Optimization of panax notoginseng root extract hydrolysis by cordyceps militaris derived glycosidase and bioactivities of hydrolysis products

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY SIMURABIYE JEAN BAPTISTE OPTIMIZATION OF Panax notoginseng ROOT EXTRACT HYDROLYSIS BY Cordyceps militaris DERIVED GLYCOSIDASE AND BIOACTIVITIES OF HYDROLYSIS PRODUCTS MASTER THESIS KHANH HOA – 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY SIMURABIYE JEAN BAPTISTE OPTIMIZATION OF Panax notoginseng ROOT EXTRACT HYDROLYSIS BY Cordyceps militaris DERIVED GLYCOSIDASE AND BIOACTIVITIES OF HYDROLYSIS PRODUCTS MASTER THESIS Major: Food Technology Topic allocation Decision 192 /QD-DHNT Decision on establishing the Committee: Defense date: 17/09/2020 Supervisors: Dr Nguyen Duc Doan Dr Le Thi Hoang Yen Dr Trinh Dac Hoanh Chairman: Faculty of Graduate Studies: KHANH HOA – 2020 UNDERTAKING I undertake that the thesis entitled: “optimization of Panax notoginseng root extract hydrolysis by Cordyceps militaris derived glycosidase and bioactivities of hydrolysis products” is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted Khanh Hoa, 27/08/2020 SIMURABIYE Jean Baptiste iii ACKNOWLEDGMENTS I appreciate the conditions provided by Nha Trang University, especially faculty of Food Technology for this master program to be completed I especially thank my supervisors, Dr Nguyen Duc Doan; Lecturer at Vietnam National University of Agriculture for selflessly contributing to the progress of this thesis project until completion, and Dr Le Thi Hoang Yen; Member of the Institute of Microbiology and Biotechnology of the Vietnam National University for theoretically and practically guiding me and providing all the assistance that was required in every step of this thesis I would like to extend my gratitude to Dr Trinh Dac Hoanh from the Institute of Chemistry and Material Science of Military Academy of Science and Technology for his technical and financial contribution towards this project, and Dr Duy Nhan Vu from the Institute of Chemistry and material science of Military Academy of Science and Technology for financing my whole length of stay in Hanoi In no particular order, I would also like to acknowledge the contribution of Miss Nguyen Thi Huong Nhu, Miss Hoang Anh Dong, Miss Thanh Tran Huyen and Miss Le Hong Anh and eventually express my appreciation Lastly, I dedicate this work to each of my family members for the love and support This thesis was supported by “Đề án phát triển ứng dụng công nghệ sinh học lĩnh vực công nghiệp chế biến” (grant number DT.11.19/CNSHCB) Khanh Hoa, 27/08/2020 SIMURABIYE Jean Baptiste iv CONTENTS UNDERTAKING iii ACKNOWLEDGMENTS iv CONTENTS v LIST OF SYMBOLS viii LIST OF ABBREVIATIONS xii LIST OF TABLES xiii LIST OF FIGURES xiv ABSTRACT xvi Chapter 1: INTRODUCTION 1.1 Overview 1.2 Objectives 1.2.1 Overall Objective 