Characterization of Bioactive Compounds from Garcinia

mangostana L. obtained by Supercritical Fluid

Carbon dioxide Process

A thesis submitted to the

University of Mysore

In fulfillment of the requirements for the degree of

Doctor of Philosophy

in

BIOTECHNOLOGY

By

A.S. ZARENA

Under the supervision of

Dr. K. Udaya Sankar

Scientist-G

Food Engineering Department

Central Food Technological Research Institute

Mysore-570 020, INDIA

February 2011

Characterization of Bioactive Compounds from

Garcinia mangostana L. obtained by Supercritical

Fluid Carbon dioxide Process

A thesis submitted to the

University of Mysore

In fulfillment of the requirements for the degree of

Doctor of Philosophy

in

BIOTECHNOLOGY

By

A.S. ZARENA

Under the supervision of

Dr. K. Udaya Sankar

Scientist-G

Food Engineering Department

Central Food Technological Research Institute

Mysore-570 020, INDIA

February 2011

CERTIFICATE

I, A.S. Zarena, certify that this thesis is the result of research work done by me under

the supervision of Dr. K. Udaya Sankar, Scientist-G in the Food Engineering

Department, Central Food Technological Research Institute, Mysore-20. I am

submitting this thesis for possible award of Doctor of Philosophy (Ph.D) degree in

Biotechnology of the University of Mysore.

I, further certify that this thesis has not been submitted by me for award of any

other degree/diploma of this or any other University.

Signature of Doctoral candidate

Signed by me on

Signature of Guide Date :

Date :

Counter signed by

Signature of Chairperson/Head of Department

Institution with name and official seal.

Dr. K. Udaya Sankar

Scientist- G

Food Engineering Department

Central Food Technological Research Institute

Mysore-570 020

E-mail: udaya@cftri.res.in

Certificate

I, hereby certify that the thesis entitled, “Characterization of bioactive compounds

from Garcinia mangostana L. obtained by supercritical fluid carbon dioxide

process” submitted for the degree of Doctor of Philosophy in Biotechnology to the

University of Mysore is the result of the work carried out by Ms. A.S. Zarena under

my guidance in the Food Engineering Department, Central Food Technological

Research Institute, Mysore during the period 2007-2010.

Date: 2011 (Dr. K.Udaya Sankar)

Place: Mysore Research Guide

mailto:udaya@cftri.res.in

A. S. ZARENA

CSIR-Senior Research Fellow

Food Engineering

Central Food Technological Research Institute

Mysore – 570 020, India

Declaration

I, hereby declare that the thesis entitled “Characterization of bioactive compounds

from Garcinia mangostana L. obtained by supercritical fluid carbon dioxide

process” which is submitted herewith for the degree of Doctor of Philosophy in

Biotechnology of the University of Mysore, is the result of the research work carried

out by me under the supervision of Dr. K. Udaya Sankar, Scientist-G, Food

Engineering Department, Central Food Technological Research Institute, Mysore

during the period 2007- 2010.

I further declare that the results of this work have not been previously submitted

for any other degree or fellowship.

Date: 2011 (A.S. Zarena)

Place: Mysore

Dedicated to my parents for their enduring love

v

Abstract

Mangosteen fruits usually grown in Southeast Asia, have found international market in

recent years because of their growing knowledge in the pharmaceutical and food

industry. To sum up, this work highlights the importance of mangosteen pericarp which

have been traditionally used as an indigenous medicine as a rich source of health

benefits. Supercritical carbon dioxide (SC-CO2) extractions were carried with and

without ethanol as modifier. The use of ethanol as an entrainer in SC-CO2 increased the

overall yield and the xanthone recovery, comparing well with the Soxtec™ method.

Response surface methodology (RSM) proved to be extremely useful in predictive

modeling and optimization of extraction conditions such as pressure, temperature,

solvent to material ratio and time on the extracts yield. Analytical tools such as RP-

HPLC-DAD, LC-ESI-MS and 1H,

13C NMR spectral techniques were useful in

screening quantification and identification of xanthones, phenolic acids, flavonoids and

anthocyanin compounds. A one-step enzymatic glycosylation of α-mangostin in SC-

CO2 was successfully employed for the synthesis of α-mangostin-D-glucoside using

amyloglucosidase. The conversion yield was optimized using central composite

rotatable design. The results on long-term stabilities of mangosteen extract in oil-in-

water (MIO/W) emulsions have shown to possess important implications for the design

of whey protein concentrate stabilized emulsions for development of functionally

bioactive compounds for health benefits. An overall result of the investigation are

highly encouraging and adds to the current knowledge in the pharmaceutical and food

industries for the possible commercial application of SC-CO2 in the extraction of

bioactive compounds from mangosteen pericarp.

vi

Synopsis

There is an increasing public awareness of the health, environment safety and hazards

associated with the use of organic solvents in food processing and the possible solvent

contamination of the final products. The high cost of organic solvents and the

increasingly stringent environmental regulations together with the new requirements of

the pharmaceutical and food industries for ultra-pure and high added value products

have emphasized the need for the development of new and clean technologies for the

processing of food products. Supercritical carbon dioxide (SC-CO2) as a solvent has

provided an excellent alternative to chemical solvents. Over the past three decades, SC-

CO2 has been used for the extraction and isolation of valuable compounds from natural

products. Carbon dioxide is particularly suitable solvent for food processing

applications, because it’s moderate critical temperature (31.1 °C) and critical pressure

(7.38 MPa) enables the extraction of thermally labile food compounds in near natural

form. Additionally, it is non-toxic, environmentally acceptable and relatively

inexpensive. Compared with conventional solvents CO2 does not leave any harmful

solvent residue after extraction.

Mangosteen (Garcinia mangostana L.) belongs to the family of Guttiferae and is

named ‘‘the queen of fruits”, it is a slow-growing tropical evergreen tree mainly found

in India, Myanmar, Sri Lanka and Thailand. It bears dark red to purple rounded fruits

of 5-7 cm in diameter. The edible portion of fruit (aril) is white, soft with a slightly

sour taste. The pericarp of mangosteen fruit is 6-10 mm in thickness and has been used

in Thai indigenous medicine for the treatment of skin infections, wounds and diarrhea

vii

for many years. The major secondary metabolites of mangosteen have found to be

prenylated xanthone and oxygenated xanthones, tannin, isoflavone, flavone and other

bioactive substances. Xanthones have a variety of biological activity, for example

antioxidant, antibacterial, antifungal, antitumor, antiplatelet aggregation,

antithrombotic, prevention of oxidative damage of LDL, and inhibition of HIV-1

protease.

