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The effect of various dehydration techniques on ginsenoside recovery from North American ginseng and… Purnama, Monica 2009

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THE EFFECT OF VARIOUS DEHYDRATION TECHNIQUES ON GINSENOSIDE RECOVERY FROM NORTH AMERICAN GINSENG AND BIOACTIVE PROPERTIES ON 3T3-L1 ADIPOSE TISSUE CELL LINE  by  MONICA PURNAMA B.Sc., The University of Manitoba, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Food Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2009  © Monica Purnama, 2009  ABSTRACT Three dehydration techniques, applied to North American ginseng roots (NAG), were evaluated to determine the subsequent pore characteristics, recovery of the ginsenoside content, and the related bioactive properties on a cultured 3T3-L1 fat cell line. Fresh North American ginseng roots were shredded prior to air drying (AD), freeze drying (FD), and vacuum-microwave drying (VMD) processes. Total porosities and pore distribution measurements were determined using a mercury porosimeter. Among samples, FD ginseng obtained the highest total porosity followed by VMD and AD, respectively. All dehydrated samples showed a porous structure with pore sizes ranged from 0.002 µm to 172 µm. Dried ginseng root matrices, regardless of dehydration methods, were mainly constituted by macropores (>1.5µm). Pore characteristics of dried ginseng roots may affect the ginsenosides exposure with extraction solvent. As total porosity increases, the total ginsenoside content may also increase. Therefore, ginsenoside content of dried NAG following methanolic extraction was determined to evaluate the relationship between dried NAG pore characteristics and the subsequent ginsenoside extraction. Ginsenoside composition was determined using High Performance Liquid Chromatography (HPLC). Total ginsenoside content, derived from the seven individual ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3), were affected by the dehydration methods employed. AD process recovered the lowest amount of total ginsenosides, followed by VMD and FD processes, which recovered relatively similar amounts of total ginsenosides. The comparable total ginsenoside amounts of VMD and FD ginseng root ii  extracts suggests that the relationship between pore characteristics and the subsequent extraction of bioactive component was highly dependent on the dried food material. The bioactivity potential of all ginseng extracts on 3T3-L1 adipose tissue cells was exhibited following AD, VMD, and FD processes. Dried North American ginseng root extracts (NAGEs), regardless of dehydration techniques, affected the viabilities of pre-confluent and post-confluent preadipocytes as well as mature adipocyte cells. Moreover, dried NAGEs at non-toxic levels were able to inhibit adipogenesis when added to both pre- and post-confluent preadipocytes. These results, therefore, showed that dehydration techniques affected the pore characteristics and the subsequent ginsenoside recoveries of NAG, and the dried NAGEs exhibited bioactive potential on 3T3-L1 adipose tissue cells.  iii  TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii TABLE OF CONTENTS................................................................................................... iv LIST OF TABLES............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF ABBREVIATIONS............................................................................................. x ACKNOWLEDGEMENTS.............................................................................................. xii DEDICATION................................................................................................................. xiii GENERAL INTRODUCTION........................................................................................... 1 RESEARCH HYPOTHESES AND OBJECTIVES....................................................... 4 RESEARCH ACTIVITIES............................................................................................. 6 Experiment I: Drying and Recovery of Ginsenosides in Shredded Dehydrated North American Ginseng Roots.................................................................................... 6 Experiment II: Assessment of Bioactive Potential of Recovered Ginsenosides in Dehydrated North American Ginseng Roots on 3T3-L1 Fat Cell Line ............. 7 CHAPTER 1: LITERATURE REVIEW ............................................................................ 8 1.1 History and Cultivation of Ginseng .......................................................................... 8 1.2 Types of Ginseng .................................................................................................... 11 1.2.1 Asian Ginseng.................................................................................................. 11 1.2.2 North American Ginseng ................................................................................. 12 1.3 Ginsenosides ........................................................................................................... 12 1.4 Composition Analysis of Ginseng .......................................................................... 16 1.4.1 Thin Layer Chromatography (TLC) ................................................................ 17 1.4.2 High-Performance Liquid Chromatography (HPLC) ...................................... 18 1.4.3 Liquid Chromatography/Mass Spectrometry (LC/MS) ................................... 20 1.5 Bioactivities of Ginseng.......................................................................................... 21 1.5.1 Antioxidative Properties .................................................................................. 21 1.5.2 Antidiabetic Properties..................................................................................... 22 1.5.3 Antihypercholesterolemic Properties............................................................... 24 1.5.4 Antiobesity Properties...................................................................................... 25 1.6 Drying Methods ...................................................................................................... 27 1.6.1 Air Drying........................................................................................................ 28 1.6.2 Vacuum Microwave Drying ............................................................................ 29 1.6.3 Freeze-Drying .................................................................................................. 31 1.7 Cell Culture Activities for Modelling Fat Metabolism........................................... 32 iv  1.7.1 Preadipocyte Cell (3T3-L1) ............................................................................. 32 CHAPTER 2: COMPARISON OF DIFFERENT DEHYDRATION TECHNIQUES ON POROUS STRUCTURE AND GINSENOSIDE RETENTION OF NORTH AMERICAN GINSENG ......................................................................................................................... 35 2.1 INTRODUCTION .................................................................................................. 35 2.2 MATERIALS AND METHODS............................................................................ 37 2.2.1 Drying of North American Ginseng Roots ...................................................... 37 2.2.1.1 Air-Drying................................................................................................. 37 2.2.1.2 Vacuum Microwave-Drying ..................................................................... 38 2.2.1.3 Freeze-Drying ........................................................................................... 38 2.2.2 Extraction of North American Ginseng Roots................................................. 39 2.2.3 North American Ginseng Roots Characterization ........................................... 39 2.2.3.1 Moisture Content ...................................................................................... 39 2.2.3.2 Total Porosity and Pore Distribution ........................................................ 40 2.2.4 High Performance Liquid Chromatography Analysis of Recovered Ginsenoside Content from Dehydrated North American Ginseng Roots ........ 41 2.2.5 Statistical Analysis........................................................................................... 42 2.3 RESULTS ............................................................................................................... 42 2.3.1 Dehydration of North American Ginseng Roots ............................................. 42 2.3.2 Effect of Dehydration Methods on Total Porosity and Pore Distributions of North American Ginseng Roots ....................................................................... 46 2.3.3 Identification and Quantification of Ginsenosides by High Performance Liquid Chromatography (HPLC) Analysis .................................................................. 48 2.4 DISCUSSION ......................................................................................................... 52 2.5 CONCLUSION....................................................................................................... 57 CHAPTER 3: COMPARISON OF DIFFERENT DEHYDRATION TECHNIQUES ON THE BIOACTIVE POTENTIAL OF NORTH AMERICAN GINSENG ON CULTURED 3T3-L1 FAT CELL LINE .......................................................................... 59 3.1 INTRODUCTION .................................................................................................. 59 3.2 MATERIALS AND METHODS............................................................................ 61 3.2.1 Extraction of North American Ginseng Roots................................................. 61 3.2.2 3T3-L1 Cell Culture Analyses ......................................................................... 62 3.2.2.1 Cell Proliferation....................................................................................... 62 3.2.2.2 Cell Viability Assay.................................................................................. 62 3.2.2.3 Adipogenesis Procedural Assay................................................................ 64 3.2.2.4 Oil-Red-O Staining Assay ........................................................................ 65 3.2.2.5 Lactate Dehydrogenase (LDH) Assay ...................................................... 65 3.2.2.6 Effect of Individual Ginsenoside Standards on 3T3-L1 Cell Adipogenesis ............................................................................................................................... 66 3.2.3 Statistical Analysis........................................................................................... 67 3.3 RESULTS ............................................................................................................... 67 3.3.1 Effect of Different Dehydration Methods of North American Ginseng Roots on 3T3-L1 Preadipocyte Cell Viability ................................................................. 67  v  3.3.2 Effect of Different Dehydration Methods of North American Ginseng Roots on 3T3-L1 Mature Adipocyte Cell Viability......................................................... 73 3.3.3 Effect of Different Dehydration Methods of North American Ginseng Roots on Adipogenesis of 3T3-L1 Cell ........................................................................... 76 3.4 DISCUSSION ......................................................................................................... 82 3.5 CONCLUSION....................................................................................................... 85 GENERAL CONCLUSION ............................................................................................. 87 REFERENCES ................................................................................................................. 90 APPENDIX A: HPLC METHOD FOR DETERMINATION OF GINSENOSIDE AMOUNT RECOVERED IN DRIED NORTH AMERICAN GINSENG.................... 109 A.1 North American Ginseng Roots........................................................................... 109 A.2 Chemicals and Equipment ................................................................................... 109 A.3 Determination of HPLC Method Parameters....................................................... 110 A.4 Ginsenoside Concentration in Dried North American Ginseng Root Extract ..... 113 APPENDIX B: IC50 DETERMINATION OF DRIED NORTH AMERICAN GINSENG ROOT EXTRACT ON 3T3-L1 FAT CELL................................................................... 114 B.1 Determination of IC50 Parameters ........................................................................ 114 B.1 Determination of IC50 of Dried North American Ginseng Root Extract.............. 115 APPENDIX C: LDH ACTIVITY DETERMINATION OF DRIED NORTH AMERICAN GINSENG ROOT EXTRACT ON 3T3-L1 FAT CELL.......................... 116 C.1 Determination of LDH Activity Parameters ........................................................ 116 C.2 Determination of LDH Activity of Dried North American Ginseng Root Extract ..................................................................................................................................... 117  vi  LIST OF TABLES Table 1.1 Individual ginsenosides of 20(s)-protopanaxadiol and 20(s)-protopanaxatriol groups................................................................................................................................ 14 Table 1.2 Ginsenoside of oleanolic acid group................................................................. 15 Table 2.1 Moisture content and water activity of fresh and dried North American ginseng root .................................................................................................................................... 45 Table 2.2 Total porosity (%) and pore distribution (%) of NAG after various drying methods ............................................................................................................................. 48 Table 2.3 Individual ginsenoside (mg/g dry solid) of North American ginseng extract (NAGE) recovered in ginseng root following various dehydration methods ................... 52 Table 3.1 IC50 of NAGE on pre-confluent preadipocyte 3T3-L1 cells............................. 72 Table 3.2 IC50 of NAGE on post-confluent preadipocyte 3T3-L1 cells ........................... 72 Table 3.3 IC50 of NAGE on mature adipocyte 3T3-L1 cells ............................................ 76 Table 3.4 The effect of NAGE on adipogenesis of pre-confluent preadipocyte 3T3-L1 cells measured by Oil-Red-O staining assay .................................................................... 79 Table 3.5 The effect of NAGE on adipogenesis of post-confluent preadipocyte 3T3-L1 cells measured by Oil-Red-O staining assay .................................................................... 79 Table 3.6 The effect of individual ginsenosides on 3T3-L1 cell adipogenesis ................ 81  vii  LIST OF FIGURES Figure 1.1 Basic structures of 20(s) - protopanaxadiol and 20(s)-protopanaxatriol ginsenosides ...................................................................................................................... 14 Figure 1.2 Basic structure of oleanolic acid ginsenoside.................................................. 15 Figure 2.1 Micromeritics mercury porosimeter Autopore™ IV 9500 Series (A) and a 5ccbulb volume and a 1.131cc-stem volume powder penetrometer (B) ................................ 40 Figure 2.2 Physical appearances of whole, fresh NAG roots (A) and shredded, dehydrated NAG roots after air drying (B), vacuum-microwave drying (C), and freeze drying (D) processes ......................................................................................................... 44 Figure 2.3 Total porosity (%) and pore distribution of NAG due to various drying methods ............................................................................................................................ 47 Figure 2.4 HPLC chromatograms showing separation of individual ginsenosides in dried North American ginseng .................................................................................................. 50 Figure 2.5 Total ginsenosides recovered following various dehydration methods ......... 51 Figure 3.1 The effect of NAGE on pre-confluent 3T3-L1 preadipocyte cells viability measured by MTT assay .................................................................................................. 70 Figure 3.2 The effect of NAGE on post-confluent 3T3-L1 preadipocyte cells viability measured by MTT assay .................................................................................................. 71 Figure 3.3 Lactate dehydrogenase (LDH) activity measured in pre-confluent preadipocyte 3T3-L1 cells after 24, 48 and 72 h of treatment at 1.5mg/mL NAGE ............................. 73 Figure 3.4 The effect of NAGE on 3T3-L1 mature adipocyte cells viability measured by MTT assay ....................................................................................................................... 75 Figure 3.5 Morphologies of mature 3T3-L1 adipocyte cell after stained by Oil-Red-O . 78 Figure 3.6 The effect of NAGE on adipogenesis of 3T3-L1 cells measured by Oil-Red-O staining assay ................................................................................................................... 80 Figure 3.7 The effect of individual ginsenoside standards on 3T3-L1 cell adipogenesis 81 Figure A.1 Calibration curve for ginsenoside Rg1 ......................................................... 110 Figure A.2 Calibration curves for ginsenosides (A) Re, (B) Rb1, and (C) Rc ............... 111 viii  Figure A.3 Calibration curves for ginsenosides (A) Rb2, (B) Rd, and (C) Rg3............. 112 Figure B. 1 Viability of pre-confluent 3T3-L1 preadipocyte cells as measured by MTT assay after treatment by AD – BC ginseng root extract.................................................. 114 Figure C. 1 Absorbance change per minute curve of untreated 3T3-L1 cells (i.e. control) ......................................................................................................................................... 116 Figure C. 2 Absorbance change per minute curve of air-dried North American ginseng root extract treatment on 3T3-L1 cells............................................................................ 