhancer, which may have wide implications in the food industry.

Introduction

The human gut microbiota consists of more than 400 species of bacteria, with Bifidobacteria and Lactobacilli being the predominant members of the intestinal microbiota that have beneficial effects on health [1]. Prebiotics are defined as substrates that are selectively utilized by the intestinal microbiota and confer health benefits to the host [2]. Prebiotics are important for maintaining the gut microflora balance but only a few carbohydrate compounds, including fructooligosaccharide, inulin, and galactooligosaccharide, can be classified as prebiotics [3]. Recently, many studies have focused on evaluating the prebiotic activities of polysaccharides extracted from plant sources, including bamboo shoots [4], Platycodon grandiflorus [5], and Codonopsis pilosula roots [6].

Polysaccharides, which are common in plants, are important biological macromolecules with relatively low toxicity and can be used as therapeutics [7,8]. In addition, many studies have reported that plant-extracted polysaccharides exert strong immune-enhancing activities [9,10]. Immunoglobulin A (IgA), an immunomodulatory indicator, is induced in Peyer's patches (PP) against specific mucosal antigens and is modulated by transforming growth factor (TGF)-β, interleukin (IL)-6, and IL-10 [11].

Molokhia (Corchorus olitorius L.) is a member of the family Malvaceae and is mostly cultivated in Egypt and Southeast Asian countries as a fibre crop [12]. Molokhia leaves are well-known for being rich in nutrients such as phenolic compounds, and especially are high in water-soluble mucilage polysaccharide [13]. Many studies have shown that molokhia leaves have anti-inflammatory, hepatoprotective, and antioxidant effects [14,15]. Furthermore, we have recently shown that water extracts of molokhia leaves exert beneficial effects on gut health [16]. We hypothesized that the effects on gut microbiota and intestinal health could be greater in polysaccharide extracts than in water extracts. However, the intestinal immune-enhancing and prebiotic activities of polysaccharides extracted from molokhia leaves have not been evaluated. Therefore, we investigated and characterized the prebiotic activity and immune-enhancing effects of molokhia leaf polysaccharides.

Section snippets

Preparation of molokhia leaf polysaccharide fraction

Molokhia leaves were cultivated in Hongcheon-gun, Republic of Korea. The leaf powder (100 g) was extracted in 2 L of distilled water for 3 h at 80 °C. After filtration and concentration using a vacuum rotary evaporator (R-114; Buchi Labortechnik, Flawil, Switzerland), the leaf powder was precipitated with four volumes of ethanol for 16 h at 4 °C. After centrifugation (3000 ×g, 30 min), the precipitates were dialyzed for two days at 4 °C using a dialysis membrane (molecular weight cut-off:

Chemical composition of MPF

The chemical composition of MPF is summarized in Table 1. The neutral sugar and uronic acid contents were 47.1% and 44.7%, respectively. MPF mainly consisted of sugars, such as rhamnose (22.4%), galactose (22.1%), galacturonic acid (22.1%), glucuronic acid (17.9%), and glucose (5.7%). Trace amounts of arabinose (2.6%), xylose (0.5%), and mannose (0.4%) were detected. The high rhamnose and galacturonic acid contents suggested that MPF is a pectic polysaccharide containing the rhamnogalacturonan

Conclusion

MPF was obtained from molokhia leaves, and structural analysis of its chemical composition, GPC, and methylation revealed that it is a polysaccharide of rhamnogalacturonan-I structure composed of several side chains, such as galactans and linear glucans. MPF demonstrated prebiotic activity on probiotic strains, such as Lactobacillus and Bifidobacterium by increasing its prebiotic scores and concentrations of total SCFAs. In addition, MPF demonstrated intestinal immune-enhancing activity in the

CRediT authorship contribution statement

Hye-Bin Lee: Investigation, Data curation, Writing – original draft. Seung-U Son: Investigation. Jang-Eun Lee: Methodology. Sang-Hoon Lee: Software, Validation. Chang-Ho Kang: Investigation. Young-Soo Kim: Conceptualization. Kwang-Soon Shin: Writing – review & editing. Ho-Young Park: Conceptualization, Methodology, Writing – original draft.