The objectives of the present investigation were:

1. Isolation and screening of mangosteen pericarp extract for their bioactive

components obtained by SC-CO2 and by conventional Soxtec™ extract.

2. Characterization and evaluation of the extracts for their anti-oxidant properties

in vitro assay.

3. Formulation of SC-CO2 extracted bioactive compounds as deliverable forms.

The thesis consisted of seven chapters each containing a brief introduction,

methodology, results and discussion.

Chapter 1: General introduction and scope of the present investigation

This chapter begins with a general introduction on SC-CO2 extraction of natural

bioactive compounds, followed by an overview of current literature in detail. Besides,

the extractions of active components, the advantages of carrying out enzymatic

glycosylation reactions in SC-CO2 media are discussed. The chapter also highlights the

objectives and scope of the present investigation.

viii

Chapter 2: Extraction and characterization of bioactive compounds in mangosteen

pericarp obtained by SC-CO2

The highest extract yield was obtained in Soxtec™ method with acetone water

(80:20) (22.0 wt %) and the lowest was in hexane extract (7.7 wt %). With reference to

SC-CO2 highest extract yield of 15.4% was obtained with 5% ethanol as modifier at 280

bar, 50 ºC and with pure SC-CO2, the yield was 7.6 wt% at 300 bar and 60 ºC. Total

phenolic content (TPC) ranged from 135 to 431 mg gallic acid equivalent/g (GAE/g) of

extract depending on the solvent/solvent mixture used for extraction, with the lowest

being in hexane and the highest in methanol extract. In SC-CO2 +5% ethanol extracts,

the TPC ranged from 53.7 to 85.9 mg GAE/g, while in pure SC-CO2 the TPC ranged

from 16.0 to 53.3 mg GAE/g. The identification and composition of the extracts were

evaluated by HPLC and LC-MS. As many as, 13 xanthones were identified and

quantified in the SC-CO2 extract. The extracts showed both quantitative and qualitative

differences in the xanthone composition depending on the extraction conditions. The

total xanthone content in decreasing order was SC-CO2 +5% ethanol > pure SC-CO2 >

Soxtec™ extract. SC-CO2 extraction was highly selective resulting in better purity of

the desired active compounds.

Chapter 3: Antioxidant activity of mangosteen pericarp extracts

This chapter deals with the comparison of antioxidant activity of plant extracts

obtained by SC-CO2 with extracts obtained by Soxtec™ method. SC-CO2 extracts with

modifier showed maximum antioxidant activity and free radical scavenging activity in

vitro conditions. Antioxidant activity was found to be marginally higher for extracts

ix

obtained by SC-CO2 +5% ethanol than with SC-CO2 alone, in spite of the fact that the

main compounds were same in both the extracts but their composition were higher in

the former. The xanthone composition of the SC-CO2 +5% ethanol was 75 wt% (50 ºC,

280 bar) and in pure SC-CO2 xanthone content was 65 wt% (60 ºC and 300 bar). The

antioxidant activity of these extracts was compared with known synthetic antioxidants,

such as BHA and α- tocopherol. The correlation of TPC with the antioxidant activity of

SC-CO2 extracts was higher than the Soxtec™ extracts.

Chapter 4: Optimization of SC-CO2 extraction parameters on extract yield in

mangosteen pericarp

Basing on Box-Behnken design, response surface methodological (RSM) study

was investigated on the extraction yields of mangosteen pericarp using SC-CO2. The

total yield in SC-CO2 extraction depended on the solute’s vapor effect. From response

surface plots, the extraction of xanthones exhibited independent and interactive effects

of pressure, temperature and solvent to material ratio. At optimum condition, the

xanthone yield was 8.0 wt% at 60 oC, 300 bar and solvent to material ratio of 300 kg/kg,

while experimental yield was 7.6 wt%. Another set of experiments were carried out

with SC-CO2 + 5% ethanol as co-solvent. With entrainer a maximum yield of 15.4 wt%

was obtained at 50 oC, 280 bar with process time of 8h. In the presence of co-solvent,

higher extraction yields were obtained, while with SC-CO2 alone it was not possible

with same pressure and temperature. Scanning electron microscope was used to

elucidate the morphological changes of matrix during extraction. The plant matrix

extracted by SC-CO2 showed marked swelling indicating the pressurized solvent at

x

supercritical state had a greater ability to diffuse through the ultra fine complex matrix

than conventional solvents.

Chapter 5: Characterization of phenolic acids and anthocyanins

This chapter deals with the phenolic acids and anthocyanins profile of

mangosteen pericarp. A HPLC procedure for separating 15 polyphenols was used for

the determination of phenolic acids and flavanoid content in the pericarp of the

mangosteen. Sequential hydrolysis of the pericarp showed phenolic acids was released

by hydrolyzing the mangosteen pericarp under basic or acidic conditions; however, the

former was more efficient than the latter. Phenolic acid such as caffeic acid (15.4%),

mandelic acid (14.4%), t-cinnamic acid (7.5%), vanillic acid (7.2%), sinapic acid

(7.2%) and flavanoids such as epicatechin (7.9%) and quercetin (23.1%) were the most

predominant components in the base hydrolysed fraction. The base hydrolysed sample

showed potent antioxidant and radical scavenging activity compared to acid or

unhydrolysed extract.

A new anthocyanin pelargonidin 3-glucoside along with two known

anthocyanins; cyanidin 3-sophoroside and cyanidin 3-glucoside were characterised from

the acidified, methanolic extract of mangosteen pericarp by preparative HPLC after

purification by partition against ethyl acetate and amberlite XAD-7. The structures

were elucidated by nuclear magnetic resonance spectroscopy and high-resolution

electrospray mass spectrometry. Cyanidin 3-sophoroside was detected in higher

amount (76.1%) followed by cyanidin 3- glucoside (13.4 %) and pelargonidin 3-

xi

glucoside (6.2%). The total monomeric anthocyanin content as measured by the pH-

differential method was 13.2 mg/L.

Chapter 6: Central composite rotable design for glucosylation of α-magostin in SC-

CO2 media

The ability of SC-CO2 as a reactions media was explored with respect to

enzymatic glycosylation of the α-mangostin. A central composite rotatable design

(CCRD) involving 32 experiments of five variables at five levels was employed to

study the glucosylation reaction catalyzed by amyloglucosidase. Reactants employed

were: α-mangostin (0.25 mmol) and carbohydrate (D-glucose 0.75 mmol). The

variables studied were: pressure (80-160 bar), temperature (35-75 °C), enzyme

concentration (15-45 mg), buffer pH (4.0-8.0) and buffer volume (1.0-5.0 mL).