117  ix  LIST OF ABBREVIATIONS AAPH AD -Af AMPK -Ap AQH aw t-BAQ BMI CAT CCAAT C/EBPα C/EBPβ C/EBPδ cGMP CPM DMEM DNA DPPH EDTA ERK ESI-MS FD FMOC FOXO1 -G -GA GE(s) GLP-1 GLUT2 GLUT4 GSPx HCl HPLC HPLC-UV HPTLC IBMX IC50 LC/ESI-MS LC/MS LC/MS/MS LDH  2,2’-azobis(2-amidinopropane) dihydrochloride Air drying Arabinofuranose Adenosine monophosphate-activated protein kinase Arabinopyranose 9,10-dihydroxyantracene derivative Water activity 2-tert-butylanthraquinone Body mass index Catalase Cytidine-cytidine-adenosine-adenosine-thymidine CCAAT/enhancer-binding protein-α CCAAT/enhancer-binding protein-β CCAAT/enhancer-binding protein-δ Cyclic guanosine monophosphate Cyclophosphamide Dulbecco’s modified Eagle’s medium Deoxyribonucleic acid 1,1-diphenyl-2-picrylhydrazyl Ethylenediaminetetraacetic acid Extracellular signal-regulated kinase Electrospray ionization to mass spectrometry Freeze-dried 9-fluorenylmethoxycarbonyl Forkhead transcription factor Forkhead box O1 Glucopyranose Glucuronic acid Ginseng extract(s) Glucagon-like peptide-1 Glucose transporter-2 Glucose transporter-4 Glutathione peroxidase Hydrochloric acid High-performance liquid chromatography Ultraviolet high-performance liquid chromatography High-performance thin layer chromatography 3-isobutyl-1-methylxanthine 50% inhibition concentration Liquid chromatography/electrospray ionization – mass spectrometry Liquid chromatography/mass spectrometry Liquid chromatography/mass spectrometry/mass spectrometry Lactate dehydrogenase x  LDL-C LPL MAPK MEK m-Rb1 m-Rb2 m-Rc m-Rd mRNA MTT NADH NAG(s) NAGE(s) NO NPY ODS PBS PD PEPCK PI3-kinase PPARγ PT PTFE -R Rt R1 R2 R3 SCD1 SCFA SDS ±SEM SOD TAG TFA TLC VMD VMD0.8kW VMD1.3kW VMD1.8kW WAT  Low density lipoprotein – cholesterol Lipoprotein lipase Mitogen-activated protein kinase MAPK/ERK kinase malonyl-ginsenoside Rb1 malonyl-ginsenoside Rb2 malonyl-ginsenoside Rc malonyl-ginsenoside Rd Messenger ribonucleic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Nicotinamide adenine dinucleotide – H North American ginseng(s) North American ginseng root extract(s) Nitric oxide Neuropeptide Y Octadecyl silane Phosphate buffer saline 20(s)-protopanaxadiol Phosphoenolpyruvate carboxykinase Phosphoinositide 3-kinase Peroxisome proliferators-activated receptor-γ 20(s)-protopanaxatriol Polytetrafluoroethylene Rhamnopyranose Retention time Region 1 Region 2 Region 3 Stearoyl-CoA-desaturase-1 Short-chain fatty acid Sodium duodecyl sulphate Standard error of mean Superoxide dismutase Triacylglycerol Trifluoroacetic acid Thin layer chromatography Vacuum-microwave drying/dried Vacuum-microwave drying/dried at 0.8kW Vacuum-microwave drying/dried at 1.3kW Vacuum-microwave drying/dried at 1.8kW White adipose tissue  xi  ACKNOWLEDGEMENTS  Firstly, I would like to thank Father almighty and the Holy Spirit for giving me strength and guidance through all my life and especially through the process of becoming a MSc. at the University of British Columbia. I am deeply indebted to my graduate supervisor, Dr. David Kitts, who gave me the opportunity to work for his research team and who supervised my studies and research project, and helped me in all the time of research and writing of this thesis. I, furthermore, thank my committee members: Dr. Tim Durance, Dr. Chris Scaman, and Dr. Vivien Measday, who gave me valuable advices for the progress of my thesis research. My “family” at the UBC Food Science program who have supported me through this graduate study. Endless gratitude to Dr. Parastoo Yaghmaee, Dr. Charles Hu, Dr. Pedro Aloise, Dr. Aneta Kopec, Valerie Skura, Xiumin Chen, Ingrid Elisia, Katie Du, Steve Tomiuk, Imelda Cheung, Melanie Lam, Jina, Patrick Leung, and Tram Nguyen. I have made friends, better yet family, with all of you and I cannot thank you enough. If I have made some silly mistakes along the way, please forgive my limited human capacity. A special thank to all the people who have believed in me from the beginning of this journey, during the process, until this very end: Dr. Trust Beta, Dr. Susan Arntfield, Dr. Arnie Hydamaka, Dr. Harry Sapirstein, Dr. Gustaaf Sevenhuysen, Ms. Rina Syah, and my family at St. Paul’s College, Winnipeg, MB. My very supportive best friend/housemate: Elaine Linaksita, my endless supporters: Pavle Vrljicak and Antonia Manda, and the whole St. Mark’s crowds: Thank you! Edwin, I would not be able to get through all these without you. Your patience and wisdom are my rock, my foundation, my strength. I love you. Finally, I would like to give my special thanks to my mum and dad, my brother Dr. Dharmawan Ardi Purnama, and my sister Farra Purnama whose patient love and encouragement enabled me to complete this important stage of my life. This is for you all, I love you. xii  DEDICATION  For Papa: For your faith in me at the beginning, now, and always  xiii  GENERAL INTRODUCTION Various diseases and afflictions have been suggested in traditional herbal folklore to be prevented, managed, or treated by individual herbs or herbal cocktails. This belief has contributed to the remarkable popularity of complementary and alternative medicines in the Western world, with herbal remedies outgrowing other alternative treatment methods (Ernst and White, 2000; Eisenberg et al., 1998). Consumers have embraced herbs and other natural health products as natural alternatives to prevent and treat a diverse group of diseases. The herbal and natural health product market has responded to this interest with its collective worth being estimated to be over $4.4 billion in 2005 (Blumenthal, 2006). Moreover, the total 2005 sale of ginseng dietary supplements in the United States was estimated to be approximately US$12 million (Blumenthal, 2006). Ginseng, as a perennial herb, is one of the oldest and most well known traditional herbal ingredients. Moreover, native North Americans have already had a long history of North American ginseng usage as part of their traditional medicinal practice (Court, 2000; Dixon, 1976). This popularity has led to a rejuvenation of ginseng usage in the Western world and has also developed a vast interest in ginseng research. Scientific research on ginseng already started early with the first modern compositional study on ginseng being published in 1854 (Shoji, 1985). To date, ginseng is commonly formulated into herbal supplements and nutraceuticals as capsules, powders, tinctures, teas, and as a beverage ingredient. These formulations are based on specific health expectations that focus on a broad range of effects such as immune stimulatory, antioxidative, antidiabetic, antihyperglycemic, antihypercholesterolemic, and hypotensive.  1  The bioactive potentials of ginseng have been associated with the main active components of ginseng called ginsenosides. Over 30 different individual ginsenosides have been identified and are associated with certain therapeutic properties. The six common individual ginsenosides include Rb1, Rb2, Rc, Rd, Re, and Rg1. The percentages of these six fingerprint ginsenosides usually make up the total ginsenoside compositions. The high moisture content of fresh ginseng can adversely affect the quality of the ginseng root as well as the quantity of the ginsenosides. Thus, ginseng roots are typically sold as dried roots, with air-drying processing the most common used technique to preserve ginseng roots. Freeze-drying (FD) has also been explored to preserve ginseng roots as FD retains the initial food products characteristics and usually produces high quality chemicals, medicines, and other food products (Shishehgarha et al., 2002). However, FD process is technically complicated, slow, and the final products require additional care during handling and storage. Consequently, other dehydration process, such as vacuum-microwave drying (VMD), should be explored in order to improve the preservation of ginseng root. Vacuum-microwave drying (VMD) is a novel dehydration technique which combines the advantages of both vacuum and microwave drying. It allows rapid mass transfer at reduced boiling points by utilizing the mechanisms of microwave heating combined with the low-pressure environment created by the vacuum (Scaman and Durance, 2005). Since VMD allows dehydration to occur over a short amount of time, it has been proposed as a potentially more economical drying technique than FD for obtaining high quality food and/or herbal products (Farrell et al., 2005; Cui et al., 2004).  2  Moisture removal through dehydration may affect the matrix structure of the respective food product. A useful parameter to evaluate the dehydration effect on food product structure is porosity. Porosity provides the volume fraction of total pores compared to the total volume of the dried food product (Rahman et al., 2005). Information on the pore characteristics of the dried food products can be utilized for the estimation of several food properties, such as thermal conductivity, density, moisture diffusivity, as well as bioactive component extractability (Rahman and Sablani, 2003). Pore characteristics of a dried food product may affect the food bioactive components exposure to an extraction solvent. Consequently, as total porosity increases, the extracted amount of the bioactive component may also increase. Information on the pore characteristics and the extraction of ginsenosides related to the pore characteristics of dried ginseng roots following AD, VMD, and FD processes has not been widely explored. Moreover, the bioactive properties of North American ginseng (Panax quinquefolium; NAG) as antihyperglycemic and antihypercholesterolemic agents have brought about the potential of NAG as an antiobesity agent. Therefore, the purpose of this thesis research was to specifically determine the effect of AD, VMD, and FD processes on the pore characteristics of dried NAG roots and furthermore to relate the changes in root matrix structure to the retention of individual ginsenosides, measured by high performance liquid chromatography (HPLC), and the subsequent bioactivities. The latter objective was accomplished using viability and differentiation criteria for cultured 3T3-L1 fat cell line both in preadipose and mature adipose stages exposed to dried ginseng extracts.  3  RESEARCH HYPOTHESES AND OBJECTIVES The primary objective of the present study was to determine the effects of different drying treatments on ginsenoside recovery and the retained bioactivity potentials of North American ginseng (NAG). This study hypothesized that: 1. Vacuum-microwave drying (VMD) processing produced enhanced extraction and/or recovery of NAG ginsenosides compared to air-drying (AD) and freezedrying (FD). 2. VMD processing at 1.8 kW microwave energy level produced enhanced total porosity and the subsequent recovery of NAG ginsenosides compared to VMD processing at 1.3 kW and 0.8 kW microwave energy levels. 3. Drying-specific recoveries of NAG ginsenosides parallel changes in bioactivity as observed in 3T3-L1 cells that exist as: i. Preadipocyte cells ii. Differentiated adipocyte cells  The objectives of this research are as follows: 1. To reduce the initial moisture content of fresh ginseng root samples to a shelfstable level that enabled subsequent comparison of different drying methods (e.g. conventional air drier, vacuum-microwave drier, and freeze drier protocols); 2. To measure and compare the porosity of dried NAG from all drying methods, using a mercury porosimeter;  4  3. To determine and compare the recovery of saponins from dried NAG among all drying methods, using high-performance liquid chromatography (HPLC) analysis; 4. To determine the relative effects of dried NAG root on the viability of preadipocyte and differentiated adipocyte cells. 5. To determine the relative effects of dried NAG root on the differentiation of preadipocyte cells.  5  RESEARCH ACTIVITIES Experiment I: Drying and Recovery of Ginsenosides in Shredded Dehydrated North American Ginseng Roots Fresh Ginseng Roots  VMD  AD  Phosphorus pentoxide dehydration (MC ≤ 1%)  FD  Extraction  • Pre-soaked in methanol overnight • Soxhlet extraction for 9 hours • Vacuum concentrated  Porosity  Identification & Quantification by HPLC  Statistical analysis 6  Experiment II: Assessment of Bioactive Potential of Recovered Ginsenosides in Dehydrated North American Ginseng Roots on 3T3-L1 Fat Cell Line  Fresh Ginseng Roots  AD  VMD  FD  Extraction  • Pre-soaked in methanol overnight • Soxhlet extraction for 9 hours • Vacuum concentrated to dryness  Reconstitution (Concentrated crude extracts in culture medium – DMEM, 10% calf serum, 100U penicillin, 100µg/ml streptomycin)  3T3-L1 Preadipocytes  Adipogenesis Assay • Initiation medium • Progression medium • Maintenance medium  Viability Assay  3T3-L1 Adipocytes  Oil-Red-O Staining Assay  Statistical analysis  7  CHAPTER 1: LITERATURE REVIEW 1.1 History and Cultivation of Ginseng The name ‘ginseng’ refers to several species in the plant family Araliaceace and the genus Panax, which is indigenous to Korea and China (Panax ginseng C.A. Meyer), North America (Panax quinquefolium), Himalaya (Panax pseudo-ginseng), Vietnam (Panax vietnamensis), and Japan (Panax japonicus) (Kennedy and Scholey, 2003; Yun, 2001; Kitts and Hu, 2000). The genus name Panax originated from the Greek words: pan (all) and akos (remedy) indicating that the plant is believed to be a universal remedy (Lee et al., 2005). The ginseng plant consists of a precious root that can grow for many years, a stem that can reach approximately 25 – 50 cm tall, which bears 3 – 5 petioles that possess a compound leaf with five leaflets and a single stalk that sustains bright red ginseng berries (Court, 2000; Thompson, 1987; Dixon, 1976). Ginseng roots are thick, fleshy, spindle-shaped, and cream to pale yellowish in colour. They can grow up to 20 cm in length and 2.5 cm in thickness, and are frequently irregularly branched according to the soil environment (Court, 2000). Heavy stony soils cause short and thick primary roots with many thick secondary roots while light sandy soils produce longer, lean, and straight roots with less secondary roots (Court, 2000). The rhizome bears stem scars which indicate the age of the plant since the stem begins to shoot in the spring and dies off in the autumn of the same year and develops again the following spring (Court, 2000; Dixon, 1976). In late April or early May of the first year, ginseng seeds develop to a single 7.5 – 10 cm tall shoot with a composite leaf of three small, oval leaflets (Court, 2000; Dixon, 1976). This aerial growth dies down in the autumn while the roots and rhizomes remain growing underground. In the spring of the second year, two or three compound leaves  8  arise and this growth also dies in the autumn (Court, 2000; Dixon, 1976). The stem reaches 20 – 25 cm in height, together with three compound leaves each subdivided into five leaflets, three large upper and two small lower leaflets, in the spring of the third year. The plant will also bear flowers for the first time. The greenish-white flowers will develop into bright red berries in the beginning of autumn. These berries bear some seeds that fall off to germinate new plants and from now on, the plant will continue to grow up to 60 cm in height and will produce seeds most years (Court, 2000; Dixon, 1976). Although ginseng leaves and berries have been widely sold in the market, ginseng roots are still the most sought after. Ginseng roots found in the market are either Asian ginseng, which is originated from China or Korea, or North American ginseng, found in USA and Canada. Siberian or Russian ginsengs (Eleutherococcus senticosus) which are also commonly sold in the herbal market should not to be confused with true ginseng since Siberian ginseng is a different genus than ginseng. The earliest written description of ginseng was Shen Nung’s The Book of Herbs (Shen Nung Pen Ts’ao Ching) dated 196 A.D. (Lee et al., 2005; Dixon, 1976). This Chinese emperor stated that ginseng was used medicinally in China and Tibet, from approximately 3000 – 2000 B.C. Panax ginseng, which originated from the mountain valleys of Shantung and Liaotung in South of Manchuria (Dixon, 1976); has been used medicinally as a tonic or known as an adaptogen in most alternative medicine literatures (Kennedy and Scholey, 2003; Kiefer and Pantuso, 2003; Kitts and Hu, 2000). It can be used especially to strengthen the five senses, calm the soul, improve cognitive learning, as well as revitalize the body and prolong life (Hou, 1978; Dixon, 1976). These benefits of ginseng are in compliance with the Chinese belief that ginseng roots are able to restore  9  any ill part of the human body since ginseng roots bear a shape of a man with a head, hands, and feet. In accordance with Chinese belief, a plant resembling a human part is the cure for the particular part (Kennedy and Scholey, 2003). This follows the Asian medical approach which sees the body as a whole and therefore, to cure illness is to restore the holistic balance of the body so that it will cure itself. This philosophy is quite different from the Western medical approach which sees the body as a machine and thus, to cure a disease requires the action of diagnosing followed by treating the disease (Lee et al., 2005; Dixon, 1976). North American ginseng (Panax quinquefolium) was first sought out in the 18th century by a Jesuit priest, Father Joseph Francis Lafitou, who worked among the Iroquois Indian tribe in Sault Saint Louis, near Montreal, Canada (Dixon, 1976; Harriman, 1973). This successful search was first initiated by newsletters sent by a fellow Jesuit priest, Father Pierre Jartoux, who made a detailed evaluation of the Panax ginseng plant, also suggested ginseng might be found in areas of Canada where the mountainous, forested habitat closely resembled that in China (Dixon, 1976; Harriman, 1973). The finding, therefore, initiated the international trade of these Canadian ginseng roots to be as far as China and Hong Kong in the period of 1720 – 1750. However, this international trade soon diminished since the quality of the root was incomparable to the prices they were sold at, and the supply of the Canadian wild ginseng was also depleted. This situation initiated George Stanton, a retired New York tinsmith, to set up a Chinese ginseng farm in 1886 (Court, 2000; Dixon, 1976). Stanton’s successful attempt was a result of imitating the natural growth condition of ginseng. Stanton used woodland soil for the beds, provided artificial shade that resembled the shady forest condition, sufficient  10  drainage and ventilation, as well as used leaf molds for fertilizer (Court, 2000; Dixon, 1976; Harriman, 1973). Although North American ginseng cultivation have risen and fallen in the past, research has steadily increased the cultivation of this indigenous plant both in Canada and the United States.  1.2 Types of Ginseng 1.2.1 Asian Ginseng Asian ginseng usually refers to the roots of Panax ginseng C.A. Meyer. Typically, these roots are divided into either white or red ginseng based on the processing techniques. Whereas white Asian ginseng refers to the root of Panax ginseng C.A. Meyer that has been air- or sun-dried, red ginseng refers to Chinese or Korean ginseng that has been subjected to a steam heating processing prior to any dehydration techniques. Steaming the ginseng produces a red tint compared to the usual light brown colour of the air-dried roots. Although compositional studies indicate only minor differences in content of the red and white ginsengs, red ginseng is typically believed to have stronger bioactive potencies (Kim et al., 2000). Traditional Chinese medicinal uses of Asian ginseng are for a resemblance to stimulate the ‘yang’, an ancient Asian concept that explains the balance of the world which is associated to masculinity, energy, light, and heat. Chinese traditional medicine practitioners have prescribed Asian ginseng to counter the effects of aging, cold climates, stress, and hormonal changes for the purpose of improving conditions such as asthma, depression, and heart, liver, nervous system, digestive as well as circulatory system problems (Li et al., 1996; Banthorpe, 1994).  11  1.2.2 North American Ginseng North American ginseng (Panax quinquefolium) is commonly grown in the northeastern states of USA and in the Canadian provinces of British Columbia and Ontario. However, this ginseng species can sometime be found as far south as Missouri, Georgia, and Alabama, as well as scarcely in the states west of and bordering to the Mississippi river (Court, 2000; Dixon, 1976). Whereas Panax ginseng C.A. Meyer is commonly sold as either red or white ginseng, Panax quinquefolium is typically sold as air- or sun-dried (white) ginseng at a price which is 5 to 10 times higher than that of the Asian ginseng (Lu et al., 2008; Wang et al., 2001). Both physical characteristics and composition properties of the active ingredients of North American ginsengs (NAGs) are not significantly different from its Asian counterpart. However, NAG is thought to have the opposite effect of Asian ginseng by stimulating the ‘yin’ which has a cooling effect on the body. NAG is more useful for those in warmer climates, children and young adults, and those with high blood pressure, diabetes, heart, and lung problems (Hu and Kitts, 2001; Li et al., 1996).  1.3 Ginsenosides The main bioactive components of ginseng are the triterpenoid saponins, called as ginsenosides, of which over 30 individual compounds have been identified, varying in content and proportion depending on the species of the ginseng (Gillis, 1997). In addition to the saponins, NAG contains as many as 200 different components in various amounts (Duke, 1992). These compounds include vitamins (e.g. Vitamin A and B12, niacin, and 12  folic acid), minerals (e.g. calcium and zinc), sugars (e.g. glucose and fructose), inorganic salts (e.g. sodium, and magnesium), simple organic acids (e.g. acetic acid and maltol (3hydroxyl-2-methyl-4H-pyran-4-one)) as well as high-molecular weight polysaccharides (e.g. ginsenan), phytosterols (e.g. β–sitosterol, campesterol, and stigmasterol), oligopeptides (e.g. peptidoglycans), and volatile oils (Kitts and Popovich, 2003; Court, 2000; Duke, 1992; Dixon, 1976). Ginsenosides are classified into three different groups based on the positions of the sugar moieties: 20(s)-protopanaxadiol, 20(s)-protopanaxatriol, and oleanolic acid groups (Güçlü-üstündağ and Mazza, 2007; Ma et al., 2005; World Health Organization, 1999; Attele et al., 1999). 