1.2.2 Specific Objective Chapter 2: REVIEW OF THE LITERATURE 2.1 Ginseng and ginsenosides 2.1.1 History, evolution, botanical characteristics and use 2.1.2 Ginseng medicinal compounds, their structures, types and distribution in plant parts 2.1.1 Ginsenoside metabolism 11 2.2 Bioavailability of ginsenosides 13 2.2.1 Methods of biological conversion 13 2.2.2 Ginsenosides as antimicrobial agents 14 2.2.3 Anti-cancer and immune-modulatory effects of ginsenosides 15 2.2.3.1 Anti-cancer cell mediated immune system modulation by ginsenosides 16 v 2.2 Mushroom beta glucosidase 24 2.2.1 Overview 24 2.2.2 Properties of mushroom β-glucosidase 27 Chapter 3: MATERIAL AND METHODS 30 3.1 Material 30 3.1.1 Methods 32 3.1.2 Total saponin content determination 33 3.1.3 TLC analysis 33 3.1.4 HPLC analysis 33 3.2 Mushroom β-glucosidase 34 3.2.1 Mushroom cultivation and enzyme harvest 34 3.2.2 Mushroom enzyme purification 34 3.3 Mushroom enzyme activity assay 34 3.3.1 CMCase activity of mushroom enzyme 34 3.3.2 Enzyme activity test by DNS method 35 3.4 Ginsenoside fermentation 36 3.5 Bioactivities 38 3.5.1 DPPH Scavenging activity 38 3.5.2 Antibacterial activities 39 3.5.3 Cytotoxicity assay 39 3.5.4 Data analysis 40 Chapter 4: RESULTS AND DISCUSSION 41 4.1 Ginsenoside extraction 44 4.1.1 Extraction yield 44 4.1.2 Saponin content of P notoginseng fractions and crude extract 45 vi 4.2 Ginsenoside fermentation 45 4.3 Bioactivities 58 4.3.1 DPPH scavenging activity 58 4.3.2 Antibacterial activity 60 4.3.3 Cytotoxic activity 62 REFERENCES 65 APPENDICES I vii LIST OF SYMBOLS (NH4)2SO4: Ammonium sulphate ∙OH: Hydroxyl radical Araf: Arabinofuranose Arap: Arabopyranose Bcl-XL: B cell lymphoma-extra large C: Carbon C-20 : Carbon 20 C-3 : Carbon C-6: Carbon C-20: Carbon 20 Ca : Calcium CaCl2: Calcium chloride CaCO3: Calcium carbonate cAMP: Cyclic adenosine monophosphate CCL2: c-c motif ligand CCl5: Chemokine ligand C-FLIP: Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein CHK1: Checkpoint kinase 1gene CHK2: Checkpoint kinase 1gene Ck: Ginsenoside Compound K CMC: Carboxymethylcellulose CMCase: Carboxymethyl cellulase CO2: Carbon dioxide CT26: A murine colorectal carcinoma cell line Cu: Copper DCs: Dendritic cells DNA: Deoxy-ribonucleic acid F1: Ginsenoside F1 F2: Ginsenoside F2 Fe: Iron viii FeSO4: Iron sulphate Glc: Glucose GM-CSF: Granulocyte-macrophage colony-stimulating factor H2Ax: H2Ax gene HOCl Hydrochloric radical IBD: Inflammatory Bowel Disease IFN- Interferon Gamma IL-1: Interleukin-1 IL-10: Interleukin-10 IL-12 : Interleukin-12 IL-1:  Interleukin-1 IL-23: Interleukin-23 IL-35: Interleukin-35 IL-6: Interleukin-6 iNOS: Indicible nitric oxide synthase IP-10: Inducible protein-10 (also CCL10) K: Potassium K2HPO4: Dipotassium phospkhate KCl: Potassium chloride LOX : Lysyl oxidase LPS: Lipopolysaccharide M1: 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol MCP-1: Monocyte chemo-attractant protein-1 (also CCL2) MCs: Mast cells MDSC: Myeloid-derived suppressor cells MgSO4.7H0: Magnesium sulphate heptahydrate MHC-1: Major Histocompatibility class MHC-2: Major Histocompatibility class MiR-146a: MicroRNA-146a MMP-2: Matrix metalloproteinase-2 MMP-7: 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0.0051x - 0.0082 R² = 0.9985 Absorbance (530 nm) 0.5 0.4 0.3 0.2 0.1 0 -0.1 20 40 60 80 100 120 Aescin concentration (mg/L) 0, 20, 40, 60, 80, 100, and 120 mg/L concentrations of aescin were mixed in volumes of 25µL with 25µL vanillin solution and 50 µL sulphuric acid solution Saponin content was expressed as equivalent of aescin I Appendix 3: DNS data NO Mushroom 741 MPG14 Hy01 Gano2 F2 10 11 COTK 12 13 Than 14 EXPERIMENT Absorbance Reducing Sugar content medium sample control sample control 0.656 0.922 0.943914 0.665059231 0.675 0.972 0.996331 0.684977461 0.412 0.528 0.530873 0.409267219 0.647 0.747 0.760457 0.655624279 0.654 0.831 0.848517 0.662962575 0.693 0.879 0.898836 0.703847363 0.686 0.805 0.82126 0.696509068 0.417 0.477 0.477409 0.414508858 0.669 0.81 0.826502 0.678687493 0.501 0.72 0.732152 0.502568403 0.646 0.882 0.901981 0.654575951 0.685 0.729 0.741587 0.69546074 0.644 0.702 0.713282 0.652479296 0.556 0.667 0.676591 0.560226439 activity 1.239357 1.383793 0.540471 0.465924 0.824685 0.866618 0.554449 0.279554 0.656952 1.020373 1.09958 0.205006 0.270236 0.517175 EXPERIMENT Absorbance NO Mushroom medium sample control 741 0.486 0.242 2 0.8 0.48 MPG14 0.634 0.504 0.672 0.563 Hy01 0.634 0.462 0.778 0.608 Gano2 0.511 0.388 0.479 0.433 F2 0.63 0.504 10 0.587 0.38 11 COTK 0.72 0.501 12 0.583 0.524 13 Than 0.662 0.613 14 0.699 0.591 II Reducing sugar content sample control 0.486805 0.231051473 0.815954 0.480553517 0.641946 0.505713387 0.681779 0.567564734 0.641946 0.461683615 0.792893 0.614739491 0.513011 0.384107349 0.479467 0.431282105 0.637753 0.505713387 0.592678 0.375720725 0.732095 0.502568403 0.588485 0.526679945 0.671296 0.61998113 0.710082 0.596917916 activity 1.136683 1.49067 0.605476 0.507618 0.801164 0.791793 0.572907 0.214157 0.586841 0.964255 1.020117 0.274689 0.228068 0.502949 Absorbance EXPERIMENT Reducing Sugar content sample control sample 0.3507 0.113 0.345005 0.427 0.107 0.424992 0.315 0.204 0.307579 0.312 0.199 0.304434 0.39 0.198 0.386204 0.348 0.19 0.342174 0.371 0.238 0.366286 0.296 0.252 0.287661 0.229 0.112 0.217423 0.399 0.201 0.395639 0.454 0.243 0.453297 0.352 0.297 0.346368 0.273 0.229 0.26355 0.465 0.335 0.464829 control 0.095817172 0.089527204 0.191215012 0.185973372 0.184925045 0.176538421 0.226858161 0.241534752 0.094768844 0.188070028 0.232099801 0.288709508 0.21742321 0.328545969 activity 1.107500204 1.49095526 0.517175106 0.526493576 0.894573156 0.736159159 0.61967828 0.205006348 0.545130517 0.922528567 0.983098624 0.256257935 