Experimental data fitted the second-order polynomial equation, (R2=0.93). Various

contour plots were generated to describe the relationship between operating variables

and the conversion yields. The optimal enzymatic conversion within the experimental

range of the variables reached 20.3% at a pressure of 120 bar, temperature of 55 °C,

enzyme concentration of 30 mg, buffer volume of 3 mL and pH 6.0. The formation of

glucosides was confirmed by HPLC, MS, IR and NMR data.

Chapter 7: Formulation of mangosteen in oil-in-water emulsions

This chapter deals with the formulation of the improved shelf-stable emulsions

rich in xanthones. The physical properties of mangosteen extract in oil-in-water

(MIO/W) emulsions stabilized by whey protein concentrate (WPC) were examined for

xii

rheological properties and physical stability. Rheological study of these emulsions

showed near Newtonian characteristics. The apparent viscosity of the emulsions

increased appreciably for 20 wt% MIO/W to that of 10 wt% MIO/W. The increase in

NaCl concentration (0, 50, 100 and 200 mM) and heat treatment (70 ºC) on 10 wt %

MIO/W emulsion showed significant increase in the apparent viscosity. Viscosity

increase imposed a destabilizing effect which was confirmed by creaming index and

microstructure of the emulsions as observed by phase contrast microscopy. The 10 wt%

MIO/W emulsions stored at 25 ºC and at 4 ºC appeared to be homogenous in nature; the

droplets were nearly spherical in shape and were evenly distributed and were less prone

to creaming and aggregation.

The thesis ends with a comprehensive summary, bibliography and outcome of

the present investigation.

xiii

ACKNOWLEDGEMENTS

I am greatly indebted to my advisor Dr. K. Udaya Sankar for his valuable advice during

the course of this study.

I am especially grateful to the academic advice of Dr. Suvendu Bhattacharya,

Dr. N.M. Sachindra, Mr. B. Manohar, Dr. P. Srinivas, DR. S. Subramanian and Dr. S.

Divakar. I would like to thank the other members of the advisory committee, for their

helpful and valuable suggestions during my research work.

I would thank the Council of Scientific and Industrial Research, India for the

award of Senior Research Fellowship. My thanks to the Director, Central Food

Technological Research Institute, for providing an opportunity to work in this esteemed

institute. I wish to express my gratitude to Dr. K.S.M.S. Raghava Rao, Head, FED.

I express my heartfelt gratitude to my friends Ms. Hemavathi, for her

innumerable effort in lending me her helping hand when most needed. Special thanks

are to Ms. Swapna Sonale, Ms. Dhanalakshmi and Ms. Roopa the knowledge you

shared with me and your support are gratefully acknowledged. Thanks are also

extended to Mr. Mukund (CIFS) and Mr. Girish (Pilot plant, FED).

To all my colleagues: I am deeming myself very fortunate in having had the

possibility of working with you. I must concede that there was lot more who helped me

directly or indirectly in the completion of this work, whose name I could not mention. I

hope that each and every one of you knows how much I appreciate your company and

friendship for making every moment enjoyable and meaningful.

(A.S.Zarena)

xiv

CONTENTS

Page No.

Certificate ii–iii

Declaration iv

Abstract v

Synopsis vi–xii

Acknowledgements xiii

List of Tables xvi–xviii

List of Figures xix–xxiii

List of Abbreviations xxiv

CHAPTER 1 General introduction 1–28

CHAPTER 2 Extraction and characterization of bioactive compounds

in mangosteen pericarp obtained by SC-CO2

29–80

2A. Preliminary study on the influence of SC-CO2 extraction

of bioactive compounds

30–51

2B. SC-CO2 extraction of xanthones: Characterization by

HPLC/ESI-MS/ NMR

52–69

2C. Influence of co-solvents on selective extraction of

xanthones with SC-CO2

70–80

CHAPTER 3 Antioxidant activity of mangosteen pericarp extracts 81–111

3A. In vitro antioxidant activity of extract obtained by

Soxtec™ method

82–98

3B. In vitro antioxidant activity of xanthones enriched SC- 99–111

xv

CO2 extracts

CHAPTER 4 Optimization of SC-CO2 extraction parameters on

extracts yield in manogsteen pericarp

112–150

4A. Response surfaces of extracts yield in SC-CO2 113–129

4B. Optimization of extracts yield in ethanol modified SC-

CO2 and antioxidant activity

130–150

CHAPTER 5 Characterization of phenolic acids and anthocyanins 151–180

5A. Phenolic acids profile and antioxidant activity 152–166

5B. Isolation and identification of anthocyanins 167–180

CHAPTER 6 Central composite rotable design for glucosylation of α-

magostin in SC-CO2 media

181–199

CHAPTER 7 Formulation of mangosteen in oil-in-water emulsions 200–216

Summary of the work 217–218

Bibliography 219–233

Outcome of the research work 234–236

Reprints of the publication 237

xvi

LIST OF TABLES

Table No. Title Page No.

1.1 Selected physicochemical properties of liquids, gases

and supercritical fluids

24

1.2 Critical properties of selected solvents 25

1.3 Summary of the extraction of bioactive compounds

from plants by SC-CO2

26

2A.1 LC–ESI–MS of SC–CO2 extract at 300 bar and 50C

with 2% EtOH as entrainer

47–49

2A.2 Total extraction yield in different solvent extracts

(Soxtec™) of mangosteen pericarp

50

2A.3 Composition of the mangosteen extract as obtained by

Soxtec™ extract

51

2B.1 Identification and quantification of the compounds

extracted by SC-CO2 alone under different operational

conditions

64

2B.2 LC-MS of the identified compounds extracted from

mangosteen pericarp by SC-CO2

65

2B.3 1H NMR and

13C NMR data of compounds (1-5)

extracted by SC-CO2

66–69

2C.1 Identification and quantification of the compounds

extracted by SC-CO2 +5% ethanol under different

operational conditions.