20(s)-protopanaxadiol refers to ginsenosides with a steroid backbone containing 17 carbon atoms arranged in four trans-rings with sugar moieties at position C-3, while 20(s)-protopanaxatriol has sugar residues attached to position C-6 (Figure 1.1). However, ginsenoside Ro, the only oleanolic acid ginsenoside, has a different structure with a pentacyclic backbone and sugar moieties at position C-3 and C28 (Figure 1.2) (Peng et al., 2004; Attele et al., 1999). The ginsenoside composition, which is usually expressed as the percentage of six individual ginsenosides commonly found in all ginseng species, varies considerably dependent on the plant species, growing region and condition, time of harvesting, age of the plant, subterranean parts used among other factors (Mudge et al., 2004; Court, 2000; Yang et al., 1989). The fingerprint ginsenosides include Rb1, Rb2, Rc, Rd, Re, and Rg1. Each ginsenoside has been associated with certain therapeutic properties and therefore, can influence the overall bioactive properties of ginseng to include anticarcinogenic (Wang et al., 2007; Li et al., 2006; Popovich and Kitts, 2004), antihyperglycemic (Li et al., 2004; Attele et al., 2002;  13  Vuksan et al., 2000) antihypercholesterolemic (Zhou et al., 2004), as well as membranepermeabilizing potential and cytotoxicity (Popovich and Kitts, 2002).  Figure 1.1 Basic structures of 20(s) - protopanaxadiol and 20(s)-protopanaxatriol ginsenosides Table 1.1 Individual ginsenosides of 20(s)-protopanaxadiol and 20(s)-protopanaxatriol groups 20(s)-protopanaxadiol 20(s)-protopanaxatriol R1  R2  R3  R1  R2  Rb1  -G[2→1]G  -H  -G[6→1]G  Rb2  -G[2→1]G  -H  Rc  -G[2→1]G  Rd  R3  Re  -H  O-G[2→1]R  -G  -G[6→1]Ap  Rg1  -H  O-G  -G  -H  -G[6→1]Af  Rf  -H  O-G[2→1]G  -H  -G[2→1]G  -H  -G  Rg3  -G[2→1]G  -H  -H  Rh2  -G  -H  -H  PD  -H  -H  -H  PT  -H  -OH  -H  Abbreviations represent G: glucopyranose, Ap: arabinopyranose, Af: arabinofuranose, R: rhamnopyranose. R1: region 1, R2: region 2, R3: region 3. PD: aglycone of 20(s)protopanaxadiol, PT: aglycone of 20(s)-protopanaxatriol.  14  Figure 1.2 Basic structure of oleanolic acid ginsenoside Table 1.2 Ginsenoside of oleanolic acid group R2 R1 Ro  -GA[2→1]G  -G  Aglycone  -H  -H  Abbreviations represent G: glucopyranose, GA: glucuronic acid. R1: region 1, R2: region 2. In addition to the oleanolic acid ginsenoside Ro, there are four more acidic ginsenosides, commonly called malonyl ginsenosides (Du et al., 2004). These malonyl ginsenosides are malonic acid derivatives which includes malonyl(m)-Rb1, m-Rb2, mRc, and m-Rd (Awang, 2000). The malonyl ginsenosides are more polar and water soluble than their neutral counterparts (Awang, 2000). Consequently, malonyl ginsenosides are more susceptible to hydrolysis to the respective neutral ginsenosides (Court et al., 1996).  15  1.4 Composition Analysis of Ginseng Ginseng extracts (GEs) can be prepared in many different ways and the extraction methods heavily influence the final composition and concentration of the ginsenosides in the extract (Yoshikawa et al., 1998; Court et al., 1996; Li et al., 1996; Saxena et al., 1994; Soldati and Sticher, 1980). Ginseng extractions may utilize hot water, organic solvents, or water-solvent mixtures, as well as a supercritical fluid (CO2) (Zhang et al., 2006; Popovich and Kitts, 2004; Hu and Kitts, 2001). These liquid extractions of the ginseng roots may be assisted by heat reflux, Soxhlet extraction, microwave assisted extraction, ultrasonic extraction as well as solid phase extraction (Lou et al., 2006; Popovich and Kitts, 2004; Shu et al., 2003; Kaufmann and Christen, 2002; Hu and Kitts, 2001). Although there have been many ginseng extraction methods investigated, the most common extraction method is the soxhlet extraction with aqueous methanol (Zhang et al., 2006; Popovich et al., 2005; Hu and Kitts, 2001; Court et al., 1996; Nho and Sohn, 1989). Soxhlet extraction requires a solid-liquid contact to remove one or more compounds from the ginseng plant by dissolving the compounds into a refluxing liquid solvent. This method prevents the extraction solvent from becoming saturated with the extractable material by exposing the sample material with fresh solvent repeatedly and therefore, enhancing the removal of the compounds from the matrix (Zhang et al., 2006). An effective extraction method does not only focus on increasing the extraction yield, while keeping the extraction time minimal, but it also generates consistent compositional yield. This is difficult to achieve as different parts of the ginseng plant contain various levels of ginsenosides in addition to other factors. This, therefore, has contributed to the inconsistencies of ginsenoside content as identified and quantitatively  16  analyzed by thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and liquid chromatography/mass spectrometry (LC/MS).  1.4.1 Thin Layer Chromatography (TLC) TLC is a rapid technique for identifying bioactive compounds of various plant materials and extracts including ginsenosides in ginseng root, used because of its versatility, low cost, and minimal analysis time. TLC involves the passage of a suitable solvent as the mobile phase across a uniform layer of a finely divided insoluble adsorbent, which acts as a stationary phase (Court, 2000). Individual compounds generally migrate across the TLC plate according to their respective polarities (Shibata et al., 1965). Furthermore, ginsenosides are named according to the distances migrated on a TLC plate. This explains the name of individual ginsenoside Rx as the capital R refers to the root and the lower case x relates to the relative positions of the separated neutral saponin spots on the thin-layer chromatograms, with ginsenoside Ro being the least polar to migrate the furthest among other ginsenosides (Shibata et al., 1965). However, ginsenosides Re and Rd can reverse respective order of migration according to the choice of solvent (Shibata et al., 1985). Different solvent systems that have been applied to separate ginsenosides include upper phase of η-butanol: ethyl acetate: water (4: 1: 5), lower phase of chloroform: methanol: water (13: 7: 2), chloroform: methanol: ethyl acetate: water (2: 2: 4: 1; lower phase), or η-butanol: chloroform: methanol: water (8: 4: 3: 2; lower phase) (Fuzzati, 2004; Shibata et al., 1985; Betz et al., 1979). The chromatographic plate, depending on the solvent system of choice, is usually precoated with silica gel, silica gel F254, or CM-  17  cellulose (Fuzzati, 2004; Court et al., 2000; Betz et al., 1979). The most common solvent systems used for identifying ginsenosides is the combination of η-butanol: ethyl acetate: water (4: 1: 5; upper phase) and chloroform: methanol: water (13: 7: 2; lower phase) on a two-dimensional, silica gel plate TLC (Fuzzati, 2004). TLC-densitometric methods have been developed to quantitatively analyze ginsenosides in ginseng roots and ginseng dry extracts (Corthout et al., 1999). Ginsenosides Ra, Rb1, Rb2, Rc, Re, Rd, Rg1, Rf, and Rg2 of Panax ginseng roots were quantified by high-performance TLC (HPTLC) silica gel F254, developed by solvent system of chloroform: ethyl acetate: methanol: water (15: 40: 22: 9; lower phase), visualized by anisaldehyde reagent and finally, the absorbance was measured at 535nm. This method was optimized by Vanhaelen-Fastré et al. (2000) using a HPTLC silica gel 60F254, solvent mixture of 1,2-dichloroethane: ethanol: methanol: water (56.8: 19.2: 19.2: 4.80), detected by vapours of thionyl chloride, and absorbance was measured at 275 nm while fluorescence-reflection was detected at 366 nm.  1.4.2 High-Performance Liquid Chromatography (HPLC) HPLC is an ideal technique for analyzing ginsenosides since it is fast, versatile, sensitive, and adaptable to polar and non-polar compounds. An HPLC system equipped with a C18–reversed phase column allows for the separation of individual ginsenosides. The solvent mixture commonly used for the separation is acetonitrile-water in the gradient elution mode with acetonitrile concentration increases with time (Lou et al., 2006; Wan et al., 2006; Court et al., 1996; Yamaguchi et al., 1988; Soldati and Sticher, 1980). Moreover, an acetonitrile-water-potassium dihydrogen phosphate mobile phase  18  can be used to specifically separate ginsenosides Rg1 and Re (Fuzzati, 2004). A preliminary clean-up can be obtained by using a pre-column, such as Sep-Pak C18 cartridge, to reduce the front peaks caused by impurities in the extract to provide a clearer background and a smoother baseline (Court, 2000; Corthout et al., 1999; Li et al., 1996). An appropriate solvent control program adequately separates the main ginsenosides Rb1, Rc, Re, Rb2, Rg1, and Rg1 from the more uncommon ginsenosides Rh1, Rh2, and Rg3 (Kwon et al., 2001). However, the determination of malonylginsenosides is still challenging since these compounds are highly unstable and there is a lack of suitable standards. Court et al., (1996) developed an indirect method to quantify malonyl-ginsenosides m-Rb1, m-Rb2, m-Rc, and m-Rd by hydrolyzing these acidic saponins into the respective neutral ginsenosides with aqueous potassium hydroxide. Ginsenosides are commonly detected by ultraviolet HPLC (HPLC-UV) method. However, fluorescence HPLC can also be used to determine ginsenoside content. Since ginsenosides do not posses a suitable fluorescence chromophore, they need to be derivatized prior to detection (Park et al., 1996). A quantitative determination of ginsenosides Rb1 and Rg1 by fluorescence HPLC was developed by Shangguan et al. (2001). The double bond at the C24–C25 position of the ginsenoside was converted into an aldehyde group by passing an ozone-oxygen mixture through the methanolic ginsenoside solution (ozonolysis). The aldehyde group was reacted with 9-fluorenylmethoxycarbonyl (FMOC) hydrazine to form the ginsenosides FMOC-hydrazone which were then analyzed by HPLC on a C18–reversed phase column using methanol–water–0.1% trifluoroacetic acid (TFA) gradient elution. The detection was performed by fluorescence (excitation at 270 nm, emission at 310 nm) achieving a detection limit for ginsenosides Rb1 and Rg1 of  19  2 and 1 ng, respectively. This method was developed based on reacting ginsenoside Rg1 with aqueous 2-tert-butylanthraquinone (t-BAQ) solution on a Lichrosorb NH column by Park et al. in 1996. The column effluent was passed through a 40 cm polytetrafluoroethylene (PTFE) capillary tube coiled around a 10W-UV lamp to reduce tBAQ to a highly fluorescent 9,10-dihydroxyanthracene derivative (AQH) which was then detected by a fluorescence detector (excitation: 400 nm, emission: 525 nm). Since the amount of AQH produced is proportional to the amount of the ginsenoside Rg1 reacted, the quantity of ginsenoside Rg1 can be determined by measuring the fluorescence intensity of AQH (Park et al., 1996).  1.4.3 Liquid Chromatography/Mass Spectrometry (LC/MS) LC/MS has been successfully applied to characterize different ginsenosides in the ginseng plant. A LC/MS/MS method has been developed to distinguish Asian ginseng from its North American counterpart based on the identification of ginsenoside Rf in Panax ginseng root and 24(R)-pseudoginsenoside F11 in Panax quinquefolium root (Li et al., 2000). Although this method is highly sensitive, it has poor reproducibility and does not identify the thermally unstable malonyl-ginsenoside. The introduction of electrospray ionization to mass spectrometry (ESI-MS) has provided a soft ionization technique to make the identification of thermolabile molecules possible (Fuzzati, 2004). Identifying ginsenosides can be performed by the LC/ESI-MS in positive and negative ionization modes. Negative ionization tends to produce a total ionization chromatograph with less baseline noise and a clearer total ion chromatograph (Liu et al., 2004; Ji et al., 2001). Moreover, negative ion-scan usually produces [M-H]- ion  20  fragments for all ginsenosides due to the loss of sugars from the glycosidic chains (Liu et al., 2004; Ji et al., 2001). On the other hand, positive ionization usually provides the structural information of ginsenosides. This is due to the production of cationized ions such as [M+H]+, [M+NH4]+, [M+K]+, and especially [M+Na]+ since sugar has a strong affinity to sodium ion in the gas phase (Liu et al., 2004; Miao et al., 2002).  1.5 Bioactivities of Ginseng 1.5.1 Antioxidative Properties In addition to its immune stimulatory property, ginseng has also been established as an antioxidant. Ginseng extracts (GEs) had been shown to sequester metal ions (Kitts et al., 2000), scavenge stable free radicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azobis(2-amidinopropane)dihydrochloride (AAPH), and reduce formation of lipid hydroperoxides (Kim et al., 2002; Hu and Kitts, 2001; Kitts et al., 2000). Ginseng also inhibited Cu2+-induced low-density lipoprotein oxidation as well as Fe2+-induced supercoiled DNA strand scission (Hu and Kitts, 2001). The protective mechanism of ginseng towards liver and brain cells had been shown due to the inhibition of lipid peroxidation and maintenance of the level of a potent antioxidant enzyme glutathione peroxidase (GSPx) (Shukla and Kumar, 2009; Keum et al., 2000). Ginseng also promotes the transcription of Cu/Zn superoxide dismutase (SOD) which can result in an increased removal of superoxide radicals (Kim et al., 1996). The specific ginsenoside associated with the upregulation of SOD had been ginsenoside Rb2 while ginsenoside Rb1 was associated with an increased level of catalase (CAT) activity by 47.2% (p<0.001) (Kim et al., 1996; Deng and Zhang, 1991).  21  Antioxidant activity of ginseng has also been associated with a protective effect against various vascular injuries. Ginsenosides Rb1, Rb2, Rb3, Rc, Rg1, Rg2, Re, and Rh1 had been proposed to protect free radical-induced atherosclerotic plaque formation (Zhong and Jiang, 1997). Although the antioxidant activity of individual ginsenosides was different in protecting various vascular injuries, these ginsenosides showed a synergistic effect (Liu et al., 2002; Chen, 1996). Ginseng concurrently increased 6-ketoprostaglandin F1α, a vasodilator marker, which consequently inhibited platelet adhesion and increased vaso-relaxation (Chen, 1996). Ginsenosides, particularly protopanaxatriol ginsenosides (e.g. ginsenosides Rg1, Re, and Rg3), also enhanced the release of nitric oxide (NO) and further increased the level of cyclic guanosine monophosphate (cGMP) (Gillis, 1997; Chen, 1996). This supports the evidence of an indirect antioxidant activity of ginseng on vasodilation and protection of injured pulmonary endothelium induced by reactive oxygen species (Kim et al., 1999; Chen, 1996).  1.5.2 Antidiabetic Properties Diabetes is a metabolic disorder focused on the endocrine system. The prevalence of diabetes in Canada had reached 1.3 million people in 2005, making diabetes the seventh leading cause of death in Canada. The treatment of diabetes has widely been achieved through chemical and biochemical drugs. However, the relatively high incidence of side effects with chemical drugs has provided a traditional-plant medicinal therapy as a unique alternative to diabetes treatment. Panax ginseng roots have been reported to treat type-2 diabetes in mice without evidence of toxicity or other adverse effects (Yuan et al., 2008). The proposed anti-hyperglycemic mechanisms of standardized  22  GE are to regulate the enzymatic activities related to glucose metabolism, decrease the rate of carbohydrate absorption, and promote insulin secretion (Lee et al., 1998; Kitamura et al., 1997; Ng and Yeung, 1985). Oral administration of white ginseng root and rootlet extracts, derived from Panax ginseng C.A. Meyer, to mice resulted in a reduction of fasting blood glucose levels by 40% and 37% respectively, compared to the untreated group (Chung et al., 2001). Moreover, intraperitoneal injection of GE (90 mg/kg) significantly lowered fasting blood glucose levels of two diabetic mice, male KK-CAy mice and alloxan-diabetic mice, by 76% and 62%, respectively (Kimura et al., 1999). This may be due to the inhibition of intestinal glucose absorption as well as carbohydrate absorption into portal hepatic circulation (Vuksan et al., 2000). Anti-hyperglycemic and anti-obesity effects of Panax ginseng berry extract and ginsenoside Re has been reported on obese diabetic C57BL/6J ob/ob mice (Attele et al., 2002). Following intraperitoneal injections of a ginseng berry extract and ginsenoside Re for 12 days, the blood glucose level of the ob/ob mice was normalized, which was associated to the reduction of serum insulin, and glucose tolerance was significantly improved (Attele et al., 2002). Ginsenoside Re demonstrated a significant role in antihyperglycemic action which could be due to its ability to increase glucose transporter-4 (GLUT4) protein and reduce inflammation in peripheral tissues resulting in reduction of insulin resistance (Zhang et al., 2008). Moreover, the anti-hyperglycemic property of ginseng may also be mediated by NO since a study suggested that an increase of NO was capable of increasing glucose uptake into rat skeletal muscle and adipose tissue while  23  also enhancing glucose-dependent insulin secretion in rat islet cells (Roy et al., 1998; Spinas et al., 1998).  1.5.3 Antihypercholesterolemic Properties Hypercholesterolemia has been established as an independent risk factor for the development of atherosclerosis cardiovascular disease (Steinberg, 2002; Lacoste et al., 1995). Reducing serum low density lipoprotein-cholesterol (LDL-C) level and platelet activity have been associated with the prevention of atherosclerosis. As previously mentioned, ginseng was used as an antioxidant to inhibit free radical-induced platelet adhesion as well as vascular injuries (Chen, 1996). However, Malinow et al. (1977) proposed that saponins directly interacted with cholesterol producing an insoluble complex which prevented cholesterol absorption. Saponins were also proposed to indirectly affect cholesterol metabolism by interacting with bile acids and increasing fecal bile acid excretion (Oakenfull et al., 1979), thus reducing cholesterol absorption in the small intestine. Oral administration of ginseng saponins at 0.01 g/kg for 4 weeks to hyperlipidemic rabbits was found to reduce serum triacylglycerols (TAG) as well as cyclophosphamide (CPM)-induced cholesterol (Inoue et al., 1999). Furthermore, ginseng saponins treatment recovered postheparin plasma lipoprotein lipase (LPL) activity which was previously markedly reduced by CPM treatment (Inoue et al., 1999). This study demonstrated that ginseng sustained a protected LPL activity resulting in the reduction of serum TAG and cholesterol.  24  Asian red ginseng and NAG had been shown to reduce blood cholesterol level in various animal models (Hwang et al., 2008; Banz et al., 2007; Trinh et al., 2007). It had been proposed that ginsenoside Re provided the anti-hypercholesterolemic effect of ginseng since ginsenoside Re (20 mg/kg) reduced serum total cholesterol of diabetic rats by 22% and 32% following one- and two-week treatments, respectively (Cho et al., 2006). Moreover, intraperitoneal injection of ginsenoside Rg1 was also found to prevent the elevation of serum cholesterol level (Hattori et al., 1991). Ginsenoside Rb1 was also found to decrease the total cholesterol level in the liver (Park et al., 2002). However, ginsenoside Rb1 failed to reduce the serum cholesterol level and therefore, this supported the stronger effect of 20(s)-protopanaxatriol (PT) ginsenosides compared to that of 20(s)protopanaxadiol (PD) ginsenosides in lowering serum total cholesterol (Kim et al., 2009).  