Separation and Analysis of Fructooligosaccharides in ...library.umac.mo/etheses/b31355079_toc.pdf · type fructooligosaccharides (FOS), which are a group of compounds recognized as

Separation and Analysis of Fructooligosaccharides in Medicinal

Plants

by

LI Jing

Doctor of Philosophy in Biomedical Sciences

2014

Institute of Chinese Medical Sciences

University of Macau

Separation and Analysis of Fructooligosaccharides in Medicinal

Plants

by

LI Jing

SUPERVISOR: Professor LI Shao-Ping

Doctor of Philosophy in Biomedical Sciences

2014

Institute of Chinese Medical Sciences

University of Macau

Author’s right 2014 by

LI, Jing

i

Acknowledgements

I would like to express my sincere gratitude to my supervisor Prof. Shao-Ping Li

for his kindness, patience, encouragement and untiring support. He had enlightened

me, guided me, and inspired me to finish my research work. I would also like thanks to

Prof. Yi-Tao Wang the director of our institute, Prof. Qing-Wen Zhang from our

institute and Prof. Yu Wang from Jiangsu Institute for Food and Drug Control, for

helping me overcome the difficulties I faced during my studies.

I would also like to thank the entities that funded this research work. This

research was supported by grants from Macao Science and Technology Development

Fund (059/2011/A3 to S.P. Li), and the University of Macau (MYRG085 to J. Zhao

and MYRG140 to S.P. Li). I would also like to thank the University of Macau for

providing a postgraduate studentship to me.

I would like to express my heartfelt thanks to Dr. Jing Zhao, Dr. Chun-Feng Qiao,

Prof. Ming-Yuen Lee, Prof. Ying Zheng, Prof. Ru Yan, Dr. Pui Man Hoi, Dr. Peng Li,

Dr. Jian-Bo Wan, Dr. Yuan-Jia Hu, Dr. Kun Feng, Dr. Xiao-Jia Chen, Dr. Jia Guan, Dr.

Zheng-Ming Qian, Dr. Wei-Hua Huang and Dr. De-Qiang Li for their comments and

assistance in my study. Moreover, I would like to thank all the administrative staffs in

ICMS. Special thanks to Ms. Hattie U, Ms. Chloe Lao, Ms. Carol Lam, Mr. Leon Lai,

Ms. Sandy Lao, Ms. Sio Kio Kuong, Ms. Wing Leong, Ms. Katrina Wong, Ms. Ada

Wong for their warm support during my study and staying in Macau.

I would also like to thank my friends, Lan-Zhen Meng, Lan-Ying Wang, Yi-Wen

Chen, Guang-Ping Lv, Kit-Leong Cheong, De-Jun Hu, Jing Xie, Xian-Qiang Chen,

Ding-Tao Wu, Xiao-Mei Liu, and Wan-Rong Zong, as well as other classmates who

are studying or have already graduated for helping me, and giving me a happy memory

ii

in studying and working in UM.

Last but not the least, I am forever indebted to my family for their

understanding, endless patience and support during these years.

iii

Abstract

Inulin-type fructooligosaccharides (FOS), which are a group of compounds

recognized as natural food ingredients and classified as dietary fiber in almost all

European countries, are formed by β-(2→1) linked fructofuranosyl unit on the end of

sucrose molecule. In this study, the methods for extraction, separation and purification

of FOS were investigated, and three analytical methods for the determination of FOS

in plants were developed respectively. The major achievements of this study are

summarized as follows:

(1) A fast protein liquid chromatography coupled with refractive index

detection (FPLC-RID) method was firstly developed for separation and purification of

FOS with different DP (DP3 to DP13) from burdock. Total FOS were purified on

Bio-Gel P columns eluted with water at the flow rate of 0.3 ml/min, and the

HPLC-ELSD method was used for determination of the purities of FOS. The separated

FOS were identified by their methylation analysis, MS and NMR data.