80

3A.1 Total phenolic content in different Soxtec™ extracts of

mangosteen pericarp

98

3B.1 Total phenolic content of mangosteen pericarp extract

obtained by SC-CO2

109

xvii

3B.2a Antioxidative effect of mangosteen pericarp obtained by

SC-CO2+ 5% ethanol

110

3B.2b Antioxidative effect of mangosteen pericarp obtained by

SC-CO2 alone

111

4A.1 Box-Behnken design matrix along with the

experimental and predicted values of the yield of

extracts

126

4A.2 Analysis of variance (ANOVA) for the fitted quadratic

polynomial model

127

4A.3 Regression coefficients of the fitted quadratic equation

and standard errors for the yields.

128

4A.4 Antioxidant activity of the SC-CO2 extracts by FRAP

method

129

4B.1

Uncoded and coded levels of the independent variables

for Box–Behnken design.

146

4B.2 Box–Behnken design matrix along with the

experimental and predicted values of total extraction

yield and DPPH radical scavenging activity

147

4B.3a Analysis of variance (ANOVA) of the regression

parameters for the total extract yield.

148

4B.3b Analysis of variance (ANOVA) of the regression

parameters for the DPPH scavenging activity

149

4B.4 Regression coefficient of polynomial functions of

response surfaces of total extract yield and radical

scavenging activity

150

5A.1 Relative retention times, molecular ions in the negative

ion ESI-MS of phenolic acid present in mangosteen

pericarp

166

5B.1. 1H and

13C NMR spectroscopic data of pelargonidin 3-

O-β-glucopyranoside

180

xviii

6.1 Treatment levels and coded values for each of the

independent variables used in the design of experiments.

196

6.2 CCRD design with experimental and predicted yields of

glucosylation based on response surface methodology

197

6.3 Analysis of variance (ANOVA) of the response surface model

198

6.4 Regression coefficient for main factors and their interaction

199

xix

LIST OF FIGURES

Figure No. Title Page No.

1.1 Phase diagram of SC-CO2 21

1.2 P–T diagram of CO2 demark interest at densities from

100 to 1200 g/L

22

1.3 Critical points in ternary mixtures 23

2A.1 Mangosteen fruit and structure of xanthone 43

2A.2 Schematic flow diagram of SC-CO2 extraction 44

2A.3a Recovery of mangosteen extract (g) with CO2 (kg) at a

temperature of 50 ºC and pressure of 300 bar using SC-

CO2 without entrainer P–T diagram of CO2 demark

interest at densities from 100 to 1200 g/L

45

2A.3b Recovery of mangosteen extract (g) with CO2 (kg) at a

temperature of 50 ºC and pressure of 300 bar using SC-

CO2 with ethanol as an entrainer

45

2A.3c Solvent to material ratio versus extracts recovery (%)

during mangosteen extraction with and without

entrainer using SC-CO2 at 300 bar and 50 ºC.

45

2A.4 HPLC chromatograms of various xanthones from G.

mangostana extracted by SC-CO2 + 2% EtOH at 300

bar, 50 ºC

46

2B.1a Effect of temperatures on the extraction yield of

mangosteen pericarp at constant pressure

61

2B.1b Effect of pressures on the extraction yield of the

mangosteen pericarp at constant temperature

61

2B.1c Plot showing the cross over point at 0.75 g/cm3 61

xx

2B.2 A typical HPLC chromatogram of SC-CO2 extract 62

2B.3 Chemical structure of xanthones 63

2C.1 Effect of co-solvents on the extraction yield of

mangosteen pericarp at 50 °C and 280 bar

77

2C.2 Cumulative yield of mangosteen pericarp extracted at

50 °C and 280 bar for different concentration of ethanol

77

2C.3 Effect of time on the cumulative yield of mangosteen

pericarp at 50 ºC and 280 bar

78

2C.4 Effect of temperature and pressure on the extraction

yield of mangosteen pericarp

78

2C.5 A typical HPLC chromatogram of SC-CO2 +5% ethanol

extract.

79

3A.1 Comparison of ferric reducing antioxidant power of

different extract of mangosteen pericarp

95

3A.2 Comparison of Trolox equivalent antioxidant capacity

of mangosteen pericarp extract by ABTS method

95

3A.3 Comparison of IC 50 value of different extract of

mangosteen pericarp by DPPH assay

96

3A.4 Comparison of reducing power of mangosteen pericarp on

Fe3+

to Fe2+

ion

97

3A.5 Chelating effects of mangosteen pericarp extracts on

Fe2+

ion

97

3B.1 Phosphomolybdate complex assay of (a) SC-CO2 + 5%

ethanol & (b) SC-CO2 alone extracts of mangosteen

pericarp

106

3B.2. Reducing power of (a) SC-CO2 + 5% ethanol & (b) SC-

CO2 alone extracts of mangosteen pericarp

107

3B.3. Iron chelating of (a) different SC-CO2 + 5% ethanol &

SC-CO2 alone extracts of mangosteen pericarp.

108

4A.1 Observed values vs. predicted values for xanthone 122

xxi

yields

4A.2 Response surface plots showing effect of pressure and

temperature on the extraction yield at solvent material

ratio of 300 kg/kg

123

4A.3 Response surface plots showing effect of temperature

and solvent to material ratio on extraction yield at

pressure of 300 bar

124

4A.4 Response surface plots showing effects of pressure and

solvent to material ratio on extraction yield at

temperature of 60 oC

125

4B.1 Plot of observed vs. predicted (a) Total extract yield (b)

DPPH EC50

140

4B.2a Counter plot showing the effects of pressure and time

on the total extraction yield.

141

4B.2b Contour plot showing the effects of pressure and

temperature on the total extraction yield.

141

4B.2c Contour plot showing the effects of temperature and

time on the total extraction yield.

142

4B.3a Contour plot showing the effects of pressure and time

on radical scavenging activity.

143

4B.3b Contour plot showing the effects of pressure and

temperature on the radical scavenging activity.

143

4B.3c Contour plot showing the effects of temperature and

time on the radical scavenging activity.

144

4B.4 Comparison of SEM images (1000X magnification) of

mangosteen pericarp powder

145

5A.1a. HPLC chromatogram of BHPA fraction in mangosteen

pericarp detected at 280 nm

162

5A.1b. High-performance liquid chromatogram profile of

phenolic acids extracted from sequential hydrolysis of

163

xxii

mangosteen pericarp.

5A.2. ESI-MS fingerprints of BHPA fraction of mangosteen

pericarp

164

5A.3. DPPH• radical scavenging (%) activity and inhibition

(%) of lipid peroxidation of different mangosteen

pericarp fractions.