1.5.4 Antiobesity Properties Obesity treatment currently involves chemical drugs, such as sibutramine, orlistat, and rimonabant (Kim et al., 2009). Although these drugs modestly promote weight loss, adverse effects of these drugs are not negligible. Sibutramine, a norepinephrine-serotonin reuptake inhibitor, was approved in the USA in 1997 with a typical dose of 10-15 mg once daily (Padwal and Majumdar, 2007). Three randomized double-blind, placebocontrolled weight loss trials of 1 year in 929 overweight or obese patients suggested that sibutramine reduced weight by 4-5 kg/year but was associated with increases in blood pressure and heart rate (Rucker et al., 2007). Orlistat, a gastric and pancreatic lipase inhibitor, reduced weight by 2-7 kg/year in a 4-year double-blind, placebo-controlled randomized study of 3305 Swedish obese patients (Torgerson et al., 2004). Orlistat was  25  approved in 1998 with a typical dose of 120 mg three times daily (Padwal and Majumdar, 2007). However, orlistat has been associated with numerous gastrointestinal adverse effects, such as fatty and oily stool, faecal urgency, and oily spotting (Padwal and Majumdar, 2007). Rimonabant, the first of the endocannabinoid receptor antagonists, at 20 mg daily reduced weight by 4-5 kg/year in 1222 overweight or obese patients (PiSunyer et al., 2006). Adverse effect of rimonabant includes an increased incidence of mood-related disorders (Padwal and Majumdar, 2007). The need for a better tolerated antiobesity treatment through therapeutic herbs, which have far fewer side effects, is expanding. Intraperitoneal injection of 200 mg/kg crude saponin of red Korean ginseng was able to reduce body weight, food intake, and fat content of obese Sprague-Dawley rats (Kim et al., 2005). This study reported that leptin, an adipocyte-derived protein that circulates at levels proportional to body fat content, was significantly reduced in the rats fed by high-fat diet after crude saponin treatment (Kim et al., 2005). The neuropeptide Y (NPY), which regulates energy homeostasis by increasing food intake, reducing thermogenic capacity, and reducing the oxidation of dietary fat, was also reduced after three weeks of crude saponin treatment (Kim et al., 2005). The same worker investigated and compared the antiobesity activity of 20(s)-protopanaxadiol (PD) and 20(s)protopanaxatriol (PT) type ginsenosides (Kim et al., 2009). Although PT ginsenosides showed a stronger effect than PD ginsenosides in reducing serum total cholesterol, PT ginsenosides reduced weight gain, total food intake, and body fat accumulation to a lesser extent, compared to PD ginsenosides (Kim et al., 2009).  26  The stronger effect of PD ginsenosides in reducing body fat accumulation may be attributable to the adipogenesis inhibitory activity of ginsenoside Rg3, which is a PD type ginsenoside (Hwang et al., 2009; Lim et al., 2009). These workers reported that ginsenoside Rg3 inhibited adipogenesis of a 3T3-L1 fat cell line by inhibiting the transcription of peroxisome proliferators-activated receptor-γ (PPARγ), which is responsible for adipocyte differentiation (Hwang et al., 2009). In addition, ginsenoside Rg3 activated adenosine monophosphate-activated protein kinase (AMPK)-dependent signalling pathways which also inhibited adipocyte differentiation (Hwang et al., 2009).  1.6 Drying Methods Changes during harvesting, handling, storage, and preparation potentially affect the quality of ginseng roots as well as the quantity of the ginsenosides. Preservation, therefore, is essential to maintain ginseng’s quality especially since it typically undergoes long transport times to reach consumers. Dehydration is the most effective method of ginseng preservation to-date. Furthermore, sun-drying is the oldest preservation method of Asian ginseng since the sun in the Orient is hot enough to dry the plants efficiently by harvesting time in autumn. However, the same condition does not apply in North America and therefore, other dehydration processes have been explored for the purpose of preserving these valuable plants. Drying is a water-removal process that prevents the growth of spoilage microorganisms as well as inhibits adverse chemical reactions (Rodriguez et al., 2004). Drying  27  also induces changes in physical, chemical, and biological properties of food products as well as modifies phytochemical characteristics. Removing water from food products lowers the moisture content as well as the water activity (aw) and thus, lowers the water availability for microorganisms to grow and chemical reactions to occur (Fennema, 1996). Being defined as the ratio of water vapour pressure of a food product to water vapour pressure of pure water at the same temperature, aw measures the amount of moisture that is available to support microbial growth and chemical activity (Fennema, 1996). Most bacteria, molds, and yeasts do not grow below the aw range of 0.70 – 0.90 (Heidelboug and Karel, 1975) while the rate of non-enzymatic browning and lipid oxidation is reduced below the aw range of 0.60 – 0.80 and 0.20 – 0.40, respectively (Drapron, 1985).  1.6.1 Air Drying Air drying (AD) is the simplest and cheapest dehydration method that has been widely used to dry herbs, spices and some fruits (Consuelo Díaz-Maroto et al., 2002; Kim et al., 2000). Several different conventional air driers such as the continuous hot-air drier, cabinet drier, conveyor drier, rotary drier, and spray drier are built to accommodate different characteristics and quantities of food products as well as drying mechanisms. Conventional air driers are equipped with heating elements and fans to circulate the hot, dry air around the food. The dry air evaporates the moisture into the air stream by convection while transferring the moisture to the food surface by conduction (Brennan, 2006). As the drying process continues, the moisture diffusion rate to the food surface slows down creating a high surface tension which results in the collapse of the matrix  28  structure (Brennan, 2006). Moreover, the moisture carries soluble materials, such as sugars and salts, to the food surface and as moisture evaporates, the soluble materials accumulate at the drying surface and therefore, results in case hardening (Brennan, 2006). This collapse of the porous structure and case hardening adversely affect the texture, rehydration rate as well as the rehydration capacity of the food products (Kim et al., 2000; Karathanos et al., 1996). During rehydration, the water movement around the food depends on the water diffusivity, shrinkage properties, and the cell walls of the respective food (Brennan, 2006; Karathanos et al., 1996). AD often causes further damage such as an increased rate of oxidation, hydrolysis of glycosides, or a reduction in nutrients and volatile compounds in addition to case hardening and collapse of the food matrix (Karabulut et al., 2007; Consuelo Díaz-Maroto et al., 2002). Undesirable browning is often found in air-dried products since AD facilitates enzymatic oxidation of phenolic compounds by polyphenol oxidase (Karabulut et al., 2007; Krokida et al., 2001). AD also induces vaporization of heat-sensitive nutrients as well as volatile compounds since the AD process involves long exposure of high temperature and oxygen (Cui, 2004; Yousif et al., 2000; Yongsawatdigul and Gunasekaran, 1996). However, drying temperature of the hot-air driers is controllable and drying time is comparatively much shorter than freeze-drying (Kim et al., 2000).  1.6.2 Vacuum Microwave Drying Vacuum-microwave drying (VMD) combines the advantages of both vacuum and microwave drying. It allows dehydration to occur over a short amount of time by associating the dipole excitation and ion migration, which underlies the mechanisms of  29  microwave heating, as well as evaporation at reduced boiling points in a low-pressure chamber created by the vacuum (Scaman and Durance, 2005; Kaensup et al., 2002). Since the boiling temperature of water is reduced from 100°C (212°F) at atmospheric pressure of 760 mm Hg to lower temperatures at pressures below 760 mm Hg, VMD allows rapid evaporation at a low temperature under vacuum (Yousif et al., 1999; King et al., 1989; Hsu and Beuchat, 1986). Microwaves are electromagnetic waves ranging from 1 mm (infrared) to 1 m (radio waves), corresponding to frequencies between 0.3 and 300 GHz (Drouzas and Schubert, 1996; Salunkhe et al., 1991). Microwave heating of foods usually occurs at a frequency of 2.45 GHz or a wavelength of 1.2 x 10-1 m (Drouzas and Schubert, 1996). Similar to visible light, microwaves can be reflected, polarized, scattered, diffracted, and absorbed at atmospheric pressure. As food absorbs microwave energy, the polar molecules within the food material, such as water molecules, attempt to align themselves following the electromagnetic field direction. Heat is then created by the intermolecular friction. As mentioned earlier, the major advantage of using the VMD process is the fast mass transfer at a low temperature caused by the vacuum. The vacuum also reduces the air-exposure during the VMD process resulting in inhibition of oxidation as well as retention of the natural appearances of food (Kim et al., 2000). Consequently, the VMD process allows improved energy efficiency and product quality compared to AD. VMD is normally used for sensitive materials that can be damaged at high temperature and has been studied as a potentially low-cost means for obtaining high quality dried meat, dairy products, fruits, vegetables, and spices (Farrell et al., 2005; Cui et al., 2004; Kaensup et  30  al., 2002; Kiranoudis et al., 1997). However, VMD may cause a non-uniform heating and over-drying as the process is fast. VMD process can be as economical as AD since VMD process reduces the dehydration time significantly despite the high capital cost. Moreover, the short dehydration time of VMD process making VMD more economical than freeze-drying (FD) process (Owusuansah, 1991).  1.6.3 Freeze-Drying Freeze-drying (FD) is the best dehydration method to produce fine quality chemicals, medicines, and food products (Shishehgarha et al., 2002; Bruttini et al., 2001). It retains the initial product physicochemical characteristics that impact on appearance, structure, flavour, as well as chemical and biological activities (Shishehgarha et al., 2002; Krokida et al., 2001). FD also maintains the high rehydration capacity of the product as heat-damage imposed on the product does not occur (Oetjen and Haseley, 2004; Carpenter et al., 1993; Salunkhe et al., 1991; Hellman et al., 1983). FD processing involves freezing of the food product, followed by drying under a vacuum to moisture levels around 1 – 4% (Shishehgarha et al., 2002; Hammami et al., 1999). Ice within the food products is sublimed into water vapour without melting by applying sufficient heat under reduced pressure (Salunkhe et al., 1991). As the FD process is generally performed at a low absolute pressure, flavour volatiles may be significantly lost during the process, often resulting in a tasteless product (Flink, 1975). Futhermore, the sublimation process provides the characteristic porous structure of FD products which makes the food product sensitive to temperature and oxygen (Oetjen and  31  Haseley, 2004; Kim et al., 2000; Jiang and Nail, 1998). This porous structure also requires additional care during handling and storage of the food materials. The freezing phase of FD processing is very critical since the product can be easily spoiled if freezing is poorly done. Although large ice crystals are relatively easy to freeze-dry, cell walls of food products will rupture. Therefore, optimal freezing rates of different foods need to be determined to initiate nucleation and produce small ice crystals without causing stress to the food matrix that will result in splitting or cracking of the tissues (Fellows, 2000; Paine, 1992). The FD process is technically complicated, slow, and requires high capital and maintenance costs and therefore, is only suitable for high value-added products (Hammami et al., 1999).  1.7 Cell Culture Activities for Modelling Fat Metabolism 1.7.1 Preadipocyte Cell (3T3-L1) Obesity is a ubiquitous clinical problem that contributes to chronic diseases such as Type 2 diabetes, dyslipidemia, hypertension, and atherosclerosis, gastroesophageal reflux, ischaemic stroke, and some types of cancer (Flegal et al., 2005; Gregg et al., 2005; Douketis et al., 1999). Obesity has also been associated with a significant reduction in life expectancy (Alfieri et al., 1995). Tjepkema (2006) reported that the prevalence of obesity in Canada had doubled over the previous 25 years. Moreover, Statistics Canada revealed that almost 1 in 4 (23.1%) Canadian adults were obese (Body Mass Index (BMI) ≥ 30 kg/m2) and another 36.1% were overweight (BMI ≥ 25-29.9 kg/m2). The incidence of obesity can result as a consequence of an excess of white adipose tissue (WAT). WAT is the major energy reserve in higher eukaryotes which  32  stores and mobilizes triacylglycerols in times of energy excess and energy deprivation, respectively (Gregoire et al., 1998). WAT primarily consists of mature adipose cells and although the adipose cell and adipose tissue are formed before birth as was found in human, pig, and mice, WAT expands rapidly after birth in both cell number as well as size (Gregoire et al., 1998). In vitro systems have been used to provide a better understanding regarding the transition of undifferentiated preadipocytes to the mature adipocytes. One of the widely used adipose cell lines is 3T3-L1 which is cloned from Swiss 3T3 mouse embryos (Green and Kehinde, 1975). Undifferentiated 3T3-L1 cells have fibroblast-like structures. After treatment with 10% fetal calf serum, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), and insulin, often regarded as the induction media, the cell structure becomes more circular, which results in the accumulation of intracellular lipid droplets (Rubin et al., 1978). The development of 3T3-L1 preadipocytes can be divided into four defining stages that include: (1) preconfluent proliferation, (2) confluence growth arrest, (3) hormonal induction clonal expansion, and finally (4) permanent growth arrest terminal differentiation (Cowherd et al., 1999). The introduction of dexamethasone on cell confluence activates the transcription factor CCAAT/enhancer-binding protein-δ (C/EBPδ), while IBMX activates the related transcription factor C/EBPβ (Yeh et al., 1995; Cao et al., 1991). These transcription factors will then further induce the transcription of C/EBPα and peroxisome proliferators-activated receptor-γ (PPARγ) (Cowherd et al., 1999). C/EBPα and PPARγ are involved in the growth arrest required for activation of adipogenesis (Gregoire et al., 1998). Moreover, insulin or insulin-like  33  growth factor-1 promotes adipocyte differentiation by activating phosphoinositide 3kinase (PI3-kinase) and AKT activity. Modulation of the activity of the forkhead transcription factor forkhead box O1 (FOXO1) appears to be necessary for insulin to promote adipocyte differentiation (Nakae et al., 2003). Following the growth arrest, preadipose cell lines re-enter the cell cycle and undergo one or two rounds of mitotic clonal expansion for 24 – 36 h (Cowherd et al., 1999). Finally, the adipose cells withdraw from the cell cycle and terminally differentiate. C/EBPα and PPARγ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as lipoprotein lipase (LPL), fatty acid binding protein adipocyte protein-2 (aP2), GLUT4, leptin, adipsin, and adiponectin (Gregoire et al., 1998). The identification of mechanisms underlying adipogenesis provides the possibility of preventing or treating obesity through pharmacological means. PPARγ and C/EBPα have received particular attention as targets, as they are essential for the final phase of adipocyte differentiation and also as pharmacological targets for various antidiabetic drugs (Lehman et al., 1995). Moreover, inhibitors of these transcription factors’ activities have been identified to inhibit adipogenesis, and might serve as the basis for development of effective anti-obesity agents (Cowherd et al., 1999).  34  CHAPTER 2: COMPARISON OF DIFFERENT DEHYDRATION TECHNIQUES ON POROUS STRUCTURE AND GINSENOSIDE RETENTION OF NORTH AMERICAN GINSENG 2.1 INTRODUCTION Dehydration is one of the oldest and most efficient preservation methods in the food processing industry. The basic objective in drying food products is to remove water resulting in the prevention of growth of spoilage micro-organisms as well as the level of oxidation (Rodriguez et al., 2004). Changes in physical, chemical, and biological properties of food products are directly proportional to the time and temperature involved in a drying process. Conventional AD has the major disadvantage of inducing thermal degradation of important flavour and nutritional compounds. The high temperature and long time processes associated with conventional hot-air drying most often adversely affect texture, colour, flavour, and the nutritional value of the product (Karabulut et al., 2007; Brennan, 2006). Although FD can be applied to avoid heat damage and obtain excellent structural retention, it is a slow and costly process and is only practically suitable for high value products. Moreover, freeze-dried products usually acquire the characteristic porous structure which requires additional care during handling and storage (Oetjen and Haseley, 2004; Kim et al., 2000). VMD has been successfully used to prevent significant losses in product quality due to thermal degradation (Scaman and Durance, 2005; Yousif et al., 1999). Due to radiant energy transfer, VMD allows an accelerated removal of moisture at a lower temperature than conventional drying. This reduces the air-exposure resulting in inhibition of oxidation as well as retention of the natural appearances of food (Kim et al., 35  2000). The VMD process allows improved energy efficiency resulting in greater product quality compared to AD process. Successful applications of microwave vacuum technology employed in drying of food products have been reported with many food products including meat, dairy products, fruits, vegetables, and the herb Echinacea purpurea (Farrell et al., 2005; Cui et al., 2004; Kwok et al., 2004; Kaensup et al., 2002; Kim et al., 2000; Kiranoudis et al., 1997). Porosity is a useful parameter of the effect of dehydration on food product structure. It provides the volume fraction of total pores compared to the total volume of the food sampled (Rahman et al., 2005). Information on the characteristics of individual pores and the structural properties of dried food products can be utilized for process design, food quality determination, and the estimation of other food properties, such as thermal conductivity, density, moisture diffusivity, and bioactive component extractability (Rahman and Sablani, 2003). There is limited information available on the characteristics of pores derived from ginseng root products following AD, FD, and VMD processes. Moreover, the potential effect of total porosity on the ginsenoside exposure to an extraction solvent, which consequently affecting the ginsenoside retention of the dried ginseng roots, has not been widely explored. Thus, the aim of this experiment was to evaluate and compare the effects of three drying methods: AD, VMD, and FD on the porosity changes and distribution in dried NAG root and the subsequent ginsenoside retention.  36  2.2 MATERIALS AND METHODS Ginseng roots (Panax quinquefolium) were supplied by Chai-Na-Ta Corp. (Kamloops, BC) and Great Mountain Ginseng (Niagara-On-The-Lake, ON). The 4-years old BC- and 5-years old ON-ginseng roots were harvested in June 2007, packed into separate boxes, and delivered to the Food, Nutrition, and Health building at UBC where the ginseng roots were pooled, based on places of origin, and rinsed before being shredded using a household food processor. NAG roots were shredded to allow uniform heating and drying to occur during dehydration processes. The shredded ginseng roots were then randomly vacuum-packed into five separate bags, each containing 1 kg of ginseng roots, and stored at 4°C prior to dehydration, allowing a maximum storage time of 2 days. Methanol, acetonitrile, ethanol, isopropanol, hydrochloric acid, and trishydrochloric acid were from Fisher Scientific, Inc. (Nepean, ON). Water was purified by Epure water purification unit (Barnstead Intl.; Dubuque, IA). Ginsenoside standards Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3, and Rh2 were purchased from Chromadex, Inc. (Irvine, CA).  2.2.1 Drying of North American Ginseng Roots 2.2.1.1 Air-Drying Shredded ginseng roots were evenly distributed on 15” aluminum pans before being placed on a convection dryer (Sausage Maker Co.; Buffalo, NY) that was set at 38°C/100°F. Ginseng roots were dried for approximately 12 hours at three different occasions to obtain the three drying replicates. The final water activities ranged from 0.373 to 0.619 (AquaLab®, Model Series: 3; Decagon Devices, Inc.; Pullman, WA).  