(2) A twice development HPTLC method was established for simultaneously

qualitative and quantitative analysis of seven FOS (DP3~9) in Morinda officinalis and

Arctium lappa. The analysis was performed on silica gel 60 plate with n-butanol -

isopropanol - water - acetic acid (7:5:2:1, v/v/v/v). The bands were revealed by

reaction with 1-naphthol-sulfuric acid reagent and densitometrically determined at 585

nm wavelength.

(3) An HPLC-ELSD and microwave-assisted extraction method were

developed for determination of seven FOS (DP3~9), as well as fructose, glucose and

sucrose in Morinda officinalis and Arctium lappa from different regions. The

separation was performed on a column B (4.6×250 mm id, 3.5 μm) with gradient

iv

elution.

(4) An HPLC-CAD method was developed for simultaneous determination of

FOS (DP 3 ~13) in different samples from Liliaceae, Asteraceae, Campanulaceae, and

Rubiaceae families. LC-MS/MS analysis was also carried out for qualitative analysis

of FOS in these samples.

In summary, An FPLC-RID method was developed for fast preparation and

purification of FOS (DP3-DP13) for the first time. Three analytical methods (HPTLC,

HPLC-ELSD, HPLC-CAD) for quantitative analysis of FOS in different samples were

also developed, which provided powerful tools for quality control of these plants.

v

Declaration

I declare that the thesis here submitted is original except for the source materials

explicitly acknowledged and that this thesis as a whole, or any part of this thesis has

not been previously submitted for the same degree or for a different degree.

I also acknowledge that I have read and understood the Rules on Handling

Student Academic Dishonesty and the Regulations of the Student Discipline of the

University of Macau.

vi

Table of Contents

Acknowledgements ....................................................................................................... i

Abstract ....................................................................................................................... iii

Declaration ................................................................................................................... v

Table of Contents ........................................................................................................ vi

List of Tables ............................................................................................................. viii

List of Figures ............................................................................................................ iix

List of Abbreviations ................................................................................................... xi

Chapter 1 Introduction ................................................................................................. 1

1.1 General Background ............................................................................................... 1

1.1.1 Physicochemical properties and natural occurrence of FOS ......................... 2

1.1.2 Beneficial effects of FOS .............................................................................. 3

1.1.3 Preparation of FOS ........................................................................................ 7

1.1.4 Safety of FOS ................................................................................................ 9

1.1.5 Applications of FOS .................................................................................... 10

1.2 Specific Background ............................................................................................ 10

1.2.1 Extraction of FOS ........................................................................................ 10

1.2.2 Separation and purification of FOS ............................................................. 12

1.2.3 Analysis of FOS .......................................................................................... 13

1.3 Research Goals and Objectives ............................................................................ 19

1.4 Research Methodology and Design ...................................................................... 20

1.5 Potential Contributions ......................................................................................... 20

1.6 Organization of Thesis ......................................................................................... 21

1.7 Statement of Originality ....................................................................................... 21

References .................................................................................................................. 24

Chapter 2 Extraction, Separation and Characterization of FOS (DP3 to DP13) from

Burdock ...................................................................................................................... 41

2.1 Introduction .......................................................................................................... 41

2.2 Experimental ........................................................................................................ 42

2.2.1 Chemicals and materials.............................................................................. 42

2.2.2 Extraction and decolorization of FOS ......................................................... 43

2.2.3 FPLC preparative separation and purification of FOS ................................ 44

2.2.4 HPLC-ELSD analysis of FOS ..................................................................... 45

vii

2.2.5 LC-MS/MS analysis of FOS ....................................................................... 45

2.2.6 Methylation analysis ................................................................................... 45

2.2.7 Infrared (IR) spectroscopy .......................................................................... 46

2.2.8 1H and

13C NMR test ................................................................................... 46

2.3 Results and Discussion ......................................................................................... 46

2.3.1 Optimization of sample preparation ............................................................ 46

2.3.2 Optimization of FPLC conditions ............................................................... 50

2.3.3 FPLC separation of FOS ............................................................................. 55

2.3.4 Identification of FOS .................................................................................. 57

2.4 Conclusion ........................................................................................................... 70

References .................................................................................................................. 71

Chapter 3 Qualitative and Quantitative Analysis of FOS in Different Plants............ 74