165

5B.1 Reverse-phase HPLC chromatogram recorded at 530

nm and UV/vis spectrum

178

5B.2 Electrospray mass spectrum of pelargonidin 3-glucoside in

positive-ion mode

179

Scheme 6.1 Synthesis of α–mangostin–D–glucoside 191

6.1 Schematic diagram of SC-CO2 reactor 192

6.2 Observed values vs. predicted values for –mangostin–

D–glucoside yields

193

6.3 Contour plot for yield of α–mangostin–D–glucoside as

influenced by enzyme concentration and pressure

194

6.4 Contour plot for yield of α–mangostin–D–glucoside as

influenced by buffer volume and pressure

194

6.5 Contour plot for yield of α–mangostin–D–glucoside as

influenced by pH and enzyme concentration

195

6.6 Contour plot for yield of α–mangostin–D–glucoside as

influenced by enzyme concentration and temperature

195

7.1 Rheogram for 10 and 20 wt% of mangosteen emulsions

(MIO/W)

211

7.2. Apparent viscosity as affected by the concentrations of

(a) mangosteen and (b) NaCl during storage at room

temperature

212

7.3. Effect of processing and storage of 10 wt% mangosteen

emulsions at various temperatures

212

xxiii

7.4. Creaming index of mangosteen emulsions during

storage

213

7.5. Creaming index of (a) NaCl treated and (b) heat treated

10 wt% mangosteen emulsions during storage

213

7.6. Photomicrographs of different mangosteen emulsions

during storage at 25 ºC

214

7.7. Photomicrographs of 10 wt% MIO/W during storage at

different temperatures.

215

7.8. Photomicrographs of 10 wt% MIO/W treated with

different NaCl concentration during storage

216

xxiv

ABBREVIATIONS

AA Ascorbic acid

ABTS 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)

BHA Butylated hydroxy anisole

DPPH 1,1-diphenyl-2-picrylhydrazyl

ESI Electrospray ionisation

FT-IR Fourier transform infrared

FRAP Ferric reducing antioxidant power

GAE Gallic acid equivalents

H2O2 Hydrogen peroxide

HSCCC High-speed counter current chromatography

HPLC High performance liquid chromatography

HSQC Heteronuclear single quantum coherence (NMR)

LC Liquid chromatography

MS Mass spectrometry

NMR Nuclear magnetic resonance spectroscopy

PDA Photodiode array

RP-HPLC Reversed phase HPLC

SC-CO2 Supercritical carbon dioxide

SCF Supercritical critical fluid

SF Supercritical fluid

SFE Supercritical fluid extraction

SEM Scanning electron microscopy

TBARS Thiobarbituric acid-reactive substances

TE Trolox equivalents

TEAC Trolox equivalent antioxidant capacity

Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid

TPTZ 2,4,6-tripyridyl-s-triazine

Chapter I

General Introduction

Chapter 1

2

1. General Introduction

There is an increasing public awareness of the health, environmental and safety

hazards associated with the use of organic solvents in food processing and the possible

solvent contamination of the final products. The food industry is always looking for the

best separation technology to obtain natural compounds from high purity, healthy

products that are of excellent quality (Raventós, et al., 2002). The high cost of organic

solvents, increasingly stringent environmental regulations and new requirements of the

pharmaceutical and food industries for ultra-pure and high added value products have

increased the need for the development of new and clean technologies for the

processing of food products.

Over the last few years, supercritical fluid (SCF) technology has enjoyed a much

more sustained growth in interest and has become more firmly entrenched in the field of

plant material extraction and purification with considerable work committed to

modeling, SCF kinetics and analyte solubility (Dean and Liu, 2000; Rodrigues, et al.,

2003). The availability of various hyphenated techniques, enabling linkages to various

separation systems such as supercritical fluid chromatography, gas chromatography and

mass spectrometry has further encouraged expansion of research and use of

supercritical fluid extraction (SFE ) (Smith, 1999).

Thermodynamically, SCF is a state where pressure and temperature are beyond

the critical point values. There are two interesting points of the phase diagram of a pure

substance: the triple point, where solid, liquid and gas are all in equilibrium, and the

Chapter 1

3

critical point, at which liquid and gas phases cease to have separate existence. When a

fluid is forced to a pressure and temperature above its critical point it becomes a

supercritical fluid as schematically described in a pressure-temperature phase diagram

(Fig. 1.1). Above the critical temperature, it is not possible to liquefy a gas by

increasing the pressure, but the density increases continuously with increasing pressure.

In general, when a gas is compressed to a sufficiently high pressure, it gets liquefied.

However, if the gas is heated beyond a specific temperature, called the critical

temperature, no amount of compression can liquefy the gas. The critical temperature

(Tc) is defined as that temperature above which however large a pressure is applied,

gases cannot be liquefied. The pressure corresponding to this critical temperature is

called the critical pressure (Pc). Thus, the critical temperature and critical pressure of a

fluid are the highest temperature and pressure conditions at which vapor-liquid

equilibrium can exist. The critical point lies at the end of the vaporization curve where

the gas and liquid phases merge to form a single homogenous fluid phase.

Under these conditions, various properties of the fluid are placed between those

of a gas and a liquid. Although the density of a supercritical fluid is similar to a liquid

and its viscosity is similar to a gas, its diffusivity is intermediate between the two states,

as can be seen in Table 1.1. Thus, supercritical fluid has been defined as a state in

which liquid and gas are indistinguishable from each other, or as a state in which the

fluid is compressible (i.e. similar behavior to a gas) even though possessing a density

similar to a liquid and therefore, similar solvating power.

Chapter 1

4

The region above the critical temperature and pressure is the region where

maximum solvency and the largest variations in solvent properties can be achieved with

small change in temperature and pressure. This offers attractive mass transfer

characteristics owing to the solvent‟s diffusivity, viscosity, surface tension and other

physical properties. The diffusivity is one to two orders of magnitudes higher than

those of other liquids. The liquid like density of a SCF solvent provides high solvency

whereas the gas like viscosity and diffusivity, together with zero surface tension in part

excellent transport properties which enhances the rate of transfer from the original

botanical substrate to the SCF solvent in comparison to liquid solvents. Thus the gas

like characteristics of a supercritical fluid provides ideal conditions for extraction of

solutes with a high degree of recovery in a short period of time. The low viscosity and

surface tension enables the solvent to posses superior dissolving properties of a liquid

hence can easily penetrate the matrix from which the active component is to be

extracted from a complex mixture (Kiran and Sengers, 1994).