0.205006348 0.605700574 Average activity STD 1.161180013 0.069257 1.455139414 0.061788 0.554374213 0.045763 0.500011829 0.030993 0.840140638 0.048585 0.798189938 0.065464 0.582344642 0.033623 0.232905911 0.040657 0.596307818 0.056509 0.969051883 0.049098 1.034264936 0.059515 0.245317839 0.036107 0.234436645 0.033078 0.541941649 0.055673 Appendix 4: Antioxidant results NO OD exp Activity 0.65 27.77778 0.63 30 0.634 29.55556 0.59 34.44444 0.6 33.33333 0.66 26.66667 0.7 22.22222 0.645 28.33333 0.599 33.44444 10 0.711 21 11 0.701 22.11111 12 0.699 22.33333 13 0.773 14.11111 14 0.694 22.88889 15 0.709 21.22222 16 0.689 23.44444 17 0.689 23.44444 18 0.699 22.33333 19 0.698 22.44444 20 0.688 23.55556 Antioxidant activity OD OD exp2 Activity exp3 0.66 26.74806 0.655 0.62 31.11111 0.625 0.64 28.88889 0.637 0.6 33.33333 0.595 0.605 32.77778 0.6025 0.654 27.33333 0.657 0.71 21.11111 0.705 0.64 28.88889 0.6425 0.61 32.22222 0.6045 0.719 20.11111 0.715 0.689 23.44444 0.695 0.71 21.11111 0.7045 0.779 13.44444 0.776 0.701 22.11111 0.6975 0.703 21.88889 0.706 0.694 22.88889 0.6915 0.7 22.22222 0.6945 0.713 20.77778 0.706 0.707 21.44444 0.7025 0.699 22.33333 0.6935 III Activity 27.46401 30.55556 29.22222 33.88889 33.05556 27 21.66667 28.61111 32.83333 20.55556 22.77778 21.72222 13.77778 22.5 21.55556 23.16667 22.83333 21.55556 21.94444 22.94444 average 27.32995 30.55556 29.22222 33.88889 33.05556 27 21.66667 28.61111 32.83333 20.55556 22.77778 21.72222 13.77778 22.5 21.55556 23.16667 22.83333 21.55556 21.94444 22.94444 Std 0.527788 0.555556 0.333333 0.555556 0.277778 0.333333 0.555556 0.277778 0.611111 0.444444 0.666667 0.611111 0.333333 0.388889 0.333333 0.277778 0.611111 0.777778 0.5 0.611111 21 22 24 25 26 27 28 29 30 31 32 0.597 0.611 0.662 0.664 0.694 0.692 0.6 0.721 0.599 0.731 0.71 0.752 33.66667 32.11111 26.44444 26.22222 22.88889 23.11111 33.33333 19.88889 33.44444 18.77778 21.11111 16.44444 0.61 0.623 0.673 0.673 0.708 0.699 0.607 0.727 0.609 0.721 0.72 0.747 32.22222 30.77778 25.22222 25.22222 21.33333 22.33333 32.55556 19.22222 32.33333 19.88889 20 17 IV 0.6035 0.617 0.6675 0.6685 0.701 0.6955 0.6035 0.724 0.604 0.726 0.715 0.7495 32.94444 31.44444 25.83333 25.72222 22.11111 22.72222 32.94444 19.55556 32.88889 19.33333 20.55556 16.72222 32.94444 31.44444 25.83333 25.72222 22.11111 22.72222 32.94444 19.55556 32.88889 19.33333 20.55556 16.72222 0.722222 0.666667 0.611111 0.5 0.777778 0.388889 0.388889 0.333333 0.555556 0.555556 0.555556 0.277778 Appendix 5: Antibacterial results P.aeruginosa No 10 11 12 13 14 15 16 17 18 19 20 21 22 Abs Run % inhibition 0.667 10.58981233 0.695 6.836461126 0.718 3.753351206 0.735 1.474530831 0.713 4.423592493 0.821 -10.0536193 0.725 2.815013405 0.705 5.495978552 0.679 8.981233244 0.693 7.104557641 0.705 5.495978552 0.68 8.847184987 0.803 -7.64075067 0.704 5.63002681 0.716 4.021447721 0.696 6.702412869 