3.1 Introduction .......................................................................................................... 74

3.2 Qualitative and Quantitative Analysis of FOS (DP 3 ~ DP 9) in Morinda officinalis

and Arctium lappa using Twice Development Thin Layer Chromatography ............ 75

3.2.1 Experiment .................................................................................................. 76

3.2.2 Results and Discussion ................................................................................ 80

3.3 Quantitative Analysis of FOS (DP 3 ~ DP 9) in Morinda officinalis and Arctium

lappa using HPLC-ELSD .......................................................................................... 91

3.3.1 Materials and methods ................................................................................ 91

3.3.2 Results and discussion ................................................................................ 95

3.4 Qualitative and quantitative analysis of FOS (DP 3 ~ DP 13) in medicinal plants

using HPLC- CAD ................................................................................................... 104

3.4.1 Materials and methods .............................................................................. 105

3.4.2 Results and discussion .............................................................................. 110

3.5 Conclusion ......................................................................................................... 121

References ................................................................................................................ 122

Chapter 4 Conclusions ............................................................................................. 127

4.1 Conclusions ........................................................................................................ 127

4.2 Limitations of current study ............................................................................... 128

4.3 Perspectives for future work .............................................................................. 129

References ................................................................................................................ 131

Appendix .................................................................................................................. 133

Curriculum Vitae ...................................................................................................... 142

viii

List of Tables

Table 1.1 Concentrations of FOS in natural foods ....................................................... 3

Table 2.1 Design Matrix Based on L9(34) orthogonal array and measured responses

.................................................................................................................... 49

Table 2.2 The purities and recoveries of FPLC purified oligosaccharides (DP3-DP9) at

different flow rates ..................................................................................... 54

Table 2.3 The contents of the fructooligosaccharides (DP3 to DP13) separated by

FPLC-RID………………………………………………………………..57

Table 2.4 The MS characteristics of the fructooligosaccharides (DP3 to DP10) ...... 61

Table 2.5 The results of methylated analysis of purified oligosaccharides DP3-DP13

................................................................................................................... 62

Table 2.6 1H and

13C NMR chemical shifts of the isolated oligosaccharides (DP3-13)

from roots of burdock using D2O as solvent, δ in ppm ............................. 70

Table 3.1 Regression, LOD and LOQ for the investigated compounds .................... 85

Table 3.2 Stability, regression, precision, LOD and LOQ for investigated compounds

................................................................................................................... 86

Table 3.3 Repeatability and accuracy for the investigated compounds ..................... 87

Table 3.4 The contents of seven FOS (DP3~9) in Morina officinalis and Arctium lappa

................................................................................................................... 89

Table 3.5 Linear regression, LOD and LOQ for the investigated compounds .......... 97

Table 3.6 Precision and stability of the investigated compounds .............................. 99

Table 3.7 Repeatability and recoveries of the investigated compounds .................. 100

Table 3.8 Contents (mg/g) of the ten analytes in the tested samples ....................... 102

Table 3.9 The characteristics of the analyzed samples ............................................ 106

Table 3.10 Linear regression, LOD and LOQ of the FOS (DP3 to DP13) .............. 114

Table 3.11 Precision (intra- and interday) of the FOS (DP3 to DP13) .................... 114

Table 3.12 Repeatability, stability and recovery of the FOS (DP3 to DP13) .......... 115

Table 3.13 Contents (mg/g) of the eleven analytes in the tested samples ................ 119

ix

List of Figures

Figure 2.1 The raw materials of Burdock. ................................................................. 43

Figure 2.2 Schematic outline of the FPLC-RID system. ........................................... 44

Figure 2.3 Effect of extraction solvent (A) and sample pretreatment method (B) on the

extraction efficiency of FOS with different DP in burdock .................... 48

Figure 2.4 FPLC-RID chromatograms of FOS separated on (A) Sephadex G-25, (B)

Sephadex LH-20, (C) Bio-gel P-2 ............................................................ 52

Figure 2.5 Effect of flow rate on the separation of FOS on Bio-gel P-2 ................... 53