37  2.2.1.2 Vacuum Microwave-Drying Three hundred grams of shredded ginseng roots were placed in the bucket for drying in a vacuum microwave dryer (EnWave Inc, Vancouver, BC). The minimum quantity of ginseng roots required for a vacuum microwave drying process was determined to be 300 grams. Less than 100 grams of dried root remaining in the bucket after drying will potentially result in roots overheating, thus damaging the bucket and the vacuum microwave dryer. The empty bucket as well as the sample weights were determined and recorded. After placing the bucket into the vacuum microwave to begin the drying process under reduced pressure at 43.7 mm Hg with three different microwave powers of 0.8, 1.3, and 1.8kW employed, respectively. The drying process was performed at each microwave power, in triplicate, for 26, 12, and 10 minutes respectively. The drying process was also performed at three different times to obtain the three drying replicates. The final water activities were within a range of 0.219 to 0.694 determined using an AquaLab®, Model Series: 3 (Decagon Devices, Inc.; Pullman, WA).  2.2.1.3 Freeze-Drying Shredded ginseng roots were placed in foil-covered 8” pans, stored in the -18°C cold room overnight, and dried under reduced pressure (0.2mmHg) for 4-7 days. Three drying replicates were obtained by freeze-drying the shredded ginseng roots at three different occasions. The dried roots were stored in desiccators at 4°C until further analyses were performed. The final water activities of the freeze-dried roots ranged from  38  0.713 to 0.251 and were also determined using an AquaLab®, Model Series: 3 (Decagon Devices, Inc.; Pullman, WA).  2.2.2 Extraction of North American Ginseng Roots Dried North American ginseng roots were pre-soaked in 70% methanol overnight before extracted for 9h in 70% methanol using a Soxhlet extractor (Labconco Co.; Kansas City, MO) with the ratio of sample:methanol of 1:20 (Hu and Kitts, 2001). Methanolic extracts were filtered through Whatman no. 4 filter paper (Maidstone, UK) and further vacuum-concentrated (~40°C) by a rotary evaporator (Rotavapor®, Model Series: R-124; Büchi Co.; New Castle, DE).  2.2.3 North American Ginseng Roots Characterization 2.2.3.1 Moisture Content Moisture contents of both fresh and dried ginseng roots were determined using a vacuum oven (AOAC, 1955). Samples were weighed into pre-dried, pre-weighed, labelled aluminum weighing pans and then dried in a vacuum oven at 70°C and a vacuum pressure of 716.3mmHg, overnight. Upon completion of the drying, the pans were cooled and weighed. The moisture content of the sample was then calculated from the difference between the wet and dry weights divided by the dry weight of the sample. All moisture content analyses were performed in triplicate. % Moisture content =  Initial weight - Final weight x 100% Initial weight  39  2.2.3.2 Total Porosity and Pore Distribution Dried samples were further dehydrated in a phosphorus pentoxide-containing desiccator prior to taking the porosity measurement. Upon completion, the respective samples were placed in a powder penetrometer with a sample cup of 5cc-bulb volume and a 1.131cc-stem volume (Figure 2.1B). Total porosity and pore distribution of the samples were determined by introducing the penetrometer to the low-pressure analysis port followed by the high-pressure analysis port of the mercury porosimeter (Figure 2.1A) (Autopore™ IV 9500 Series; Micromeritics Instrument Co.; Norcross, GA).  B A  Figure 2.1 Micromeritics mercury porosimeter Autopore™ IV 9500 Series (A) and a 5ccbulb volume and a 1.131cc-stem volume powder penetrometer (B)  40  2.2.4 High Performance Liquid Chromatography Analysis of Recovered Ginsenoside Content from Dehydrated North American Ginseng Roots A gradient high performance liquid chromatography (HPLC) method was used to identify and quantify the ginsenosides recovered from the dehydrated North American ginseng roots, as described by Hu and Kitts (2001). Concentrated methanolic ginseng extracts were filtered through 0.45µm syringe filters (Pall Co.; East Hills, NY) and 10µL of the filtered extract was injected into a Hewlett-Packard 1100 series of HPLC system, equipped with an ODS column (250x4.6mm, 5µm) and diode-array detector operated at 203nm. The mobile phase used was HPLC-grade acetonitrile and water at a flow rate of 1mL/min and column temperature of 39°C. The solvent gradient used was 80% water (A) and 20% acetonitrile (B) at time 0, changing to 50% (A) and 50% (B) after 15 minutes, followed by 20% (A) and 80% (B) at minute 40, and 100% (B) at minute 45, and finally 80% (A) and 20% (B) at 55 minutes. The identity of individual ginseng saponins was determined based on the retention time of pure ginsenoside standards. Ginsenoside standards, Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3, and Rh2, with at least 95% purity were used to make standard stock solutions. Stock solutions containing 2.5mg/mL of individual ginsenoside standard was prepared by dissolving 5mg of each standard in 2mL of 50% acetonitrile, respectively. Calibration standard ginsenoside working solutions of 100µg/mL were prepared by diluting the stock solution with water in appropriate quantities. Standard curves were constructed by injecting the ginsenoside standard working solutions at different volumes. The amounts of ginsenosides recovered from the samples were calculated accordingly.  41  2.2.5 Statistical Analysis Experiments were done on the drying triplicates and repeated, in exception of the total and individual ginsenoside content determination, to obtain sub-sample replications. Sub-sample replicates were then averaged and analyzed using the one-way analysis of variance (ANOVA), with significance of difference defined at p<0.05 by a statistical software (SAS v 9.1; Cary, NC). Tukey’s significant difference test was used for pairwise comparison of means. Results were expressed as mean ± SEM (n=6; 3 replicates, 2 places of origin). Since ginseng roots were supplied by one farm per locations of origin (e.g. BC and ON) and preliminary results demonstrated no significant difference between the two places of origin, pooled average of the ginseng root samples was collected over the two locations of origin for each experiment.  2.3 RESULTS 2.3.1 Dehydration of North American Ginseng Roots The effect of various dehydration methods on the recovery of ginsenoside was evaluated by subjecting NAG roots to AD, FD, and VMD processes. The physical appearances of fresh and shredded and dehydrated NAG roots following AD, VMD, and FD are shown in Figure 2.2. From visual observations, a more prominent browning occurred in samples dried by AD compared to VMD and FD samples, respectively. AD ginseng root samples exhibited an apparent collapse of matrix structure, compared to the VMD and FD samples, which could be explained by the high surface tension that occurs in samples when water evaporates through its surface when exposed to AD processing (Karabulut et al., 2007; Consuelo Díaz-Maroto et al., 2002).  42  Table 2.1 shows the relative moisture content and water activity of both fresh and dried ginseng roots. The initial moisture content of the BC- and ON-ginseng roots was 77.3 %(wb) and 78.3 %(wb), respectively, corresponded to water activities of 0.97 and 0.99, respectively. Regardless of location of sample origin, the initial moisture content and water activities obtained were relatively similar. Following various dehydration processes, FD samples had the lowest moisture content and water activity (p<0.05) compared to that of AD and VMD, respectively.  43  A  C  B  D  Figure 2.2 Physical appearances of whole, fresh NAG roots (A) and shredded, dehydrated NAG roots after air drying (B), vacuummicrowave drying (C), and freeze drying (D) processes 44  Table 2.1 Moisture content and water activity of fresh and dried North American ginseng root Place of Drying Method Moisture content (%wb) Water activity (aw) origin BC  ON  Fresh  77.3 ± 1.24  0.972 ± 0.001  Air drying  8.60 ± 1.16a  0.496 ± 0.050a  VMD 0.8kW  8.70 ± 1.50a  0.456 ± 0.097ab  VMD 1.3kW  9.49 ± 0.52a  0.507 ± 0.042a  VMD 1.8kW  10.2 ± 0.36a  0.558 ± 0.022a  Freeze drying  2.01 ± 0.49b  0.214 ± 0.016b  Fresh  78.3 ± 0.81  0.990 ± 0.005  Air drying  8.31 ± 0.41a  0.458 ± 0.033a  VMD 0.8kW  7.33 ± 1.71a  0.347 ± 0.108ab  VMD 1.3kW  9.07 ± 0.21a  0.523 ± 0.031a  VMD 1.8kW  9.54 ± 0.22a  0.533 ± 0.066a  Freeze drying  1.96 ± 0.09 b  0.215 ± 0.014b  Data represent mean ± SEM (n=3) VMD0.8kW = vacuum-microwave drying at 0.8kW microwave energy VMD1.3kW = vacuum-microwave drying at 1.3kW VMD1.8kW = vacuum-microwave drying at 1.8kW a,b Within each column, treatments denoted by different superscripts are significantly different (p<0.05).  45  2.3.2 Effect of Dehydration Methods on Total Porosity and Pore Distributions of North American Ginseng Roots The method of dehydration of ginseng root had a significant effect on altering root matrix structure, as assessed quantitatively by porosity changes (Figure 2.3). Among samples, FD-ginseng roots had the highest total porosity (p<0.05) at 77.2% followed by VMD- and AD-ginseng roots at approximately 40% and 31.1%, respectively. VMDginseng roots exhibited intermediate total porosities, with increasing values ranging from 37.9% to 41.1% as the microwave energies increased from 0.8 kW to 1.8 kW, respectively. The increase in total porosity was significant (p<0.05) for VMD 0.8 kW compared to both 1.3 kW and 1.8 kW. Although the total porosities increased by increasing microwave energies from 1.3 kW to 1.8 kW, the increase was not found to be significant. All dehydrated samples showed a porous structure with a wide range (0.002 µm – 172 µm) of pore sizes. FD-ginseng roots were characterized by having significantly larger pores (>1.5 µm) (p<0.05) compared to samples processed by AD and VMD, respectively. The presence of large pores in FD-ginseng roots was 91.1% of the total porosity, which corresponded to 70.3% of the root matrix structure. A similar trend was also found in AD- and VMD-ginseng roots, although to a lesser extent. AD-ginseng roots produced 77.7% large pores, which corresponded to 24.1% of the root matrix. In contrast, VMD-ginseng roots produced approximately 28.0% pores that were large; this being equivalent to 67.0% of total porosity. The occurrence of medium-size pores (0.5 - 1.5 µm) and micro-size pores (< 0.5 µm) in FD-ginseng roots also differed significantly (p<0.05) from AD- and VMD-samples. FD-ginseng roots produced low amounts of medium and micro pores at approximately 4.00% of total porosity while AD- and VMD46  ginseng roots produced significantly higher amounts (p<0.05) of medium and micro pores in the root matrix structure. This could be due to the inability of AD and VMD to prevent the collapse of root matrix structure thus resulting in a higher proportion of smaller size pores (Karathanos et al., 1996; Huang and Clayton, 1990).  90  c 80 70  Porosity (%)  60 50  b  b  VMD1.3kW  VMD1.8kW  ab 40  a  30 20 10 0 AD  VMD0.8kW  FD  Figure 2.3 Total porosity (%) and pore distribution of NAG due to various drying methods. Data represent mean (n=6; 3 replicates, 2 places of origin). AD = air drying, VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy, VMD 1.3 kW = vacuum-microwave drying at 1.3 kW, VMD 1.8 kW = vacuum-microwave drying at 1.8 kW, FD = freeze-drying. large pores (>1.5 µm), medium pores (0.5 – 1.5 µm), micro pores (< 0.5 µm). a,bDifferent superscript letters indicate significant difference of total porosity (p<0.05)  47  Table 2.2 Total porosity (%) and pore distribution (%) of NAG after various drying methods Pore Distribution (%) Drying Total Method  Porosity (%)  > 1.5 µm  0.5 - 1.5 µm  < 0.5 µm  Air drying  31.08 ± 1.19a  24.13 ± 1.05a  2.88 ± 0.33a  4.07 ± 0.40a  VMD 0.8 kW  37.89 ± 1.76ab  25.35 ± 1.40b  5.82 ± 0.52b  6.72 ± 0.67b  VMD 1.3 kW  40.67 ± 1.66b  28.09 ± 1.16b  6.21 ± 0.69b  7.07 ± 0.57ab  VMD 1.8 kW  41.14 ± 1.78b  28.07 ± 1.72b  6.02 ± 0.64b  6.75 ± 0.58ab  Freeze-drying  77.15 ± 0.93c  70.28 ± 0.97c  3.68 ± 0.30c  3.18 ± 0.32c  Data represent mean ± SEM (n=6; 3 replicates, 2 places of origin) VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW a,b Within column, different superscript letters indicate significant difference (p<0.05).  2.3.3 Identification and Quantification of Ginsenosides by High Performance Liquid Chromatography (HPLC) Analysis Maintaining bioactivity potential of ginseng roots following dehydration is essential since the most consumed form of ginseng root is in dehydrated form. The method of dehydration was shown to have a significant effect on altering the ginseng root matrix structure. Moreover, altering the root matrix may affect the recovery of the ginsenoside by affecting the ginsenoside exposure to the extraction solvent. Consequently, the retention of ginsenosides in NAG roots following dehydration becomes even more vital in generating high quality functional products for human use. HPLC analysis of dried ginseng roots showed that ginsenoside content was retained after dehydration (Figure 2.4). Quantification of retained individual ginsenosides following AD, VMD, and FD processing is shown in Figure 2.5 and Table 2.3. Although different dehydration techniques characteristically altered the ginseng root matrix  48  structure, the effect of dehydration techniques on total ginsenoside content, calculated from the seven individual ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3), had a different trend from the total porosity (Figure 2.5). AD-ginseng root extracts contained the lowest recovery of ginsenoside (21.3 mg/g dry solid), followed by VMD 1.3 kW-ginseng root extracts (26.7 mg/g), VMD 0.8 kW (30.2 mg/g), FD-ginseng root extracts (30.9 mg/g), and VMD 1.8 kW (32.7 mg/g). Total ginsenosides retained in VMD-ginseng root extracts were comparable to the FD-ginseng root extract. Moreover, the total ginsenosides retained in VMD-ginseng root extracts did not correspond to the microwave energies applied to NAG roots. Among the different VMD energies applied to ginseng root, VMD 1.3 kW-ginseng root extracts had a lower recovery compared to ginseng root extracts dried by VMD 0.8 kW and VMD 1.8 kW (p>0.05). The similar results in ginsenoside content recovered in VMD-processed roots might correspond to the lack of change in total porosity over the different energies employed. The quantification of individual ginsenosides in extracts derived from dried ginseng root indicated that different drying techniques affected the retention of specific ginsenosides differently (Table 2.3). The five individual ginsenosides (e.g. Re, Rc, Rb2, Rd and Rg3) recovered from dried NAG following AD, VMD, and FD were not significantly different between drying treatments. VMD-ginseng root extracts yielded a lower recovery of ginsenoside Rg1 compared to the AD-ginseng root extracts with the least amount recovered in samples that were dried by VMD1.3kW (p<0.05) (Table 2.3). A different trend was found for the recovery of ginsenoside Rb1. VMD retained the highest amount of Rb1 (p<0.05), followed by FD and AD, respectively. The average retained amount of ginsenoside Rb1 in ginseng root samples dried by VMD was  49  approximately 16.0 mg/g, followed by FD (15.6 mg/g), and finally AD (8.70mg/g). The high recovery of ginsenoside Rb1 in dried ginseng root, regardless of the dehydration process used corresponds to finding by Chuang et al. (1995). Those workers indicated that ginsenoside Rb1 was one of the major ginsenosides found in NAG together with ginsenoside Re and malonyl-ginsenoside Rb1 in dried product. Ginsenoside Re, which has been associated with an anti-diabetic property of ginseng plants, was recovered in a relatively similar amount among all dehydrated samples (4.14 – 6.43 mg/g). On the other hand, the most potent antioxidant and anti-carcinogenic agent of ginseng plant, notably the ginsenoside Rg3, was recovered in very low amounts (e.g. 0.31 mg/g, 0.22 mg/g, and 0.27 mg/g) for AD-, VMD-, and FD-ginseng root extracts, respectively. )  B  mAU  DE F  1750 1500 1250 1000 750 500 250 0 5  7.5  G  10  12.5  15  B  17.5  20  22.5  7.5  10  12.5  15  17.5  20  22.5  27.5  min  VMD 1.8kW G  A  10  25  C DE F  7.5  min  G  B  1750 1500 1250 1000 750 500 250 0  27.5  Air Dried  A  mAU  25  C DE F  1750 1500 1250 1000 750 500 250 0  5  Freeze-Dried  A  mAU  5  C  12.5  15  17.5  20  22.5  25  27.5  min  Figure 2.4 HPLC chromatograms showing separation of individual ginsenosides in dried North American ginseng. VMD 1.8 kW = vacuum-microwave drying at 1.8 kW. A = ginsenoside Rg1 (retention time (Rt) = 11.4 minutes). B = ginsenoside Re (Rt = 11. 7 min). C = ginsenoside Rb1 (Rt = 15.5 min). D = ginsenoside Rc (Rt = 15.7 min). E = ginsenoside Rb2 (Rt = 16.1 min). F = ginsenoside Rd (Rt = 16.6 min). G = ginsenoside Rg3 (Rt = 20.2 min). Unidentified peaks represent unknown compounds 50  40  Total ginsenoside (mg/g dry ginseng)  b b  35  b  b 30  25  a  20  15  10  5  0 Air drying  VMD 0.8kW  VMD 1.3kW  VMD 1.8kW  Freeze drying  Figure 2.5 Total ginsenosides recovered following various dehydration methods. Data represent mean ± SEM (n=6; 3 replicates, 2 places of origin). VMD 0.8 kW = vacuummicrowave drying at 0.8 kW microwave energy, VMD 1.3 kW = vacuum-microwave drying at 1.3 kW, VMD 1.8 kW = vacuum-microwave drying at 1.8 kW  51  Table 2.3 Individual ginsenoside (mg/g dry solid) of North American ginseng extract (NAGE) recovered in ginseng root following various dehydration methods Air drying VMD 0.8 kW VMD 1.3 kW VMD 1.8 kW Freeze-drying PD Rg1  0.19 ± 0.03b  0.11 ± 0.01ab  0.08 ± 0.01a  0.15 ± 0.02ab  0.15 ± 0.02ab  Re  5.52 ± 1.58  4.14 ± 0.23  4.15 ± 0.68  4.87 ± 0.53  6.43 ± 0.80  Rb1  8.70 ± 1.55a  15.8 ± 2.18b  13.8 ± 1.41ab  17.8 ± 2.41b  15.6 ± 2.51ab  Rc  2.68 ± 0.38  3.04 ± 0.36  2.93 ± 0.21  3.63 ± 0.35  3.50 ± 0.38  Rb2  1.49 ± 0.15  1.96 ± 0.45  1.49 ± 0.09  1.70 ± 0.25  1.75 ± 0.12  Rd  3.47 ± 0.63  4.99 ± 0.68  4.61 ± 0.56  4.38 ± 0.44  4.89 ± 0.75  Rg3  0.31 ± 0.05  0.21 ± 0.04  0.22 ± 0.04  0.23 ± 0.01  0.27 ± 0.02  PT  Data represent mean ± SEM (n=6; 3 replicates, 2 places of origin) VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW PD: 20(s)-protopanaxadiol PT: 20(s)-protopanaxatriol a,b Within individual ginsenosides, different superscript letters indicate significant difference (p<0.05).  2.4 DISCUSSION Physicochemical attributes of ginseng root were altered significantly following various dehydration processes. The long exposure of ginseng roots with oxygen during AD process might facilitate enzymatic oxidation to occur resulting in the brown colour of the AD ginseng roots (Karabulut et al., 2007; Brennan, 2006; Krokida et al., 2001).  