0.721 3.351206434 0.742 0.536193029 0.74 0.804289544 0.739 0.938337802 0.762 -2.144772118 0.737 1.206434316 at hour At 20th hour Abs AVG Abs Abs AVG Run2 % inhibition inhibition Run % inhibition Run2 % inhibition inhibition 1.248 -73.09292649 -31.25155708 0.807 9.630459127 0.824 10.5320304 10.08124476 0.772 -7.073509015 -0.118523945 0.799 10.52631579 0.828 10.09771987 10.31201783 0.696 3.46740638 3.610378793 0.805 9.854423292 0.825 10.42345277 10.13893803 0.609 15.53398058 8.504255707 0.821 8.062709966 0.841 8.686210641 8.374460304 0.668 7.350901526 5.887247009 0.89 0.335946249 0.92 0.108577633 0.222261941 0.659 8.599167822 -0.72722574 0.812 9.070548712 0.831 9.771986971 9.421267841 0.622 13.73092926 8.272971335 0.861 3.583426652 0.876 4.885993485 4.234710069 0.572 20.66574202 13.08086029 0.814 8.846584546 0.833 9.554831705 9.200708126 0.621 13.86962552 11.42542938 0.808 9.518477044 0.838 9.01194354 9.265210292 0.638 11.51178918 9.308173411 0.86 3.695408735 0.891 3.25732899 3.476368862 0.581 19.41747573 12.45672714 0.864 3.247480403 0.88 4.451682953 3.849581678 0.631 12.48266297 10.66492398 0.851 4.70324748 0.883 4.125950054 4.414598767 0.593 17.75312067 5.056184998 0.863 3.359462486 0.878 4.668838219 4.014150353 0.604 16.22746186 10.92874433 0.808 9.518477044 0.836 9.229098806 9.373787925 0.636 11.78918169 7.905314707 0.885 0.895856663 0.908 1.411509229 1.153682946 0.611 15.25658807 10.97950047 0.811 9.182530795 0.827 10.2062975 9.694414149 0.654 9.292649098 6.321927766 0.891 0.223964166 0.911 1.08577633 0.654870248 0.662 8.183079057 4.359636043 0.821 8.062709966 0.862 6.406080347 7.234395157 0.61 15.39528433 8.099786936 0.84 5.935050392 0.876 4.885993485 5.410521939 0.721 0.469168901 0.875 2.015677492 0.893 3.040173724 2.527925608 0.731 -1.386962552 -1.765867335 0.842 5.711086226 0.86 6.623235613 6.16716092 0.718 0.416088766 0.811261541 0.811 9.182530795 0.847 8.034744843 8.608637819 V 23 24 25 26 27 28 10 11 12 13 14 15 16 17 18 0.76 -1.876675603 0.667 10.58981233 0.712 4.557640751 0.803 -7.64075067 0.746 0.746 Abs % Run inhibition 1.006 8.211679 1.042 4.927007 1.006 8.211679 1.037 5.383212 1.059 3.375912 0.98 10.58394 1.013 7.572993 0.965 11.95255 0.991 9.580292 1.011 7.755474 1.018 7.116788 1.073 2.09854 1.013 7.572993 1.112 -1.45985 1.047 4.470803 0.973 11.22263 1.033 5.748175 1.058 3.467153 0.759 0.662 0.63 0.817 0.721 0.654 -5.270457698 8.183079057 12.62135922 -13.3148405 -3.57356665 9.386445695 8.589499987 -10.47779558 0.845 0.864 0.852 0.89 0.893 0.85 5.375139978 3.247480403 4.591265398 0.335946249 0.873 0.892 0.877 0.913 0.921 0.801 5.211726384 3.148751357 4.777415852 0.868621064 5.293433181 3.19811588 4.684340625 0.602283656 S.aureus at hour 20th hour Abs Abs Abs % Run2 % inhibition average Run % inhibition Run2 inhibition average STD 1.058 10.1104503 9.161064565 1.049 20.6505295 1.096 21.43369 21.0421106 0.39158113 