Figure 2.6 The chromatograms of FOS separated by FPLC-RID and HPLC-ELSD 56

Figure 2.7 The LC-MS/MS spectra of the ten separated FOS ................................... 59

Figure 2.8 The GC-MS spectrum of the methylated FOS.......................................... 62

Figure 2.9 IR spectrum of FOS .................................................................................. 63

Figure 2.10 1H NMR spectrum of FOS (400 MHz, D2O) .......................................... 64

Figure 2.11 13

C NMR spectrum of FOS (100 MHz, D2O) ......................................... 67

Figure 2.12 The structures of the inulin-type fructooligosaccharides (FOS) ............. 70

Figure 3.1 The raw materials of Morinda officinalis and Arctium lappa................... 77

Figure 3.2 Optimazation the mobile phase in separate ethanol extracts of Morinda

officinalis .................................................................................................. 81

Figure 3.3 Separation of mixed standards and ethanol extracts of Morinda officinalis

using (A) once and (B) twice development on silica gel 60 TLC plate ... 82

Figure 3.4 Typical HPTLC chromatograms (A, B) and their densitograms (C, D)

colored with 1-naphthol- sulfuric acid reagent (A, C) and diphenylamine -

aniline - phosphoric acid reagent (B, D) .................................................. 83

Figure 3.5 The absorption spectrum of tested compounds ........................................ 84

Figure 3.6 Calibration curves of investigated compounds ......................................... 85

Figure 3.7 HPTLC chromatograms and their typical HPTLC densitograms of FOS

standards and tested samples detected at 585 nm after colorized ............ 90

Figure 3.8 The structures of the investigated ten sugars ............................................ 93

Figure 3.9 HPLC-ELSD chromatograms of different commercial columns ............. 96

Figure 3.10 Calibration curves of the investigated compounds ................................. 98

Figure 3.11 HPLC-ELSD chromatogram of the mixed standards (A), Morinda officin-

alis (B) and Arctium lappa (C) extracts. ............................................ 103

Figure 3.12 Effect of extraction time on the extraction efficiency of FOS with different

x

DP........................................................................................................ 111

Figure 3.14 Calibration curves of the investigated compounds. .............................. 112

Figure 3.15 HPLC-CAD chromatograms of the mixed standards (A) and the samples

of Asteraceae (B), Campanulaceae (C) and Rubiaceae (D) ............ 118

xi

List of Abbreviations

ACN acetonitrile

AEX anion exchange chromatography

Ca calcium

CAD charged aerosol detector

DAD diode array detector

DAP diphenylamine - aniline - phosphoric acid reagent

DP degree of polymerization

DV daily value

ELSD evaporative light scattering detector

ESI electrospray ionization

EtOH ethanol

FDA Food and Drug Administration

FOS fructooligosaccharides

FPLC fast protein liquid chromatography

Fru fructose

FTases fructosyltransferase

GAP Good Agricultural Practice

GC gas chromatography

Glu glucose

GI gastrointestinal

HILIC hydrophilic interaction liquid chromatography

HPAEC high performance anion-exchange chromatography

(HPAEC) HPLC high performance liquid chromatography

HPTLC high performance thin-layer chromatography

i.d. internal diameter

IEC ion exchange chromatography

IR infrared

LC liquid chromatography

LOD limits of detection

xii

LOQ limits of quantification

MALDI-MS matrix-assisted laser desorption/ionization mass

spectrometry (MALDI-MS) MeOH Methanol

Mg Magnesium

MS mass spectrometry

m/z mass-to-charge

N.D. not detected

NDOs non-digestible oligosaccharides

NP normal phase

NS 1-naphthol - sulfuric acid reagent

P Phosphorus

PAD pulsed amperometric detector

PGC porous graphitized carbon

RID refractive index detector

RP reversed phase

RSD relative standard deviation

SCFAs short chain fatty acids

SEC size-exclusion chromatography

S/N signal-to-noise ratio

TCMs traditional Chinese medicines

TDF total dietary fibre

TFA trifluoroacetic acid

TLC thin layer chromatography

USDA united states department of agriculture

UV Ultraviolet