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      An L213A variant of β-glycosidase from Sulfolobus solfataricus with increased α-L-arabinofuranosidase activity converts ginsenoside Rc to compound K

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          Abstract

          Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β- d-glucopyranoside in ginsenosides Rc, C-Mc 1, and C-Mc, but not α- l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α- l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α- l-arabinofuranosidase activity. A L213A variant β-glycosidase with increased α- l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α- l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α- l-arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

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          Ginseng compounds: an update on their molecular mechanisms and medical applications.

          Ginseng is one of the most widely used herbal medicines and is reported to have a wide range of therapeutic and pharmacological applications. Ginsenosides, the major pharmacologically active ingredients of ginseng, appear to be responsible for most of the activities of ginseng including vasorelaxation, antioxidation, anti-inflammation and anti-cancer. Approximately 40 ginsenoside compounds have been identified. Researchers now focus on using purified individual ginsenoside to reveal the specific mechanism of functions of ginseng instead of using whole ginseng root extracts. Individual ginsenosides may have different effects in pharmacology and mechanisms due to their different chemical structures. Among them the most commonly studied ginsenosides are Rb1, Rg1, Rg3, Re, Rd and Rh1. The molecular mechanisms and medical applications of ginsenosides have attracted much attention and hundreds of papers have been published in the last few years. The general purpose of this update is to provide information of recently described effects of ginsenosides on antioxidation, vascular system, signal transduction pathways and interaction with receptors. Their therapeutic applications in animal models and humans as well as the pharmacokinetics and toxicity of ginsenosides are also discussed in this review. This review concludes with some thoughts for future directions in the further development of ginseng compounds as effective therapeutic agents.
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            A comparison of the ancient use of ginseng in traditional Chinese medicine with modern pharmacological experiments and clinical trials.

            Panax ginseng C.A. Meyer is a well-known medicinal herb native to China and Korea, and has been used as a herbal remedy in eastern Asia for thousands of years. However, there is different evidence of ginseng efficacy between traditional Chinese medicine (TCM), modern pharmacological experiments and clinical trials. In TCM, ginseng is a highly valued herb and has been applied to a variety of pathological conditions and illnesses such as hypodynamia, anorexia, shortness of breath, palpitation, insomnia, impotence, hemorrhage and diabetes. Modern pharmacological experiments have proved that ginseng possesses multiple constituents (ginsenosides, polysaccharides, peptides, polyacetylenic alcohols, etc.) and actions (central nervous system effects, neuroprotective effect, immunomodulation, anticancer, etc.), ginsenosides as the active ingredients, especially, having antioxidant, antiinflammatory, antiapoptotic and immunostimulant properties. Recently, ginseng has been studied in a number of randomized controlled trials investigating its effect mainly on physical and psychomotor performance, cognitive function, immunomodulation, diabetes mellitus, cardiovascular risk factors, quality of life, as well as adverse effects. Equivocal results have been demonstrated for many of these indications. Because of the poor quality of most clinical trials on ginseng, reliable clinical data in humans are still lacking. Therefore, a broader understanding of medical knowledge and reasoning on ginseng is necessary.
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              Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation--the key step of inflammation.

              In the preliminary study, ginsenoside Rb1, a main constituent of the root of Panax ginseng (family Araliaceae), and its metabolite compound K inhibited a key factor of inflammation, nuclear transcription factor κB (NF-κB) activation, in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. When ginsenoside Rb1 or compound K were orally administered to 2,4,6-trinitrobenzene sulfuric acid (TNBS)-induced colitic mice, these agents inhibited colon shortening, macroscopic score, and colonic thickening. Furthermore, treatment with ginsenoside Rb1 or compound K at 20mg/kg inhibited colonic myeloperoxidase activity by 84% and 88%, respectively, as compared with TNBS alone (p<0.05), and also potently inhibited the expression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6, but increased the expression of IL-10. Both ginsenoside Rb1 and compound K blocked the TNBS-induced expressions of COX-2 and iNOS and the activation of NF-κB in mice. When ginsenoside Rb1 or compound K was treated in LPS-induced murine peritoneal macrophages, these agents potently inhibited the expression of the proinflammatory cytokines. Ginsenoside Rb1 and compound K also significantly inhibited the activation of interleukin-1 receptor-associated kinase-1 (IRAK-1), IKK-β, NF-κB, and MAP kinases (ERK, JNK, and p-38); however, interaction between LPS and Toll-like receptor-4, IRAK-4 activation and IRAK-2 activation were unaffected. Furthermore, compound K inhibited the production of proinflammatory cytokines more potently than did those of ginsenoside Rb1. On the basis of these findings, ginsenosides, particularly compounds K, could be used to treat inflammatory diseases, such as colitis, by targeting IRAK-1 activation. Copyright © 2011 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Writing – original draft
                Role: Data curation
                Role: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 January 2018
                2018
                : 13
                : 1
                : e0191018
                Affiliations
                [001]Department of Bioscience and Biotechnology, Konkuk University, Seoul, Republic of Korea
                Inha University, REPUBLIC OF KOREA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-6886-7589
                Article
                PONE-D-17-30023
                10.1371/journal.pone.0191018
                5764348
                29324789
                b06103f4-3e17-4382-98cb-e60a0e0c9efe
                © 2018 Choi et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 August 2017
                : 27 December 2017
                Page count
                Figures: 6, Tables: 3, Pages: 13
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003627, Rural Development Administration;
                Award ID: PJ01222601
                Award Recipient :
                This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ01222601), Rural Development Administration, Republic of Korea.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Physical Sciences
                Chemistry
                Chemical Reactions
                Hydrolysis
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Alignment
                Research and Analysis Methods
                Chromatographic Techniques
                Liquid Chromatography
                High Performance Liquid Chromatography
                Biology and Life Sciences
                Organisms
                Archaea
                Sulfolobus
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Mutagenesis and Gene Deletion Techniques
                Site-Directed Mutagenesis
                Research and Analysis Methods
                Molecular Biology Techniques
                Mutagenesis and Gene Deletion Techniques
                Site-Directed Mutagenesis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
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                Pyrococcus
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