1.087 7.64655905 6.286783174 0.98 25.8698941 1.027 26.37993 26.1249112 0.25501711 1.079 8.32625319 8.268966009 1.012 23.44931921 1.059 24.08602 23.7676704 0.31835115 1.052 10.6202209 8.00171629 0.928 29.80332829 0.963 30.96774 30.3855351 0.58220682 1.075 8.66610025 6.021006332 1.027 22.31467474 1.098 21.29032 21.8024987 0.51217608 1.036 11.9796092 11.28177539 0.943 28.66868381 0.976 30.03584 29.3522631 0.68357924 1.04 11.6397621 9.606377404 1.04 21.33131619 1.12 19.71326 20.5222889 0.80902727 0.997 15.2931181 13.62283642 1.001 24.28139183 1.033 25.94982 25.1156063 0.83421448 983 -83417.4172 -41703.9184 0.949 28.21482602 0.984 29.46237 28.8385958 0.62376979 0.985 16.3126593 12.03406688 1.081 18.22995461 1.131 18.92473 18.5773429 0.34738828 1.23 -4.50297366 1.30690733 1.104 16.49016641 1.148 17.70609 17.0981298 0.60796339 1.029 12.5743415 7.336440846 1.116 15.58245083 1.159 16.91756 16.2500068 0.66755595 0.708 39.8470688 23.71003076 1.09 17.54916793 1.13 18.99642 18.2727918 0.72362392 1.101 6.45709431 2.498620146 1.129 14.59909228 1.216 12.83154 13.7153168 0.88377553 1.05 10.7901444 7.630473677 1.12 15.27987897 1.161 16.77419 16.0270363 0.74715729 1.039 11.7247239 11.47367581 0.995 24.73524962 1.073 23.08244 23.9088434 0.82640617 1.069 9.17587086 7.46202302 1.059 19.89409985 1.141 18.20789 19.0509926 0.84310727 1.042 11.4698386 7.468495929 1.069 19.1376702 1.105 20.78853 19.9631003 0.82543013 VI 19 20 21 22 23 24 25 26 27 28 1.091 1.033 1.046 1.062 1.023 1.01 1.03 1.167 1.096 0.906 0.456204 5.748175 4.562044 3.10219 6.660584 7.846715 6.021898 -6.4781 1.047 11.0450297 1.074 8.75106202 1.063 9.68564146 1.068 9.26083263 1.23 -4.50297366 1.09 7.39167375 1.02 13.3389975 1.286 -9.26083263 1.177 1.1365 1.001 0.9535 5.750617058 7.249618602 7.123842628 6.181511203 1.07880514 7.619194538 9.680447631 -7.86946741 1.041 1.066 1.046 1.07 1.165 1.101 1.21 1.301 1.322 1.23 21.25567322 19.36459909 20.8774584 19.06202723 11.87594554 16.71709531 8.472012103 1.588502269 1.116 1.132 1.092 1.126 1.21 1.15 1.26 1.394 1.395 1.01 20 18.85305 21.72043 19.28315 13.26165 17.56272 9.677419 0.071685 1.3585 1.12 20.6278366 19.1088228 21.2989443 19.1725907 12.5687971 17.1399097 9.07471573 0.83009343 0.62783661 0.25577625 0.42148586 0.11056345 0.6928516 0.42281435 0.60270363 0.75840884 E coli at h 10 11 12 13 14 Abs Abs % Run % inhibition Run2 inhibition average 0.921 6.211812627 0.891 6.012658 6.11223543 0.912 7.128309572 0.924 2.531646 4.82997757 0.92 6.313645621 0.911 3.902954 5.1082996 0.915 6.822810591 0.923 2.637131 4.7299707 0.9 8.350305499 0.966 -1.89873 3.22578566 0.9 8.350305499 0.916 3.375527 5.86291646 0.857 12.72912424 0.935 1.371308 7.05021613 0.935 4.786150713 0.893 5.801688 5.29391924 0.85 13.44195519 0.926 2.320675 7.88131515 0.875 10.89613035 0.943 0.527426 5.71177825 0.905 7.84114053 0.97 -2.32068 2.76023271 0.898 8.553971487 0.996 -5.06329 