As for the solvents, there is a wide range of compounds that can be used as

supercritical fluids (Table 1.2). Carbon dioxide (CO2) is the most commonly used

because of its moderate critical temperature (31.1 °C) and pressure (73.8 bar). CO2 is a

gas at room temperature, so once the extraction is completed, and the system is

decompressed, a substantial elimination of CO2 is achieved without residues, yielding a

solvent-free extract. On an industrial scale, when CO2 consumption is high, the

operation can be controlled to recycle it. A further aspect to the potential success of

CO2 as SCF in the future is that except for water, it is probably one of the cheapest

Chapter 1

5

solvents. With a reduction in the price of CO2 and restrictions in the use of other

organic solvents, CO2 has begun to move from some marginal applications to being the

major solvent for SFE (Hurren, 1999). Commercial CO2 required for SFE is already

present in the environmental system, obtained as a byproduct from the fermentation

process or the fertilizers industry. So its use as an extractant does not cause any further

increase in the amount of CO2 present in the earth‟s atmosphere. Therefore, there is no

additional „green house effect‟ from using CO2 as the SCF solvent.

SC-CO2 is a good solvent for the extraction of nonpolar compounds such as

hydrocarbons (Gil-Vilegas, et al., 1997). To extract polar compounds, some polar

supercritical fluids (SF) such as Freon-22, nitrous oxide and hexane have been

considered. However, their applications are limited due to their unfavorable properties

with respect to safety and environmental considerations (Lang and Wai, 2001).

CO2 offers the following advantages as supercritical fluid:

Being non-toxic and physiologically harmless, it has GRAS (Generally

Regarded as Safe) status.

It is non-inflammable, non-corrosive and leaves behind no harmful residues

after extraction. It causes no environmental pollution problem with Tc=31.1 °C

and Pc=73.8 bar. SC-CO2 can be used at temperatures and pressures which are

relatively safe, convenient and particularly appropriate for extraction of a wide

range of compounds.

Chapter 1

6

Its near ambient critical temperature makes it ideally suitable for thermo-labile

natural products, especially in food and pharmaceutical applications.

It has good solvating power due to low viscosity and high diffusivity.

Due to this low latent heat of vaporization, low energy input is required for the

extract separation system, which renders the most natural odor and natural

tasting extracts.

The energy required for attaining supercritical state in CO2 is often less than the

energy associated with the distillation of conventional organic solvents.

CO2 diffuses through condensed liquid phases (e.g., adsorbents and polymers)

faster than do typical solvents, which have larger molecular sizes (Perry, et al.,

1997).

In general, the extractability of compounds with SC-CO2 depends on the

occurrence of the individual functional groups in the compound, their molecular

weights and polarity. SC-CO2 is an excellent extraction medium for non-polar species

such as alkanes and terpenes and reasonably good for moderately polar species like

benzene derivatives, such as aldehydes and alcohols. As put forth by Brunner (2005)

SC-CO2 (i) dissolves non-polar or slightly polar compounds, (ii) the solvent power for

low molecular weight compounds is high and decreases with increasing molecular

weight. Materials with molecular weight greater than 500 daltons have limited solubility

in CO2, (iii) free fatty acids and their glycerides exhibit low solubilities; low molecular

weight non-polar aliphatic hydrocarbons and small aromatic hydrocarbons are soluble,

(iv) pigments are even less soluble, (v) water has a low solubility (

Chapter 1

7

100 °C, (vi) polar organics such as carboxylic acids, fruit acids, sugar, polysaccharides,

proteins, phosphatides, glycosides and inorganic salts are relatively insoluble in dense

CO2.

1.1. Thermodynamic properties of supercritical fluids (SCFs)

The solvating power of SCF is highly dependent on its density. By controlling

pressure and temperature it is possible to control the density of the fluid allowing

selective manipulation of the solvating power of the fluid. It is reported that solute

solubility studies of individual components present in a mixture provide a means of

determining SCF process conditions that will afford selective extraction or separation of

the individual solutes (Fig. 1.2) (Mukhopadhyay, 2000). In the vicinity of the fluid

critical point, physical properties of the fluid change dramatically with small changes in

pressure and as a consequence the solubility of solutes changes dramatically some

authors have reported using this region it is possible to separate mixed solutes (Raeissi

and Peters, 2005).

In practice, SCF solvent is mostly used as an extractant in the approximate range

of temperature up to 1.2 times the critical temperature, (Tc) and pressure up to 3.5 times

the critical pressure, (Pc). Moreover, even near critical region itself, SCF exhibit large

changes in specific gravity and consequently its solvent power and other physico-

chemical properties with pressure or temperature. At the critical point, the fluid

compressibility becomes infinite meaning that the fluid specific gravity rapidly varies

with slight change in pressure at constant temperature. Most compounds exhibit a

Chapter 1

8

critical pressure in the range of 35 to 60 bar, except water (221 bar) and ammonia

(113.5 bar). Critical temperature increases with the complexity of the molecule and

very few compounds exhibit a critical temperature between 0 and 50 oC (ethane,

ethylene, CO2 and N2O). Mixtures behave in a more complex way depending on their

composition. In CO2 both density () and dielectric constant () rise sharply between 70

and 200 bar. At around 200 bar and beyond, both parameters attain values similar to

those for liquids. This explains why SC-CO2 exerts high solvent power above certain

pressure and may be used as a good solvent to replace conventional organic solvents.

The CO2 molecule has no net dipole moment, i.e., it is non polar and hence in the SCF

state, it serves as a good solvent for molecules that are non polar. However, it has a

quadrupole moment for which it can also dissolve slightly polar and polar substances at

relatively high pressures (>250 bar) (Mukhopadhay, 2000).

The initial solubility in the non-compressed gas phase is a function of the vapor

pressure of the solute; however, upon additional compression of the supercritical fluid

phase, a solubility minimum is observed. After the occurrence of the solubility

minimum, there is an exponential rise in the solubility of the solute with increasing gas

pressure and a solubility maximum is eventually attained at a pressure, which is

determined by the extraction temperature. The effect of increasing temperature in this

case results in an increase in solubility at both low and high pressures; however, at

intermediate pressures, the reverse trend may be observed. The latter region is termed

the cross over region and its occurrence permits selective fractionation of solutes into

the supercritical fluid medium. Solute diffusion coefficients in supercritical fluid have

Chapter 1

9

values between those attained in gaseous or liquid solvents. Although such data as a

function of pressure are relatively scarce, solute diffusion coefficients tend to exhibit

similar trends as recorded self-diffusivities of the extracting fluid. The knowledge of

solubility data on the liquid solvents and of the solids in SC-CO2 is mandatory for this

process for the proper selection of process temperature and pressure.