52  However, the use of vacuum during VMD and FD processes allow VMD- and FDprocessed ginseng roots to exhibit a more natural appearance, when compared to AD ginseng; a result reported by other workers (Popovich et al., 2005; Kim et al., 2000; Reynolds, 1998; Salunkhe et al., 1991). The difference in drying procedures among AD, VMD and FD processes resulted in a much lower moisture content and water activity (p<0.05) in FD ginseng root, compared to AD and VMD ginseng roots. Dehydration techniques also had a significant effect (p<0.05) on ginseng root matrix structure. This result was best shown by the highest total porosity observed in FD ginseng followed by VMD and AD ginseng, respectively. FD utilizes the principle of sublimation, which prevents the root matrix structure from collapsing, and therefore results in a highly porous end-product (Meda and Ratti, 2005; Hammami et al., 1999). Moreover, the high surface tension created as water evaporates through the food surface during AD processing resulting in a collapse of the matrix structure and consequently, reducing the total porosity characteristic (Meda and Ratti, 2005; Stanley and Tung, 1976). The higher total porosities of VMD ginseng roots compared to that of the AD samples may occur over a shorter drying time and a high vapour pressure due to the presence of vacuum during VMD process. These conditions ultimately resulted in structural expansion of the product (Sham et al., 2001). During VMD processing, as the microwave energy is absorbed by water molecules at reduced pressure, the rate of water vaporization increases rapidly to create a large difference in vapour pressure between the centre and surface structure of the root. This high pressure differential results in the expansion of the root matrix structure to yield a puffy texture that is characteristic of VMD samples (Sham et al., 2001). VMD ginseng root showed intermediate total  53  porosities with values that increased from 37.89% to 41.14% as the microwave energies increased from 0.8 kW to 1.8 kW, respectively. However, the differences in total porosities among VMD samples were not significant. Thus, increasing microwave energy from 0.8 kW to 1.8 kW did not significantly alter the total porosity. In a previous study, Durance (1997) described that the extent of puffing was dependent on the vacuum level utilized. Since the absolute pressure employed in this study was kept constant at 43.7 mm Hg, regardless of microwave energies used, this phenomenon could explain why similar total porosities were obtained for VMD-ginseng root at all microwave energies of 0.8 kW, 1.3 kW, and 1.8 kW. In addition to total porosity, pore sizes may potentially determine the rehydration capacity and the access of an extraction solvent to the bioactive components of the dehydrated product. For example, FD ginseng root matrix was characterized by having significant larger pores compared to that of AD and VMD ginseng roots. This could be explained by the preservation of the root matrix structure following FD which resulted in a markedly greater number of large pores in the FD ginseng root samples. On the contrary, the failure of AD and VMD to prevent the root matrix structure from collapsing resulted in a higher proportion of smaller pore size (Karathanos et al., 1996; Huang and Clayton, 1990). This finding may explain the more access of extraction solvent to ginsenosides from roots that were freeze-dried since a large pore size will result in a faster infiltration by various solvents (Meda and Ratti, 2005; Oetjen, 2004; Karathanos et al., 1996). To examine this phenomenon more carefully, a fingerprint analysis of the ginsenoside composition for each ginseng root treatment was performed. Ginsenoside  54  content is commonly derived from seven individual ginsenosides (Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3), which in this study were all retained in roots dried by different methods, thus confirming results of previous studies. These studies also identified the retention of ginsenosides that belonged to the protopanaxadiol (Rh2, Rg3, Rb1, Rb2, Rc, Rd) and protopanaxatriol (Rg1, Re) groups (Xie et al., 2007; Popovich and Kitts, 2005; 2004; Hu and Kitts, 2001; Yamaguchi et al., 1988). The concentrations of total ginsenoside retained in the dried NAGEs were found to be affected by the dehydration methods employed. Total ginsenoside was recovered the lowest following AD process followed by VMD and FD processes. The recovery of ginsenosides from VMD-ginseng root was comparable to that for FD samples. It may suggest that the different total porosities between VMD and FD ginseng roots did not impact on ginsenoside recovery. FD-ginseng root had a higher total porosity which reflects the affinity of FD processing to preserve the root matrix structure, more so than VMD process. However, the recovery of ginsenosides was similar between VMD and FD drying treatments. This result suggests that the recovery of ginsenosides in dried NAG root is not only affected by the change in porosity induced by the dehydration techniques employed but may also be due to other factors such as transformation, oxidation, or degradation of ginsenosides during dehydration (Popovich et al., 2005; Ren and Chen, 1999). Extractions of ginsenosides depend heavily on the correct choice of solvent and the application of heat to increase the extractability of the ginsenosides from ginseng root or leaf material. Alcohols such as methanol, ethanol, and butanol have been widely used as the extraction solvent of choice for many ginseng varieties. Due to the different polarities of these solvents, the extractability of ginsenosides and the mass transfer rate  55  are different. Zhang et al. (2006) determined the extraction yield of ginsenoside following a water-ethanol extraction was the highest compared to water-saturated n-butanol, watermethanol, and water in ultrahigh pressure extraction, respectively. Among several different water:ethanol ratios, 70% ethanol coupled with a solvent:material ratio of ~50:1 was found to produce the highest ginsenoside content (Zhang et al., 2006). This study utilized 70% methanol as the solvent to extract ginsenosides from dried NAG root with a solvent:material ratio of 20:1. The low boiling temperature of methanol (65°C) kept the degradation and evaporation of the ginsenosides at a minimal level during extraction. Although refluxing with methanol has been indicated to increase the extraction effectiveness (Nho and Sohn, 1989), soxhlet extraction was performed in this study in order to maximize the extraction efficiency. A higher number of samples were extracted at the same time using this procedure compared to the reflux method. All these factors combined could have affected the levels of ginsenosides recovered. The recoveries of seven ginsenosides from NAGE following AD, VMD, and FD are important to recognize the human health and wellness benefits of dried ginseng roots and are related to ginseng consumption. Ginsenoside Re, for example, has been associated with the management of diabetes by lowering hyperglycemia and hypercholesterolemia of ob/ob mice and diabetic rats, respectively (Attele et al., 2002; Cho et al., 2006). On the other hand, ginsenosides Rb1, Rc, and Rd can be transformed to more potent bioactive ginsenosides Rg3 and Rh2 by the application of heat (Popovich and Kitts, 2004). Although ginsenoside Rg3 was recovered in all dried NAG, ginsenoside Rh2 was not retained in NAG roots following dehydration. This may be due to the temperatures and times used to dehydrate the ginseng roots were either not sufficiently  56  high or long enough to transform ginsenosides to Rh2. This result further indicates that the processing and extraction methods may affect the retention of ginsenoside compounds in ginseng roots since ginsenoside Rh2 has been recovered in red Korean ginseng, a process that includes steaming process prior to dehydration (Court, 2000), and in NAG leaves by 100°C refluxing for 1.5h (Popovich and Kitts, 2002). Ginsenoside Rg3 has been reported to reduce prostate cancer cells proliferation (Liu et al., 2000) while ginsenoside Rh2 affects the proliferation of leukemia cells (Popovich and Kitts, 2002) and colon cancer cells (Popovich and Kitts, 2004).  2.5 CONCLUSION Dehydration methods were found to significantly affect ginseng root matrix structure by producing a porous structure that was uniquely different for the method of dehydration used. FD ginseng exhibited the highest total porosity followed by VMD and AD, respectively. All dehydrated samples had a porous structure with pore sizes that ranged from as small as 0.002 µm to 172 µm. Dried ginseng root matrices, regardless of dehydration method used, were mainly constituted by macropores (>1.5µm). The high occurrence of macropores in FD ginseng roots is expected as FD utilizes the principle of sublimation, which will prevent collapse of the root matrix structure, and therefore result in a highly porous end-product. However, the high total porosity of FD ginseng did not result in a high recovery of ginsenosides in the corresponding extract. Although the total ginsenoside recovered in FD-ginseng root extract was significantly higher than ADginseng root extract, the level was comparable to that recovered following VMD  57  processing. Moreover, dehydration methods also affected the recoveries of individual ginsenosides. Ginsenoside Rg1 was recovered the lowest following VMD 1.3 kW process followed by VMD 0.8 kW, VMD 1.8 kW, FD, while AD process retained the highest amount of ginsenoside Rg1. However, a different trend was shown in the recovery of ginsenoside Rb1. AD process retained the lowest amount of ginsenoside Rb1, followed by VMD 1.3 kW and FD processes, which recovered intermediate amounts of ginsenoside Rb1, and finally VMD 0.8 kW and VMD 1.8kW retained the highest amount of ginsenoside Rb1. This finding, therefore, suggested that the ginsenoside recovery was not necessarily affected by the differences in porosity induced by different dehydration techniques. Moreover, VMD process also showed comparable effectiveness over AD and FD technologies in the dehydration of NAG roots since VMD was not only able to retain the initial characteristics of the NAG roots, which was one of the main disadvantages of AD process, but also retained a relatively similar amount of ginsenosides compared to FD process.  58  CHAPTER 3: COMPARISON OF DIFFERENT DEHYDRATION TECHNIQUES ON THE BIOACTIVE POTENTIAL OF NORTH AMERICAN GINSENG ON CULTURED 3T3-L1 FAT CELL LINE 3.1 INTRODUCTION Obesity is a clinical disorder that contributes to chronic diseases such as Type 2 diabetes, hypertension, some types of cancer, and a significant reduction in life expectancy (Flegal et al., 2005; Gregg et al., 2005; Douketis et al., 1999). Whereas excess energy intake is the primary dietary cause of obesity, other metabolic factors, such as an excess deposition of white adipose tissue (WAT) is a factor in obesity. WAT is the major energy reserve in human body which stores triacylglycerols in energy depots that are in excess to the calories that are released with energy utilization (Gregoire et al., 1998). In vitro, cell culture systems have been used to allow a better understanding of the molecular mechanisms underlying the transition of undifferentiated fibroblast-structured preadipocytes, to mature, rounded adipocyte cells. This differentiation leads to the onset of lipid uptake by adipocytes. One of the widely used cells employed for these purposes is the 3T3-L1 murine fat cell line (Green and Kehinde, 1975). Using these cells to study adipogenesis has enabled workers to better understand how to prevent or treat obesity through natural therapeutic means. While a change in diet and physical activity towards a healthy direction is considered to be a primary treatment for obesity, pharmacotherapy and/or aggressive dietary therapy involving herbal products may also be useful to treat obesity (Lim et al., 2009; Attele et al., 2002).  59  Ginseng has been proposed to have bioactive potentials involving hyperglycemia and hypercholesterolemia (Kim et al., 2009; Vuksan et al., 2000). Ginsenoside Re demonstrated a significant role in normalizing the blood glucose level and improving glucose tolerance of ob/ob mice (Attele et al., 2002) whereas ginsenoside Rb1 was found to decrease the total cholesterol level in the liver (Park et al., 2002). The anti-obesity potential of ginseng has been proposed to inhibit the transcriptions of peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα) (Hwang et al., 2009; Kim et al., 2005). PPARγ is a transcription factor that induces growth arrest resulting in an adipogenesis initiation of confluent adipocyte cells while C/EBPα is a transcription factor assisting adipogenesis by inducing the transactivation of adipocyte genes such as aP2, SCD1, PEPCK, and leptin (Cowherd et al., 1999; Gregoire et al., 1998). Most of the anti-obesity potential studies of ginseng have been established using purified ginsenosides or Asian ginseng extracts. There has been limited research on the anti-obesity potential of crude NAG root extract. Therefore, the purpose of this study was to evaluate the effectiveness of NAG root extracts derived from different drying processes on the potential bioactivity of ginseng roots to modify adipogenesis. In particular, effort was directed at assessing the comparative effect of AD, VMD, and FD on ginseng root bioactive potential, using preadipocyte and mature adipocyte 3T3-L1 cell viability and adipogenesis biomarkers.  60  3.2 MATERIALS AND METHODS Ginseng roots (Panax quinquefolium) were supplied by Chai-Na-Ta Corp. (Kamloops, BC) and Great Mountain Ginseng (Niagara-On-The-Lake, ON). Ginsenoside standards Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3, and Rh2 were purchased from Chromadex, Inc. (Irvine, CA). Methanol, acetonitrile, ethanol, isopropanol, hydrochloric acid, and tris-hydrochloride were obtained from Fisher Scientific, Inc. (Nepean, ON). The fat cell line, 3T3-L1, was obtained from ATCC (Manassas, VA). Calf serum, fetal calf serum, and antibiotics were from Invitrogen Co. (Grand Island, NY). Tris base, ethylenediaminetetraacetic acid (EDTA), nicotinamide adenine dinucleotide-H (NADH), pyruvate, Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate buffer saline (PBS), isobutylmethylxanthine (IBMX), dexamethasone, insulin, MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SDS (Sodium Duodecyl Sulphate), Oil-Red-O stain, and Igepal CA-63 were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO). Cell culture Petri dishes, 24-well plates, and 96-well plates were purchased from Sarstedt Inc. (Montreal, QC).  3.2.1 Extraction of North American Ginseng Roots Dried shredded ginseng roots were extracted according to the method as previously described on page 39. Methanolic ginseng extracts recovered from different drying procedural treatments were used in cells culture assays. Extracts were vacuumconcentrated to dryness prior to reconstitution in 70% ethanol to a final concentration of 3 g/mL and further diluted in DMEM with calf serum (10%), penicillin (100 U), and streptomycin (100 µg/mL). The working concentration range was 0.5 mg/mL to 50 mg/mL. 61  3.2.2 3T3-L1 Cell Culture Analyses 3.2.2.1 Cell Proliferation Cells were propagated in tissue culture Petri dishes containing DMEM supplemented with calf serum (10%), penicillin (100 U), and streptomycin (100 µg/mL) and incubated at 37°C in a 5% CO2 humidified incubator. Cells were never permitted to reach confluence, trypsinized every third days, and plated at 1.1 x 105 per Petri dish. Stocks of frozen cells were prepared at the earliest passage possible and thawed cells were also used at the earliest passage possible.  3.2.2.2 Cell Viability Assay The cell viability assays of both preadipocyte and adipocyte 3T3-L1 cell lines were based on MTT reduction (Mosmann, 1983). The preadipocyte 3T3-L1 cells were seeded to each well of 96-well plates to a final concentration of 3x104 cells/mL. Cells were maintained in DMEM, supplemented with calf serum (10%), penicillin (100U), and streptomycin (100 µg/mL) and incubated at 37°C in a 5% CO2 humidified incubator. North American ginseng extracts (NAGEs) were added at different growth stages of preadipocyte 3T3-L1 cells to determine the viability of cells at both pre- and postconfluent stages. The concentrations of dried NAGEs added to pre-confluent preadipocytes were 5 mg/mL, 10 mg/mL, 25 mg/mL, and 50 mg/mL while the concentrations added to post-confluent preadipocytes were 5 mg/mL, 10 mg/mL, 20 mg/mL, 25 mg/mL, and 50 mg/mL. Preliminary results showed that ethanol contained in the dried NAGEs was less than 1% and had no effect on cell viability. Consequently, untreated cells were designated as control. Pre-confluent viability assays were performed by adding NAGEs to individual wells containing cells after two days of cells incubation.  62  In contrast, NAGEs were added to wells after five days of incubation for post-confluent viability assays. Following the addition of NAGE, cells were incubated for 24, 48, and 72 hours prior to adding MTT solution at a final concentration of 0.5 mg/mL. Cells were then incubated in the dark for another four hours before adding 100 µL of 10% SDS in 0.1 N HCl. The optical density of the 3T3-L1 cell line was read at 570 nm absorbance in a microplate reader (Bio-Rad, Cambridge, MS) following overnight incubation of the cells in the dark. The cell viability assay of the mature adipocyte cells was also done in a similar manner. Preadipocyte cells underwent induced differentiation, as indicated in the following adipogenesis assay, and were incubated for six days before the addition of NAGEs at concentrations of 5 mg/mL, 10 mg/mL, 20 mg/mL, 25 mg/mL, and 50 mg/mL. A MTT solution made to 0.5 mg/mL final concentration was added to individual wells containing cells at 24, 48, and 72 h incubation to evaluate the viability of the induced cells. Similar to the viability assay used for the preadipocyte 3T3-L1 cell line, the addition of MTT solution to cells was made four hours before adding 10% SDS in 0.1 N HCl. The optical density at 570 nm absorbance was read the following day to quantify cell viability according to the formula: % Cell Viability =  Abscells − Abs blank x 100% Abscontrol − Abs blank  where: Abscells = absorption of wells containing cells with dried NAG root extracts Abscontrol = absorption of wells containing cells without dried NAG root extracts Absblank = absorption of wells without cells.  63  The 3T3-L1 fat cell viability results were transformed to derive IC50 values of dried NAGEs using a data analysis and graphing software (Microcal Origin 6.0; OriginLab Co.; Northampton, MA) as described on appendix B (page 114).  3.2.2.3 Adipogenesis Procedural Assay The adipogenesis assay was followed according to the methods provided by the distributor, Chemicon (2006). Murine preadipocyte 3T3-L1 cells were suspended at a concentration of 3x103 cells/100µL/well in 96-well plates. NAGE at non-toxic concentrations of 1, 1.5, 2, and 2.5 mg/mL were added to the cultured cells during both pre- and post-confluent stages, respectively, similar to the cell viability assay repeated above. Untreated cells were used as the control. The preadipocyte 3T3-L1 cell growth medium1 was changed following confluency on the fifth day with an adipogenesis initiation medium2. Wells without cells, or blank wells, were always maintained in the growth medium. The culture medium was again changed to an adipogenesis progression medium3 after a three day incuation (37°C at 5% CO2 humidified incubator). Further incubation of cells was performed for another three days before the culture medium was changed to adipogenesis maintenance media4. Non-toxic concentrations of dried NAG root extracts were added to initiation, progression, and maintenance cell culture media at each stage of adipogenesis. Adipocyte cells were incubated for at least three days to allow the accumulation of intracellular lipid droplets to reach its maximum.  1  DMEM with 10% calf serum, 100U penicillin, 100 µg/ml streptomycin DMEM with 10% fetal bovine serum (FBS), 10 µg/ml insulin, 0.5 mM IBMX, 1 µM dexamethasone, 100U penicillin, 100 µg/ml streptomycin 3 DMEM, 10% FBS, 10 µg/ml insulin, 100U penicillin, 100 µg/ml streptomycin 4 DMEM, 10% FBS, 100U penicillin, 100 µg/ml streptomycin 2  64  3.2.2.4 Oil-Red-O Staining Assay The mature adipocyte 3T3-L1 cells which underwent induced adipogenesis were washed twice with PBS following removal of medium and 50 µL of Oil-Red-O solution (0.36% Oil-Red-O stain in 60% isopropanol) was added to each well. Non-specific binding was also controlled for by wells that did not contain cells but had culture medium. Following incubation for 15 minutes at room temperature, wells were washed 3 times with 100 µL 60% isopropanol, followed by the addition of 25 µL of dye extraction solution (4% Igepal CA-63). Plates were then set on an orbital shaker for 15-30 minutes prior to reading absorbance in a microplate reader (Bio-Rad, Cambridge, MS) at 520 nm. % Cell Differentiation =  Abscells − Abs blank x 100% Abscontrol − Abs blank  where: Abscells = absorption of wells containing cells with dried NAG root extracts Abscontrol = absorption of wells containing cells without dried NAG root extracts Absblank = absorption of wells without cells.  