1.74534017 1.049 -6.82281059 0.977 -3.05907 -4.9409412 0.997 -1.52749491 0.954 -0.63291 -1.0802032 Abs Run 0.935 0.915 0.915 0.867 0.85 0.922 0.837 0.926 0.87 0.889 1.002 1.06 0.961 0.89 VII at 20th hour % % inhibition ARun2 inhibition average STD 18.269231 0.913 20.05254 19.16088509 0.891654 20.017483 0.897 21.45359 20.73553636 0.718054 20.017483 0.903 20.9282 20.47283933 0.455357 24.213287 0.85 25.56918 24.8912318 0.677945 25.699301 0.866 24.16813 24.9337134 0.765587 19.405594 0.903 20.9282 20.16689528 0.761301 26.835664 0.851 25.48161 26.15863777 0.677027 19.055944 0.941 17.6007 18.32832229 0.727622 23.951049 0.888 22.24168 23.09636511 0.854684 22.29021 0.872 23.64273 22.96647092 0.676261 12.412587 1.008 11.7338 12.07319388 0.339394 7.3426573 1.046 8.406305 7.874481036 0.531824 15.996503 0.942 17.51313 16.75481917 0.758316 22.202797 0.879 23.02977 22.61628477 0.413488 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1.02 0.892 0.933 0.901 0.947 0.905 0.951 0.917 0.913 0.8 0.9 0.974 0.982 0.909 -3.86965377 9.16496945 4.989816701 8.248472505 3.564154786 7.84114053 3.156822811 6.619144603 7.026476578 18.53360489 8.350305499 0.814663951 0.904 0.951 0.981 0.938 0.938 1.283 0.923 1.119 1.014 0.9 0.86 1.016 0.948 0.841 4.64135 -0.31646 -3.48101 1.054852 1.054852 -35.3376 2.637131 -18.038 -6.96203 5.063291 9.2827 -7.173 0.965 0.875 0.38584822 4.42425688 0.75440202 4.65166241 2.30950355 -13.748206 2.89697681 -5.709415 0.03222563 11.798448 8.81650296 -3.1791659 0.991 0.789 0.865 0.925 0.943 0.941 0.933 0.915 0.942 0.911 0.891 0.967 1.144 1.028 13.374126 31.031469 24.388112 19.143357 17.56993 17.744755 18.444056 20.017483 17.657343 20.367133 22.115385 15.472028 1.007 0.768 0.882 0.911 0.957 0.956 0.945 0.928 0.957 0.924 0.903 0.981 1.142 0.988 11.82137 32.74956 22.76708 20.22767 16.19965 16.28722 17.25044 18.73905 16.19965 19.08932 20.9282 14.09807 1.143 1.008 12.59774595 31.89051535 23.5775936 19.6855137 16.8847899 17.01598533 17.84724689 19.3782684 16.9284962 19.72822493 21.52179038 14.78505076 11.81102362 0.77638 0.859047 0.810518 0.542157 0.68514 0.72877 0.596809 0.639214 0.728846 0.638908 0.593594 0.686977 From to 25 are hydrolysates of ginsenosides by C militaris, 26 is a crude extract, 27 and 28 are negative and positive control respectively VIII ...MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY SIMURABIYE JEAN BAPTISTE OPTIMIZATION OF Panax notoginseng ROOT EXTRACT HYDROLYSIS BY Cordyceps militaris DERIVED GLYCOSIDASE AND BIOACTIVITIES. .. of Graduate Studies: KHANH HOA – 2020 UNDERTAKING I undertake that the thesis entitled: ? ?optimization of Panax notoginseng root extract hydrolysis by Cordyceps militaris derived glycosidase and. .. activity of P notoginseng hydrolysis products by C militaris derived glycosidase 62 xiii LIST OF FIGURES Figure 2.1 Core structures of ginsenosides: A; PPD-type, B; PPT-type and C;