1.2. Addition of co-solvents

The application of modifiers probably is the simplest yet the most effective way

to obtain a desired polarity of CO2 based fluids. By selecting a modifier or just simply

changing the molar ratio of a modifier, one can readily manipulate the solubility

properties of the fluids. Usually, addition of a small amount of a liquid modifier can

enhance significantly the extraction efficiency and consequently, reduce the extraction

time without significantly changing the density and compressibility of the original SCF

solvent (Valcárcel and Tena, 1997).

The co-solvent mixed SC-CO2 solvent is supercritical when the critical pressure

and temperature of the mixture are above its mixture critical values for a particular

composition, which are usually not very different from the critical values of the pure

SC-CO2. When a binary mixture (Fig. 1.3) of SCF solvent (1) and a co-solvent (3) is

employed beyond its binary mixture critical pressure to solubilize a liquid-solute (2),

then the system is represented by a ternary diagram. In such cases, all three

components are usually distributed both in the liquid and in the SC-CO2 phases. The

extent of solubilization of the component in the two phases is characterized by the

Chapter 1

10

distribution coefficient which is given by the ratio of the concentration of the

component in the fluid phase as represented by two end points of a tie line

(Mukopadhaya, 2000).

The increase in solubility due to the addition of co-solvent is the result of

additional interactions between the solute and the co-solvents. Considering the

interactions possible, these co-solvents effects could be the result of several

mechanisms the addition of a co-solvent generally increases the mixture density, which

enhances the solubility as well as physical interactions like dipole-dipole, dipole-

induced dipole and induced dipole interactions (Ting, et al., 1993). On the contrary,

there are also reports were co-solvents have decreased extraction, both in terms of

quality and quantity of total flavanoids and terpenoids. Decreased efficiency can be

explained by the phenomenon that with a co-solvent higher temperatures are required to

reach the increased critical temperature. Also more polar substances are extracted

together with active compounds as the co-solvent increases their solubility (Yang, et al.,

2002).

1.3. Preparation of plant materials for super critical fluid extraction and operating

conditions

The other crucial parameters in SFE apart from temperature and pressure are

CO2 flow rate, particle size of the matrix and duration of the extraction process (time).

The proper selection of these parameters has the scope of producing the complete

extraction of the desired compounds in the shorter time. They are connected to the

Chapter 1

11

thermodynamics (solubility) and the kinetics of the extraction process in the specific

raw matter (mass transfer resistances). The proper selection depends on the mechanism

that controls the process: the slowest one determines the overall process velocity. CO2

flow rate is a relevant parameter if the process is controlled by an external mass transfer

resistance or by equilibrium: the amount of supercritical solvent feed to the extraction

vessel, in this case, determines the extraction rate (Reverchon and Marco, 2006).

Particle size plays a determining role in extraction processes controlled by

internal mass transfer resistances, since a smaller mean particle size reduces the length

of diffusion of the solvent. However, if particles are too small, they can give problems

of channeling inside the extraction bed. Part of the solvent flows through channels

formed inside the extraction bed and does not contact the material to be extracted thus

causing a loss of efficiency and yield of the process. As a rule, particles with mean

diameters ranging approximately between 0.25 and 2.0 mm are used (Reverchon and

Marco, 2006). The optimum dimension can be chosen case by case considering water

content in the matrix and the quantity of extractable liquid compounds that can produce

phenomena of coalescence among the particles thus favoring the irregular extraction

along the extraction bed. Moreover, the production of very small particles by grinding

could produce the loss of volatile compounds. Large particles may require longer time

for extraction, since the process may be controlled by internal diffusion. However, fine

powder can speed up the extraction but may also cause difficulty in maintaining a

proper flow rate. Chemat, et al. (2004) used SC-CO2 to extract Foeniculum vulgare

volatile oil from fennel fruits with different mean particle sizes 0.35, 0.55 and 0.75 mm.

Chapter 1

12

They found that a decrease in particle size decreased the total yield of the extracted oil.

Therefore, in such cases, some rigid inert materials such as glass beads and sea sand are

paced with the fine plant powder to maintain a desired permissibility of the particle bed.

Preparation of plant materials is another critical factor for SFE of nutraceuticals.

Fresh plant materials are frequently used in SFE of nutraceuticals. When fresh plant

materials are extracted the high moisture content can cause mechanical difficulties such

as restrictor clogging due to ice formation. Although water is only about 0.3% soluble

in SC-CO2, highly water-soluble solutes would prefer to partition into the aqueous

phase, resulting in low efficiency of SFE. Some chemicals such as Na2SO4 and silica

gel are mixed with the plant materials to retain the moisture for SFE of fresh materials

(Lang and Wai, 2001).

The solubility of a target compound in a SC-CO2 is a major factor in

determining its extraction efficiency. The temperature and density of the fluid control

the solubility. The choice of a proper density of a SCF such as CO2 is the crucial point

influencing solvency and selectivity and the main factor determining the extract

composition (Cherchi, et al., 2001). It is often desirable to extract the compound right

above the point where the desired compounds become soluble in the fluid so that the

extraction of other compounds can be minimized.

In extraction of Chilean hop (Humulus lupulus) ecotypes, Del Valle and

Aguilera (2003) found that a very limited increase in extraction rate was observed

Chapter 1

13

above 200 bar at a temperature of 40 ºC; rather, the increase in pressure increased the

co-extraction of undesirable compounds. Thus by controlling the fluid density and

temperature, fractionation of the extracts could also be achieved. For SC-CO2

extraction of squalene and stigmasterol from the entire plant of Spirodela polyrhiza,

Choi, et al. (1997) found the relative extraction yield of squalene was much higher than

that of stigmasterol at 100 bar and 50 or 60 °C. Their results confirmed that SC-CO2

could selectively extract substances from the plant materials by controlling the

conditions such as temperature and pressure.

The extraction time is another parameter that determines extract composition.

Process duration is interconnected with CO2 flow rate and particle size and has to be

properly selected to maximize the yield of the extraction process. Lower molecular

weight and less polar compound are more readily extracted during SC-CO2 extraction

since the extraction mechanism is usually controlled by internal diffusion (Cherchi, et

al., 2001; Poiana, et al., 2003). Therefore, the extract composition varies with

extraction time.