3.2.2.5 Lactate Dehydrogenase (LDH) Assay Preadipocyte 3T3-L1 cells were seeded at 1x106 cells/ml in 24-well plates and NAGEs (1.5 mg/mL) were added to individual wells containing cells on the second day of incubation. Untreated cells acted as control. Cells were incubated at 37°C in a 5% CO2 humidified incubator for 24, 48, and 72 h before cell-free supernatant was obtained by centrifugation (400 x g) for ten minutes. Two millilitres of Tris-EDTA-NADH buffer and 50 µL of cell-free supernatant were mixed and incubated in a 37°C water bath for ten minutes before adding 200 µL of pre-warmed (37°C) pyruvate to the mixture. The mixture was transferred to a 3 mL cuvette and the initial reaction velocity was recorded by continuous monitoring of the absorption at 340 nm for 6 minutes at 37°C (UV-160 65  Specthrophotometer, Shimadzu Co.; Kyoto, Japan). Data are expressed as % untreated LDH activity. The final concentration of each compound in the cuvette was 50 mM Tris buffer (pH 7.4, 37°C), 5 mM EDTA, 150 µM NADH, and 1.2 mM pyruvate as indicated by Kachmar and Moss (1982). LDH activity in International units at 37°C was calculated from: mU/mL =  1000 2.25 ∆A x x 0.60 x min 6.22 0.05  where: ∆A•min-1 = average absorbance change per minute 1000 = converts mmol to µmol 6.22 = milimolar absorptivity of NADH at 340 nm (unit: mL•mmol-1•cm-1) 2.25 = total volume in cuvette (unit: mL) 0.05 = volume of cell-free supernatant (unit = mL) 0.60 = Temperature correction factor at 37°C (Demetriou et al., 1974)  % untreated LDH activity =  LDH cells x 100% LDH control  where: LDHcells = LDH activity of cells following treatment with dried NAG root extracts LDHcontrol = LDH activity of untreated cells.  3.2.2.6 Effect of Individual Ginsenoside Standards on 3T3-L1 Cell Adipogenesis The adipogenesis procedural assay of the 3T3-L1 preadipocyte cells was done  similarly as described on page 64. Eight ginsenoside standards (e.g. ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3, and Rh2) were added to individual wells containing cells after two days of cell incubation at 50 µg/mL and 100 µg/mL concentrations. Untreated cells were designated as control. The ginsenoside standards were also added to initiation, progression, and maintenance cell culture media at each stage of adipogenesis. Upon  66  maximum accumulation of intracellular lipid droplet at the end of adipogenesis, the mature adipocyte 3T3-L1 cells underwent Oil-Red-O staining assay as described on page 65.  3.2.3 Statistical Analysis Experiments were done on the drying triplicates and repeated to obtain subsample replications. Sub-sample replicates were then averaged and analyzed using the one-way analysis of variance (ANOVA), with significance of difference defined at p<0.05 by a statistical software (SAS v 9.1; Cary, NC). Tukey’s significant difference  test was used for pair-wise comparison of means. Results were expressed as mean ± SEM (n=6; 3 replicates, 2 places of origin). Since ginseng roots were supplied by one farm per locations of origin (e.g. BC and ON) and preliminary results demonstrated no significant difference between the two places of origin, pooled average of the ginseng root samples was collected over the two locations of origin for each experiment.  3.3 RESULTS 3.3.1 Effect of Different Dehydration Methods of North American Ginseng Roots on 3T3-L1 Preadipocyte Cell Viability Cytotoxicity of dried NAG root extracts on the 3T3-L1 fat cell line was assessed prior to further biological analyses. Viability of 3T3-L1 preadipocyte cells was shown to be affected by dried NAG root extracts in a time- and dose-dependent manner (Figures 3.1 & 3.2). 3T3-L1 viability data was transformed to derive an IC50 at both pre-confluent  67  and post-confluent stages of cell proliferation (Table 3.1 & 3.2). The IC50 values of dried NAG root extracts treated to pre-confluent preadipocyte cells were found to be lower than that of the post-confluent cells, regardless of drying methods. Furthermore, in preconfluent preadipocyte cells, IC50 values established at 24 h incubation time were lower (p<0.05) than both 48 and 72 h incubation periods (Table 3.1). VMD 0.8 kW- and FDginseng root extracts gave the lowest IC50 values, followed by AD-, VMD 1.3 kWginseng root extracts while VMD 1.8 kW-ginseng root extract gave the highest IC50 values at all incubation periods of 24, 48, and 72 h. This results indicated that the highest toxicities on 3T3-L1 were exhibited by VMD 0.8 kW- and FD-ginseng root extracts while VMD 1.8 kW-ginseng root extract exhibited the lowest toxicity (p<0.05) to preconfluent preadipocyte cells. As previously mentioned, the toxicities of dried NAG root extracts on postconfluent preadipocyte cells were similar to that observed on pre-confluent cells (Table 3.2). However, dried NAG root extracts affected the viabilities of post-confluent preadipocytes differently as they were on pre-confluent preadipocytes. In post-confluent preadipocyte cells, FD- and AD-ginseng root extracts exhibited the lowest IC50 values (p<0.05), followed by VMD 1.8 kW-, and finally VMD 0.8 kW- and VMD 1.3 kWginseng root extracts in all incubation periods. This finding indicated that different dehydration methods affected the cytotoxicities of dried NAG root extracts when exposed to both pre- and post-confluent preadipocyte cells although different trends were found in the pre- and post-confluent preadipocyte cell viabilities. The relative toxicities of dried NAG root extracts, derived from root dried by different methods, on preadipocyte cells were also evaluated using the LDH activity  68  assay (Figure 3.3). As a functional marker of membrane integrity, LDH is released into the cytoplasm when the cell membrane is damaged (Kachmar and Moss, 1982). Although the concentration of ginseng extracts used in the LDH assay was not sufficient to kill the cells, a damaging effect towards the preadipocyte cell membrane was observed as the LDH activity was greater than 100% of the control. The LDH activities were higher than 100% after both 24 and 48 h incubation periods. FD-ginseng root extract exhibited a strong toxicity towards preadipocyte cells (p<0.05) after 48 h incubation, followed by VMD 1.8 kW- and AD-ginseng root extracts. However, the LDH activities of dried NAG root extracts was not significant, regardless of drying method used, after 72 h incubation. This finding may indicate similar cytotoxicities of dried NAG root extracts on preadipocyte cells despite the drying method used.  69  A 120 Cell Viability (%)  100 80 60 40 20 0 0  10  20 30 40 Concentration (mg/mL)  50  60  0  10  20 30 40 Concentration (mg/mL)  50  60  0  10  20 30 40 Concentration (mg/mL)  50  60  Cell Viability (%)  B 120 100 80 60 40 20 0  Cell Viability (%)  C 120 100 80 60 40 20 0  Figure 3.1 The effect of NAGE on pre-confluent 3T3-L1 preadipocyte cells viability measured by MTT assay. Each panel represents mean ± SEM (n=6; 3 replicates, 2 places of origin). air dried NAGE. vacuum-microwave dried at 0.8 kW NAGE. vacuummicrowave dried at 1.3 kW NAGE. -x- vacuum-microwave dried at 1.8 kW NAGE. freeze-dried NAGE. A=24 h, B=48 h, and C=72 h  70  A 120 Cell Viability (%)  100 80 60 40 20 0 0  10  20 30 40 Concentration (mg/mL)  50  60  0  10  20  50  60  50  60  B 120  Cell Viability (%)  100 80 60 40 20 0 30  40  -20  Concentration (mg/mL) C 120  Cell Viability (%)  100 80 60 40 20 0 0  10  20 30 40 Concentration (mg/mL)  Figure 3.2 The effect of NAGE on post-confluent 3T3-L1 preadipocyte cells viability measured by MTT assay. Each panel represents mean ± SEM (n=6; 3 replicates, 2 places of origin). air dried NAGE. vacuum-microwave dried at 0.8 kW NAGE. vacuummicrowave dried at 1.3 kW NAGE. -x- vacuum-microwave dried at 1.8 kW NAGE. freeze-dried NAGE. A=24 h, B=48 h, and C=72 h  71  Table 3.1 IC50 of NAGE on pre-confluent preadipocyte 3T3-L1 cells Drying Method 24 h 48 h 72 h Air drying  6.013 ± 0.12ab,x  5.389 ± 0.30ab,y  5.839 ± 0.15ab,y  VMD 0.8 kW  5.623 ± 0.32a,x  3.944 ± 0.68a,y  3.885 ± 0.74a,y  VMD 1.3 kW  7.024 ± 0.63ab,x  5.466 ± 0.20ab,y  3.847 ± 0.80ab,y  VMD 1.8 kW  7.003 ± 0.81b,x  6.965 ± 1.00b,y  6.203 ± 1.02b,y  Freeze-drying  5.822 ± 0.34a,x  3.967 ± 0.61a,y  4.078 ± 0.50a,y  Data represent mean ± SEM (n=6) from MTT assay at different time points. VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW a,b Within column, drying methods with different superscripts are significantly different from each other (p<0.05). x,y Within row, incubation times with different superscripts represent significant difference (p<0.05).  Table 3.2 IC50 of NAGE on post-confluent preadipocyte 3T3-L1 cells Drying method 24 h 48 h 72 h Air drying  12.14 ± 0.74a  11.87 ± 0.24a  11.02 ± 0.23a  VMD 0.8 kW  14.41 ± 1.37b  17.74 ± 0.80b  17.67 ± 0.63b  VMD 1.3 kW  15.77 ± 1.35b  15.06 ± 1.34b  14.72 ± 1.80b  VMD 1.8 kW  14.42 ± 1.23ab  13.86 ± 1.59ab  13.98 ± 1.89ab  Freeze-drying  12.02 ± 0.81a  11.62 ± 0.61a  12.81 ± 0.46a  Data represent mean ± SEM (n=6) from MTT assay at different time points VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW a,b Within column, drying methods with different superscripts are significantly different from each other (p<0.05).  72  % LDH activity related to untreated cells  250 c 200 b 150  ab  ab  ab  ab a  a  a  100  50  0 24h  48h  72h  Figure 3.3 Lactate dehydrogenase (LDH) activity measured in pre-confluent preadipocyte 3T3-L1 cells after 24, 48 and 72 h of treatment at 1.5mg/mL NAGE. Data expressed as percentage of LDH activity related to untreated cells (mean ± SEM) of six separate experiments performed in triplicates. air-dried NAGE. vacuum-microwave dried 1.8 kW NAGE. freeze-dried NAGE. a,bBars with different superscript letters are significantly different (p<0.05)  3.3.2 Effect of Different Dehydration Methods of North American Ginseng Roots on 3T3-L1 Mature Adipocyte Cell Viability Similar to the effect observed with dried NAG root extracts on the viability of preadipocyte cells, the viability of 3T3-L1 mature adipocyte cells was also affected by different dried NAG root extracts in a similar time- and dose-dependent manner (Figure 3.4). However, the individual IC50 values of different dried NAG root extracts on mature adipocyte cells were found to be higher than that of the preadipocyte cells indicating a potential greater sensitivity of preadipocyte cells to dried NAG root extracts, especially the pre-confluent preadipocyte cells (Tables 3.1, 3.2, & 3.3). NAG root extracts, derived from different drying methods used, exhibited similar toxicity effects on mature 73  adipocyte cells although VMD-ginseng root extracts exhibited the greatest toxicity effects (p>0.05) on mature adipocyte cells following 24 h incubation. VMD 0.8 kW-ginseng root extract had a considerably low IC50 value (14.9 mg/mL), followed by ginseng root extracts derived from VMD 1.3 kW (16.5 mg/mL) and VMD 1.8 kW processing (17.4 mg/mL). As the incubation period increased to 72 h, the IC50 values of VMD-ginseng root extracts increased from 16.2 mg/mL to 20.1 mg/mL. Consequently, the toxicity of VMD-ginseng root extract on mature adipocyte cells after 72 h incubation was lower compared to NAG root extracts dried by AD (15.7 mg/mL) and FD (13.8 mg/mL) (p>0.05). As mentioned earlier, the IC50 values of dried NAG root extracts was not significantly different at different incubation periods or with different dehydration methods (Table 3.3). This finding, therefore, suggest that different dehydration methods have relatively negligible effects on the cytotoxic potential of dried NAG root extracts to 3T3-L1 fat cells when cultured in mature adipose stage.  74  A120 Cell Viability (%)  100 80 60 40 20 0 0  10  20 30 40 Concentration (mg/mL)  50  60  0  10  20 30 40 Concentration (mg/mL)  50  60  0  10  20  50  60  B 120  Cell Viability (%)  100 80 60 40 20 0  C 120  Cell Viability (%)  100 80 60 40 20 0 -20  30  40  Concentration (mg/mL)  Figure 3.4 The effect of NAGE on 3T3-L1 mature adipocyte cells viability measured by MTT assay. Each panel represents mean ± SEM (n=6; 3 replicates, 2 places of origin). air dried NAGE. vacuum-microwave dried at 0.8 kW NAGE. vacuum-microwave dried at 1.3 kW NAGE. -x- vacuum-microwave dried at 1.8 kW NAGE. freeze-dried NAGE. A=24 h, B=48 h, and C=72 h  75  Drying  Table 3.3 IC50 of NAGE on mature adipocyte 3T3-L1 cells 24 h 48 h 72 h  Air drying  20.53 ± 0.49  15.96 ± 1.27  15.67 ± 0.45  VMD 0.8kW  14.92 ± 1.11  14.74 ± 2.29  20.84 ± 2.54  VMD 1.3kW  16.46 ± 2.33  16.42 ± 2.06  20.28 ± 2.15  VMD 1.8kW  17.41 ± 1.76  16.67 ± 1.83  18.91 ± 3.36  Freeze-drying  19.27 ± 0.46  13.34 ± 0.77  13.83 ± 0.34  Data represent mean ± SEM (n=6) from MTT assay at different time points VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW  3.3.3 Effect of Different Dehydration Methods of North American Ginseng Roots on Adipogenesis of 3T3-L1 Cell Following toxicity evaluation of dried NAG root extracts on 3T3-L1 preadipocyte and adipocyte cell, the effect of dried NAG root extracts on adipogenesis of 3T3-L1 cells was further determined using the Oil-Red-O staining assay (Figure 3.5). Adipogenesis was induced according to the methods previously described by Frost and Lane (1985). Adipogenesis of 3T3-L1 preadipocyte cells was affected in a dose-dependent manner by the NAG root extracts derived from different dehydration method used (Figure 3.6). Dried NAG root extracts at 2 mg/mL and 2.5 mg/mL significantly reduced adipogenesis (p<0.05), compared to lower concentrations of 1 mg/mL and 1.5 mg/mL, when added to pre-confluent preadipocyte cells (Tables 3.4). Most dried NAG root extracts did not reduce pre-confluent preadipocytes adipogenesis at 1 mg/mL, except extracts derived from VMD 0.8 kW (85.5%) and VMD 1.8 kW (96.9%). When added at 1.5 mg/mL to the pre-confluent preadipocyte cells, all dried NAG root extracts started to inhibit adipogenesis. Moreover, as the concentrations of dried NAG root extracts increased to 2  76  mg/mL and 2.5 mg/mL, the adipogenesis inhibition of dried NAG root extracts also increased. The inter-variation in responses to 3T3-L1 cells when exposed to dried NAG root extracts was too high to enable significant difference among drying methods to be observed for adipogenesis of pre-confluent preadipocyte cells. Following addition to post-confluent preadipocyte cells, ginseng root extracts derived from VMD 1.3 kW (81.9%), VMD 1.8 kW (85.4%), and AD processes (89.0%) inhibited adipogenesis at a concentration as low as 1 mg/mL, whereas VMD 0.8 kW- and FD-ginseng root extracts started to inhibit adipogenesis at 2 mg/mL. Consequently, VMD 0.8 kW- and FD-ginseng root extracts, when added to post-confluent preadipocyte cells, exhibited lower adipogenesis inhibition effects (p<0.05) at all concentrations, compared to AD-, VMD 1.3 kW-, and VMD 1.8 kW-ginseng root extracts. As the concentration of dried NAG root extracts increased to 2 mg/mL and 2.5 mg/mL, adipogenesis was significantly inhibited (p<0.05). Furthermore, since the concentrations of dried NAG root extracts used in the adipogenesis assay were much lower than the IC50 values, the inhibitory effect of NAGE on 3T3-L1 adipogenesis was not due to the cytotoxicity effect of these extracts. Individual ginsenoside standards when present at non-toxic levels were also shown to inhibit adipogenesis of 3T3-L1 cells (Figure 3.7). Most ginsenoside standards, with the exception of ginsenoside Rg1, exhibited inhibitory effects towards adipogenesis at a concentration as low as 50 µg/mL. The adipogenesis inhibitory effects of most ginsenoside standards were relatively similar at both concentrations of 50 µg/mL and 100 µg/mL, with the highest inhibitory effect (p<0.05) was found in ginsenoside Rh2 at 100 µg/mL.  77  A  B  Figure 3.5 Morphologies of mature 3T3-L1 adipocyte cell after stained by Oil-Red-O. A=untreated mature 3T3-L1 fat cell, and B=mature 3T3-L1 fat cell following treatment by VMD 0.8 kW-ginseng root extract. Arrow points to morphology and size of lipid droplets  78  Table 3.4 The effect of NAGE on adipogenesis of pre-confluent preadipocyte 3T3-L1 cells measured by Oil-Red-O staining assay % Differentiation 1 mg NAGE/  1.5 mg NAGE/  2 mg NAGE/  2.5 mg NAGE/  Drying method  mL  mL  mL  mL  Air drying  101.8 ± 16.3x  89.56 ± 12.1x  56.77 ± 6.84y  50.27 ± 7.89y  VMD 0.8 kW  85.53 ± 11.7x  80.27 ± 8.01x  51.93 ± 11.4y  47.87 ± 10.5y  VMD 1.3 kW  101.4 ± 16.0x  92.65 ± 9.78x  57.45 ± 9.22y  35.41 ± 9.50y  VMD 1.8 kW  96.86 ± 15.3x  73.90 ± 9.52x  61.64 ± 14.0y  56.69 ± 13.2y  Freeze-drying  104.7 ± 10.0x  94.98 ± 11.0x  75.93 ± 11.2y  48.67 ± 8.43y  Data represent mean ± SEM (n=6; 3 replicates, 2 places of origin) VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW x,y Within row, different superscripts indicate significant difference of concentrations (p<0.05) Table 3.5 The effect of NAGE on adipogenesis of post-confluent preadipocyte 3T3-L1 cells measured by Oil-Red-O staining assay % Differentiation 1 mg NAGE/  1.5 mg NAGE/  2 mg NAGE/  2.5 mg NAGE/  Drying method  mL  mL  mL  mL  Air drying  89.04 ± 11.1a,x  77.02 ± 12.4a,x  65.06 ± 10.1a,y  43.79 ± 7.88a,y  VMD 0.8 kW  130.7 ± 14.3b,x  112.6 ± 17.2b,x  75.89 ± 11.1b,y  61.84 ± 9.06b,y  VMD 1.3 kW  81.92 ± 13.0a,x  79.09 ± 6.61a,x  59.18 ± 9.76a,y  57.09 ± 9.12a,y  VMD 1.8 kW  85.43 ± 14.0a,x  75.87 ± 16.1a,x  60.01 ± 7.15a,y  45.91 ± 8.87a,y  Freeze-drying  116.5 ± 16.5ab,x 106.9 ± 19.0ab,x  69.74 ± 9.04ab,y 64.97 ± 13.2ab,y  Data represent mean ± SEM (n=6; 3 replicates, 2 places of origin) VMD 0.8 kW = vacuum-microwave drying at 0.8 kW microwave energy VMD 1.3 kW = vacuum-microwave drying at 1.3 kW VMD 1.8 kW = vacuum-microwave drying at 1.8 kW a,b Within column, different letters indicate significant difference of drying methods (p<0.05) x,y Within row, different superscripts indicate significant difference of concentrations (p<0.05)  79  A 140 120 % Differentiation  100 80 60 40 20 0 1  1.5 2 Concentration (mg/mL)  2.5  1  1.5 2 Concentration (mg/mL)  2.5  B 160  % Differentiation  140 120 100 80 60 40 20 0  Figure 3.6 The effect of NAGE on adipogenesis of 3T3-L1 cells measured by Oil-Red-O staining assay. Each panel represents mean ± SEM (n=6; 3 replicates, 2 places of origin). air dried NAGE. vacuum-microwave dried at 0.8 kW NAGE. vacuum-microwave dried at 1.3 kW NAGE. -x- vacuum-microwave dried at 1.8 kW NAGE. freeze-dried NAGE. A=pre-confluent stage, and B=post-confluent stage  80  140 120 % Differentiation  100 80 60 40 20 0 Rg1  Rc  Re  Rg3  Rb1  Rb2  Rh2  Rd  Figure 3.7 The effect of individual ginsenoside standards on 3T3-L1 cell adipogenesis. Each bars represents mean ± SEM (n=3). control. 50 µg/mL ginsenoside standard. 100 µg/mL ginsenoside standard  Table 3.6 The effect of individual ginsenosides on 3T3-L1 cell adipogenesis Ginsenoside standard % Differentiation 50 µg/mL  100 µg/mL  Rg1  109.4 ± 5.88c  106.7 ± 8.84c  Rc  56.47 ± 12.7b  76.47 ± 17.2bc  Re  68.24 ± 7.09b  48.63 ± 14.6b  Rg3  47.84 ± 1.57b  52.55 ± 3.49b  Rb1  80.28 ± 10.2bc  57.08 ± 12.6b  Rb2  59.40 ± 14.6b  59.86 ± 7.00b  Rh2  34.34 ± 7.29ab  6.032 ± 0.46a  Rd  29.70 ± 12.9b  48.26 ± 3.62b  Data represent mean ± SEM (n=3). a,b Different letters indicate significant difference of ginsenoside standard by concentration (p<0.05).  