Declaration of competing interest

Authors declare no conflict of interest.

Acknowledgements

This research was supported by the Main Research Program (E021600-01) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science and ICT. This work was also supported by Kyonggi University‘s Graduate Research Assistantship 2020.

References (48)

  • N. Kumari et al.

    Health-promoting properties of Corchorus leaves: a review

    J. Herb. Med.

    (2019)
  • H.-B. Lee et al.

    Molokhia leaf extract prevents gut inflammation and obesity

    J. Ethnopharmacol.

    (2020)
  • N. Blumenkrantz et al.

    New method for quantitative determination of uronic acids

    Anal. Biochem.

    (1973)
  • H.F. Zhao et al.

    Evaluation of antioxidant activities and total phenolic contents of typical malting barley varieties

    Food Chem.

    (2008)
  • J. Dai et al.

    Sugar compositional determination of polysaccharides from Dunaliella salina by modified RP-HPLC method of precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone

    Carbohydr. Polym.

    (2010)
  • H.W. Kim et al.

    Signaling pathways associated with macrophage-activating polysaccharides purified from fermented barley

    Int. J. Biol. Macromol.

    (2019)
  • Y.D. Karkhanis et al.

    A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative bacteria

    Anal. Biochem.

    (1978)
  • G.L. Sassaki et al.

    Rapid synthesis of partially O-methylated alditol acetate standards for GC-MS: some relative activities of hydroxyl groups of methyl glycopyranosides on Purdie methylation

    Carbohydr. Res.

    (2005)
  • B.L. Ridley et al.

    Pectins: structure, biosynthesis, and oligogalacturonide-related signaling

    Phytochemistry

    (2001)
  • P. Seedevi et al.

    Isolation and chemical characteristics of rhamnose enriched polysaccharide from Grateloupia lithophila

    Carbohydr. Polym.

    (2018)
  • H.R. Park et al.

    Signaling pathway and structural features of macrophage-activating pectic polysaccharide from Korean citrus, Cheongkyool peels

    Int. J. Biol. Macromol.

    (2019)
  • I.M. Sims et al.

    Structural and rheological studies of a polysaccharide mucilage from lacebark leaves (Hoheria populnea a. Cunn.)

    Int. J. Biol. Macromol.

    (2018)
  • N. Khodaei et al.

    Digestibility and prebiotic properties of potato rhamnogalacturonan I polysaccharide and its galactose-rich oligosaccharides/oligomers

    Carbohydr. Polym.

    (2016)
  • J. Wang et al.

    Cereal-derived arabinoxylans: structural features and structure-activity correlations

    Trends Food Sci. Technol.

    (2020)
  • Cited by (21)

    • Gellan gum prevents non-alcoholic fatty liver disease by modulating the gut microbiota and metabolites

      2023, Food Chemistry
      Citation Excerpt :

      The rabbit secondary antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The prebiotic activity score was determined using the modified method of Lee et al. (Lee et al., 2021). Four commercial probiotic strains were used in this study: Bifidobacterium bifidum (MG731, infant gut-origin), Bifidobacterium longum (MG723, infant gut-origin), and Lacticaseibacillus rhamnosus (MG316, infant gut-origin), which were kindly provided by Mediogen (Jecheon, Republic of Korea); Escherichia coli (KCTC2441), which was purchased from the Korean Collection for Type Cultures (Jeongeup, Republic of Korea); and Lactoccocus lactis (KF140, fermented food-origin), which was isolated from fermented kimchi at the Korea Food Research Institute.

    • Enhancing the prebiotic and antioxidant effects of exopolysaccharides derived from Cordyceps militaris by enzyme-digestion

      2022, LWT
      Citation Excerpt :

      The proportions of microbiota were calculated based on their Ct values. Prebiotic activity assays were performed using a previously described method (H.-B. Lee et al., 2021). Probiotics or enteric bacteria strains (Lactobacillus rhamnosus, KCTC 5033; L. paracasei, KCTC 3510; L. casei, KTCT 3110; L. acidophilus, KCTC 3164; L. plantarum, KCTC 3108; Bifidobacterium.

    View all citing articles on Scopus
    View full text