81  3.4 DISCUSSION Differences in the ginsenoside composition may have a potential effect on the overall ginseng bioactivity. Specific ginsenosides, such as Rg3 and Rh2, have been shown to alter cancer cell proliferation, induce apoptosis, and alter cell membrane permeability as well as integrity (Liu et al., 2000; Hwang et al., 2002; Popovich and Kitts, 2002; Kitts et al., 2007). Some ginsenosides also cause haemolysis of red blood cells resulting toxicity when absorbed into human blood stream (Harborne and Baxter, 1993; Trease and Evans, 1983). Therefore, assessing the cytotoxicity of ginseng extracts in a murine 3T3-L1 fat cell line was the first step in describing potential subsequent biological effects. The IC50 values of dried NAG root extracts to pre-confluent preadipocyte cells were lower than those of the post-confluent preadipocyte and mature adipocyte cells. The lower IC50 values in pre-confluent preadipocyte cells could be due to lower cell numbers in the pre-confluent stage and consequently more cells were exposed to the dried NAG root extracts. However, this could also indicate that the cytotoxic effects of dried NAG root extracts were potentially targeted at cell proliferation during pre-confluence. Furthermore, since DNA synthesis was limited in post-confluent preadipocyte and mature adipocyte cells (Brodie et al., 1999). This may explain the high cytotoxicities of dried NAG root extracts on the pre-confluent preadipocyte, rather than to the post-confluent preadipocyte and mature adipocyte cells, potentially target the DNA synthesis of the 3T3L1 fat cells. Moreover, the high LDH activity of pre-confluent preadipocyte cells was further evidence of a potential mechanism for ginseng toxicity on specifically inhibiting  82  proliferation of the 3T3-L1 cell line. LDH is released from the cytoplasm when the cell membrane is damaged (Kachmar and Moss, 1982). Although 1.5 mg/mL concentration of ginseng root extracts was not sufficient to kill cells, NAG root extracts were still able to damage the cell membrane as shown by the LDH activity that reached more than 100% of control. These membrane permeabilizing effects of dried NAG root extracts on preadipocytes cells likely reflects the combined bioactive properties of individual ginsenosides recovered in all extracts of dried root. Moreover, total ginsenosides recovered, as assessed by HPLC, following FD process were high. Therefore, the strong bioactive potential of FD-ginseng root extracts, as assessed by the viability of preconfluent preadipocyte cells and LDH activity assay, may further indicate the bioactive potential of ginsenosides on cultured 3T3-L1 cell line. Since the concentrations of the ginseng root extracts used in the adipogenesis assay were much lower than the IC50 values, the inhibitory effect of dried NAG root extracts on 3T3-L1 adipogenesis was likely not due to a toxic effect of the extracts per se but potentially due to the disruption of cell proliferation and the compromised cell membrane integrity that potentially interrupted cell signalling which resulted in a possible incomplete response to the adipogenesis media. Moreover, the reduction of the intracytoplasmic lipids might potentially transform the cells to resemble a preadipocyte phenotype (Gregoire et al., 1998). The cell membrane damage instigated by dried NAG root extracts, as indicated by the high percentages of LDH activity, may reduce the intracytoplasmic lipid and consequently, inhibiting the adipogenesis of 3T3-L1 cell line. The adipogenesis inhibitory potential of dried NAG root extracts may also be attributable to the ginsenoside content recovered following dehydration methods. As  83  illustrated in Figure 3.7, individual ginsenosides, with exception of ginsenoside Rg1, exhibited inhibitory effects to adipogenesis. Although ginsenoside Re had been proposed to exhibit anti-obesity potential by improving glucose tolerance and decreasing serum insulin in ob/ob mice (Attele et al., 2002), the inhibitory effects of NAG root extracts might not mainly be due to ginsenoside Re, but could also be a combine effect of many ginsenosides. Moreover, since the dried NAG roots extracts in this study recovered all of the ginsenosides listed on Figure 3.7, except ginsenoside Rh2, the adipogenesis inhibitory effects of dried NAG root extracts might be attributable to the ginsenosides as well as phytosterols that were recovered following dehydration (Court, 2000). The proposed mechanism of how American ginseng roots exhibited an antiobesity activity in a 3T3-L1 fat cell line could be explained further by the inhibition of several transcription factors that are activated leading to adipogenesis of the preadipose tissue cells. Dried NAG root extracts may potentially inhibit adipogenesis by inhibiting the transcription of PPARγ as was reported in another study showing that PPARγ transcription was inhibited following a treatment of a 3T3-L1 fat cell line by ginsenoside Rg3 (Hwang et al., 2009). PPARγ, which is transcribed before the activation of most adipocyte genes, induces adipogenesis of confluent adipocytes. Since PPARγ is a phosphoprotein that undergoes MAPK/ERK kinase (MEK) and mitogen-activated protein kinase (MAPK)-dependent phosphorylation (Gregoire et al., 1998), the inhibitory effect of ginseng extracts might be due to the phosphorylation of PPARγ into a less active form resulting in adipogenesis inhibition. Additionally, ginseng-induced modification of adipogenesis may involve a decrease in PPARγ mRNA and protein levels, and a parallel decrease in PPARγ binding activity, which results in a decrease of C/EBPα. As indicated  84  by other workers, the leptin level in obese Sprague-Dawley rats was significantly reduced following treatment with crude saponin extracted from red Korean ginseng (Kim et al., 2005). Therefore, the adipogenesis inhibition potential of dried NAG root extracts may also be attributed to the decrease of leptin level subsequent to the reduction of C/EBPα transcription.  3.5 CONCLUSION The bioactivity potential of all ginseng root extracts on the 3T3-L1 adipose tissue cell line was exhibited following AD, VMD, and FD processes. Dehydrated-ginseng root extracts affected the viability of a 3T3-L1 cell line in pre- and post-confluent, as well as mature adipocyte stages. When added to the pre-confluent preadipocytes, VMD 0.8 kW and FD-ginseng root extracts exhibited the highest toxicities among dried NAG root extracts. However, AD- and FD-ginseng root extracts exhibited the highest toxicities among dried NAG root extracts when added to the post-confluent preadipocytes. The potencies of dried NAG root extracts were quite low when added to mature 3T3-L1 adipocyte cells, as indicated by the high IC50 values. This result, therefore, may indicate the potential inhibitory effect of dried NAG root extracts on cell proliferation, especially on DNA synthesis, of 3T3-L1 cells. Dried NAG root extracts also affected the adipogenesis of 3T3-L1 cell line when added at both pre- and post-confluent stages. All sources of dried NAG root extracts exhibited a similar inhibitory effect, notwithstanding the dehydration methods applied, on adipogenesis of pre-confluent preadipocytes. However, AD-, VMD 1.3 kW-, and VMD  85  1.8 kW-ginseng root extracts exhibited higher inhibitory effect on post-confluent preadipocytes adipogenesis compared to FD- and VMD 0.8 kW-ginseng root extracts. This result does not necessarily illustrate the relationship between the total ginsenoside content and the bioactive potential of dried NAG root extracts. Consequently, this finding suggests that the bioactive potential of dried NAG root extracts on viability and adipogenesis of cultured 3T3-L1 cell line may not only be attributed to the ginsenosides recovered after dehydration but also other compounds that were co-extracted from the roots, but not quantified, such as phytosterols and non-terpenoid saponins. This experiment, therefore, showed a bioactive potential of dried NAG root extracts on adipose tissue cells and further confirmed the bioactivities of dried NAG roots following dehydration.  86  GENERAL CONCLUSION Dehydration is a water-removal process resulting in a shelf-stable product. Porous end-products can also be generated following dehydration. This phenomenon was true with dried ginseng roots since all dehydrated ginseng roots showed a porous structure after exposed to different dehydration techniques of AD, VMD, and FD. Moreover, all dehydrated samples were constituted by pore sizes that predominantly ranged from 0.002 µm to 172 µm. Among different treatments, FD ginseng had the highest total porosity and was constituted mainly by macropores (>1.5 µm). Although dried VMD and AD ginseng were also mainly constituted by macropores, the proportions of micropores (<0.5 µm) and medium pores (0.5 – 1.5 µm) in VMD and AD ginseng were significantly higher (p<0.05) than that of FD ginseng. This result was likely due to the collapse of the root matrix structure during AD and VMD processes that reduced the amount of macropores (Meda and Ratti, 2005; Stanley and Tung, 1976). On the contrary, FD prevented the root matrix structure from collapsing and consequently, resulted in a highly porous endproduct. The high total porosity of FD ginseng root, however, did not correspond to a higher recovery of ginsenosides in the subsequent extract. The total ginsenoside recovered in FD-ginseng root extract was significantly higher than AD-ginseng root extract. However, the level was comparable to that recovered following VMD processing. This suggests that the recovery of ginsenosides in dried NAG root is not only affected by the change in porosity induced by the dehydration techniques employed but may also be due to other factors such as transformation, oxidation, or degradation of ginsenosides during dehydration. Additionally, FD-ginseng root extract retained the highest amount of ginsenoside Rb1. However, the amount of  87  other ginsenosides (Rg1, Re, Rc, Rb2, Rd, and Rg3) recovered from FD- were comparable to AD- and especially, VMD-ginseng root extract. Similarly to the ginsenoside recovered following dehydration, AD, VMD, and FD processes were able to retain the bioactive potential of NAG root extracts on a 3T3-L1 adipose tissue cell line. The viability of 3T3-L1 cell line, either in pre-confluent, postconfluent preadipocyte, or differentiated adipocyte, were affected by dried NAG root extracts in a time- and dose-dependent manner. Ginseng root extracts derived from VMD processing exhibited stronger toxicities, compared to AD and FD, when exposed to the pre- and post-confluent preadipocyte cells. All sources of NAG root extracts exhibited similar toxicities to mature 3T3-L1 adipocyte cells. Moreover, all dried NAG root extracts exhibited relatively stronger potencies towards pre-confluent preadipocytes compared to both post-confluent preadipocyte and mature adipocyte cells. This result suggests the inhibitory effect of dried NAG root extracts on DNA synthesis of proliferating cells, since DNA synthesis mainly occurs during the pre-confluent stage of the cells proliferation. Dried NAG root extracts inhibited adipogenesis of 3T3-L1 cell line when added to both pre- and post-confluent preadipocyte cells. Since the bioactive properties of ginseng extract have been primarily associated to the ginsenoside content, the total ginsenoside contents of dried NAG root extracts were expected to affect their adipogenesis inhibitory effects. Moreover, the adipogenesis inhibitory effects of dried NAG root extracts were expected to be different since different dehydration techniques affected the recoveries of total ginsenosides differently. However, the inhibitory effects of dried NAG root extracts were similar despite the different dehydration methods  88  applied. This result consequently suggests that the adipogenesis inhibitory effects of dried NAG root extracts may not only be attributed to the ginsenosides recovered after dehydration but also to other compounds that were co-extracted from the roots, but not quantified, such as phytosterols and non-terpenoid saponins. Dried NAG root extracts potentially inhibited adipogenesis by compromising the cell membrane integrity, as shown by the high percentages of LDH activity, resulting in the release of intracytoplasmic lipid and the interruption of cell signalling. Consequently, the adipogenic cells resembled the preadipocyte phenotype and responded only partly to the adipogenesis media. This thesis research, therefore, showed the effects of different dehydration methods on pores characteristics of NAG roots and the subsequent retention of the ginsenosides as well as the corresponding bioactive potential on adipose tissue cells. Furthermore, this thesis research also explored the potential utilization of VMD technology in the herbal industry as VMD was shown to retain the ginseng natural characteristics as well as potential bioactivity when exposed to an adipocyte cell line. 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Journal of Cell Science, 110, 801-807.  108  APPENDIX A: HPLC METHOD FOR DETERMINATION OF GINSENOSIDE AMOUNT RECOVERED IN DRIED NORTH AMERICAN GINSENG  A.1 North American Ginseng Roots Ginseng roots (Panax quinquefolium) were supplied by Chai-Na-Ta Corp. (Kamloops, BC) and Great Mountain Ginseng (Niagara-On-The-Lake, ON). The 4-years old BC- and 5-years old ON-ginseng roots were harvested in June 2007, packed into separate boxes, and delivered to the Food, Nutrition, and Health building at UBC where the ginseng roots were pooled, based on places of origin, and rinsed before being shredded using a household food processor. The shredded ginseng roots were then randomly vacuum-packed into five separate bags, each containing 1 kg of ginseng roots, and stored at 4°C, allowing a maximum storage time of 2 days, prior to dehydration by air drying, vacuum-microwave drying, and freeze-drying according to the methods described on page 37.  A.2 Chemicals and Equipment Ginsenoside standards Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3, and Rh2 were purchased from Chromadex, Inc. (Irvine, CA). Standard stock solutions was prepared in 50% acetonitrile and kept in -20°C prior to analyses. HPLC grade methanol and acetonitrile were from Fisher Scientific, Inc. (Nepean, ON). Water was purified by Epure water purification unit (Barnstead Intl.; Dubuque, IA). Materials such as screw cap and snap cap vials (# 5182-0716; # 5182-0544) and screw and snap caps (# 5182-0717; # 51820566) were purchased from Agilent Technologies. Polyspring glass inserts (C4012-530  109  150 µL) were obtained from VWR International (Mississauga, ON). The chromatograph was a Hewlett-Packard 1100 series with Agilent Chemstation software, equipped with an Agilent Technologies ODS column (250 x 4.6 mm, 5 µm).  A.3 Determination of HPLC Method Parameters Dried shredded ginseng roots were extracted according to the method as previously described on page 39. Ginsenoside standards Rg1, Re, Rb1, Rc, Rb2, Rd, Rg3, and Rh2 stock solutions containing 2.5 mg/mL of standard were prepared by dissolving 5 mg of each standard in 2 mL of 50% acetonitrile, respectively. Calibration standard ginsenoside working solutions of 100 µg/mL were prepared by diluting the stock solution with water in appropriate quantities. Standard curves were constructed by injecting the  Area (mAU*s)  ginsenoside standard working solutions at different volumes.  5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0  y = 860.82x + 11.76 2 R = 0.9999  0  1  2  3  4  5  6  Ginsenoside Rg1 (µg)  Figure A.1 Calibration curve for ginsenoside Rg1  110  A  4000  y = 710.12x + 10.932  Area (mAU*s)  3500  2  R =1  3000 2500 2000 1500 1000 500 0 0  1  2  3  4  5  6  5  6  5  6  Ginsenoside Re (µg)  B  1600  y = 282.9x + 0.7755  Area (mAU*s)  1400  2  R =1  1200 1000 800 600 400 200 0 0  1  2  3  4  Ginsenoside Rb1 (µg)  Area (mAU*s)  C 1800 1600 1400 1200 1000 800 600 400 200 0  y = 332.16x - 1.5839 2 R =1  0  1  2  3  4  Ginsenoside Rc (µg)  Figure A.2 Calibration curves for ginsenosides (A) Re, (B) Rb1, and (C) Rc  111  A 1600 1400  y = 268.13x - 1.9326  1200  R =1  Area (mAU*s)  2  1000 800 600 400 200 0 0  1  2  3  4  5  6  5  6  5  6  Ginsenoside Rb2 (µg)  B  1200 y = 205.75x + 1.1753 2 R =1  Area (mAU*s)  1000 800 600 400 200 0 0  1  2  3  4  Ginsenoside Rd (µg)  C  3000  y = 593.13x - 289.74 2 R = 0.9755  Area (mAU*s)  2500 2000 1500 1000 500 0 0  1  2  3  4  Ginsenoside Rg3 (µg)  Figure A.3 Calibration curves for ginsenosides (A) Rb2, (B) Rd, and (C) Rg3  112  A.4 Ginsenoside Concentration in Dried North American Ginseng Root Extract The identity of individual ginseng saponins was determined based on the method described on page 40. The individual ginsenoside amount in the dried North American ginseng root extract is calculated as follow: Ginsenoside Re peak area (mAU) detected at 403 nm at 11.2 minutes = 20954.57. Dry weight of sample: Fresh weight  = 3.0003 g  Moisture content  = 11.8545%  Dry weight  = 3.0003 g * (1-0.118545) = 2.6446 g  Sample injected to HPLC: 2.6446 g mL *1000 * 10µL Injection volume = 6.0 x 10-3 g 4.4 mL MeOH µL  From ginsenoside Re standard curve: Area @ 403 nm = 710.12 (µg of Re recovered) + 10.932  Therefore in sample 1: 20954.57 = 710.12 (µg of Re recovered) + 10.932 µg of Re recovered = 29.4931 µg  Ginsenoside Re in dry weight basis (mg/g dry solid): 29.4931 µg = 4.9 mg Re/g dry solid. (1000 µg/mg) * (6.0x10 −3 g)  113  APPENDIX B: IC50 DETERMINATION OF DRIED NORTH AMERICAN GINSENG ROOT EXTRACT ON 3T3-L1 FAT CELL  B.1 Determination of IC50 Parameters Viability of 3T3-L1 cell was determined by the cell viability assay according to MTT reduction as described on page 62. The 3T3-L1 viability data was plotted using a data analysis and graphing software (Microcal Origin 6.0; OriginLab Co.; Northampton, MA) and transformed to derive an IC50.  BCADi48h BCADii48h BCADiii48h  120  100  Data: BCAD48h_BCADiii48h Model: ExpDec1  Y Axis Title  80  Chi^2 = 5.07687 R^2 = 0.99876 y0 A1 t1  60  6.81727 4296.58555 1.25211  ±0 ±6631.22083 ±0.48369  40  20  0 0  10  20  30  40  50  X Axis Title  Figure B. 1 Viability of pre-confluent 3T3-L1 preadipocyte cells as measured by MTT assay after treatment by AD – BC ginseng root extract  114  B.1 Determination of IC50 of Dried North American Ginseng Root Extract Following plotting, IC50 of dried NAG root extract was calculated using the exponential equation generated by the data analysis and graphing software: AD-BC ginseng root extract rep 1: R2 = 0.99801 y = 6.60805 + 4596.56746 e ( − x / 1.27686) 50 = 6.60805 + 4596.56746 e ( − x / 1.27686) x = 5.954 mg/mL IC50 of AD-BC ginseng root extract first replicate is 5.954 mg/mL. AD-BC ginseng root extract rep 2: R2 = 0.98654 y = 2695.23444 e ( − x / 1.59534) 50 = 2695.23444 e ( − x / 1.59534) x = 6.361 mg/mL IC50 of AD-BC ginseng root extract second replicate is 6.361 mg/mL. AD-BC ginseng root extract rep 3: R2 = 0.99876 y = 6.81727 + 4296.58555 e ( − x / 1.25211) 50 = 6.81727 + 4296.58555 e ( − x / 1.25211) x = 5.760 mg/mL IC50 of AD-BC ginseng root extract third replicate is 5.760 mg/mL. IC50 of AD-BC ginseng root extract is 6.025 ± 0.307 mg/mL.  115  APPENDIX C: LDH ACTIVITY DETERMINATION OF DRIED NORTH AMERICAN GINSENG ROOT EXTRACT ON 3T3-L1 FAT CELL  C.1 Determination of LDH Activity Parameters LDH activity of dried North American ginseng root extract on preadipocyte 3T3L1 cells were determined according to the assay described on page 65. As LDH activity in International units at 37°C was calculated from: mU/mL =  1000 2.25 ∆A x x 0.60 x min 6.22 0.05  where: ∆A•min-1 = average absorbance change per minute 1000 = converts mmol to µmol 6.22 = milimolar absorptivity of NADH at 340 nm (unit: mL•mmol-1•cm-1) 2.25 = total volume in cuvette (unit: mL) 0.05 = volume of cell-free supernatant (unit = mL) 0.60 = Temperature correction factor at 37°C (Demetriou et al., 1974). ∆A•min-1 (average absorbance change per minute) was determined using the slope of an absorbance change curve (Figures B.1 & B.2). y = -0.0106x + 0.7249 R2 = 0.9878  0.72  y = -0.0101x + 0.7221 R2 = 0.99  0.71 Absorbance  0.7  y = -0.0104x + 0.7229 R2 = 0.995  0.69 0.68 0.67 0.66 0.65 0.64 0  1  2  3  4  5  6  7  8  Tim e (m in)  Figure C. 1 Absorbance change per minute curve of untreated 3T3-L1 cells (i.e. control)  116  y = -0.0151x + 0.7251 R2 = 0.9952  0.72  y = -0.0171x + 0.7303 R2 = 0.9985  Absorbance  0.7 0.68  y = -0.0171x + 0.7256 R2 = 0.9973  0.66 0.64 0.62 0.6 0.58 0  2  4  6  8  Tim e (m in)  Figure C. 2 Absorbance change per minute curve of air-dried North American ginseng root extract treatment on 3T3-L1 cells  C.2 Determination of LDH Activity of Dried North American Ginseng Root Extract The LDH activity of air-dried North American ginseng root extract is calculated as follow:  ∆A = average absorbance change per minute (slope of curve; Figures B.1 & B.2). min LDH activity in international units at 37°C: ∆A 1000 mmol/µmol 2.25 mL x x x 0.60 mU/mL = min 6.22 mmol 0.05 mL Control Rep 1: mU/mL = 0.0106 x  1000 mmol/µmol 2.25 mL x x0.60 = 46.0 mU/mL. 6.22 mmol 0.05 mL  Control Rep 2: mU/mL = 0.0101 x  1000 mmol/µmol 2.25 mL x x 0.60 = 43.8 mU/mL. 6.22 mmol 0.05 mL  Control Rep 3: mU/mL = 0.0104 x  1000 mmol/µmol 2.25 mL x x 0.60 = 45.2 mU/mL. 6.22 mmol 0.05 mL  Control mU/mL =  46.0 + 43.8 + 45.2 = 45.0 mU/mL. 3  117  % untreated LDH activity =  LDH cells x 100% LDH control  1000 mmol/µmol 2.25 mL x x 0.60 = 65.5 mU/mL. 6.22 mmol 0.05 mL 65.5 x 100% = 146%. AD-NAGE Rep 1 % untreated LDH activity: 45.0  AD-NAGE Rep 1: mU/mL = 0.0151 x  1000 mmol/µmol 2.25 mL x x 0.60 = 74.2 mU/mL. 6.22 mmol 0.05 mL 74.2 AD-NAGE Rep 2 % untreated LDH activity: x 100% = 165%. 45.0  AD-NAGE Rep 2: mU/mL = 0.0171 x  1000 mmol/µmol 2.25 mL x x 0.60 = 74.2 mU/mL. 6.22 mmol 0.05 mL 74.2 AD-NAGE Rep 3 % untreated LDH activity: x 100% = 165%. 45.0  AD-NAGE Rep 3: mU/mL = 0.0171 x  146% + 165% + 165% 3 = 165 ± 8.57%.  AD-NAGE % untreated LDH activity =  118  

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