1.4. Application of supercritical fluid extraction

The significant benefits of SC-CO2 include their gas-like flow behavior with

good penetration capability and their liquid-like solvation power. By manipulating the

extraction pressure and temperature it is possible to fine-tune the selectivity of the SCF.

Compared to conventional extraction technique, SC-CO2 is more precise and selective,

subsequently minimizing post-extraction clean-up steps and the possibility of avoiding

Chapter 1

14

the detrimental effects of these solvents on the environment (Tservistas, et al., 2000;

Lang and Wai 2001). The use of muti-stage extraction processes further enriches the

concentration of the compound of interest.

Supercritical fluids have a higher diffusion coefficient and lower

viscosity than liquids.

Absence of surface tension allows for their rapid penetration into the

pores of heterogeneous matrices, which helps to enhance extraction

efficiencies.

Selectivity during extraction may be manipulated by varying the

conditions of temperature and pressure affecting the solubility of the

various components in the supercritical fluid.

Supercritical fluid extraction with CO2 does not leave a chemical

residue.

Supercritical fluid extractions can use carbon dioxide gas, which can be

recycled and used again as part of the unit operation (Rizvi, et al., 1994).

An important advantage of applying SC-CO2 to the extraction of active

compounds from medicinal plants is that degradation as a result of

lengthy exposure to elevated temperatures and atmospheric oxygen are

avoided.

Further, the extract‟s color, composition, odor, texture are controllable

and retains the aroma of the product.

Chapter 1

15

1.4.1. Extraction of antioxidant using supercritical carbon dioxide

SC-CO2 has been used as a solvent to extract natural antioxidants from various

plants, including carotenoids, non-polar tocopherols, terpenoids and polar phenolic

compounds such as flavonoids from oilseeds, plant leaves, labiate herbs, and spices

(Mukhopadhyay, 2000). Traditional extraction methods such as solvent extraction,

aqueous alkaline extraction and steam distillation are not selective, so antioxidant

extracts often show color (chlorophyll) and have a strong flavor. Therefore, further

purification steps are often required for the extract and final food product to remove

unwanted residuals. SC-CO2 extraction, on the other hand, inherently increases

selectivity and allows for fractionation of the extract for high added value substances,

such as antioxidants.

1.4.2. Supercritical carbon dioxide as a reaction medium

Chemical reactions in supercritical fluids are considered as good candidates for

new environment friendly technologies. There are several advantages of carrying out

chemical and biochemical reactions in supercritical media, apart from the

environmental advantages such as: reactions can be carried out in a homogeneous phase

by manipulating the pressure and temperature to control phase behavior; reaction rates

can be increased by 1 to 3 orders of magnitude because of increased diffusivities in

SCFs. Selectivity and conversion can be optimized by manipulating the pressure and

temperature; reaction rates can be enhanced by easy separation of the products from the

solvent(s) and reactants through both pressure and temperature, which increases the

control of reaction pathways and products. By using SC-CO2 an integrated production

Chapter 1

16

process can be performed, because SC-CO2 can act as solvent for the reaction and as a

separation medium after the reaction. Therefore no change from the reaction medium to

the downstream processing (separation) medium is necessary. The overall performance

of such an integrated production process can be higher than the multi media process,

using conventional organic media (Marty, et al., 1994).

1.4.3. Preparation of micro-emulsion using supercritical carbon dioxide

Particle formation using supercritical fluids involves minimal or no use of

organic solvents, while the processing conditions are relatively mild. In contrast to the

conventional particle formation methods, where a larger particle is originally formed

and then comminuted to the desired size, SC-CO2 technology involves growing the

particles in a controlled fashion to attain the desired morphology. The rigid solid

particle, once formed, does not have to undergo the thermal and mechanical stresses.

These features make supercritical fluid technology amenable to produce

biomolecules and other sensitive compounds in their native pure state. This is of

importance to many technologies, including pharmaceutical formulations, because in

most drug- delivery systems the dimensions of the particles are of critical important.

1.5. Outlook for the future

Supercritical techniques are not yet very widely used in industry and based on

the opinion of those involved it seems that the field is in some kind of lag phase at the

moment (Sihvonen, et al., 1999). Krukonis, who stated, “There is no point in doing

Chapter 1

17

something in a supercritical fluid just because it’s neat. Using the fluids must have

some real advantage” (Darr and Poliakoff, 1999).

However, the diversity of the articles and the patents in the late 1990s indicate

that there is much interest in the utilization of supercritical techniques in many areas.

Literature data presented show that supercritical fluids can provide higher conversion

rates and simple downstream processing where it is possible to separate the product

from the reactants. This interest is also reflected in the high amount of scientific papers

dealing with SFE published in recent years. Moreover, industrial applications of SFE

have experienced a strong development since the early 1990s in terms of patents

(Schütz, 2007). From a simple literature search, it can be easily deduced the impact of

SFE as sample preparation technique for the analysis of target compounds from natural

products and foods. Table 1.3 summarizes some of the recent works published on the

extraction of bioactive compounds from plant materials using SC-CO2. It is worth to

mention that SFE has been also widely used in this field for process development, that

is, to extract target (bioactive or valuable) compounds from different matrices. Some

papers have been lately published dealing with the assessment of the industrial

economical feasibility of some developed processes. Therefore, SFE can be regarded as

a possible tool not only from a laboratory point of view but also for the natural products

and food industries.

According to Perrut (2000), the four aspects that we have to consider when we are

trying to foresee what will happen in the near future are

Chapter 1

18

Regulatory issues

Quality considerations

Innovative products

Innovative processes

In recent years, the authorities have become more and more aware of the

environmental and occupational concerns of using large amounts of toxic organic

solvents. As a result, disposal regulations have become stricter, which adds to

processing costs and in many countries, most organic solvents have already been

banned in food processing (Perrut, 2000). Thus, there is pressure to invent new,

more environmentally safe processes. On the other hand, the food industry has a

strong emphasis on added value products such as high value food additives,

functional foods and nutraceuticals. In these the natural, i.e. solvent-free

preparation modes may add to market value of a product. Although supercritical

particle formation techniques have not yet been very widely used in food

applications they could be a useful tool in processing of nutraceuticals and

functional food ingredients. In the future supercritical fluid technologies can also be

applied in making new innovative products. One of the very promising areas of

research is microencapsulation of drug molecules, which are used for controlled

drug release in the human body.

The on-line coupling of SCF with supercritical-fluid chromatography (SFC) has

recently been shown to afford enhanced speed and sensitivity, as a result of the ability

Chapter 1

19

to use this technique to perform consecutive extraction, concentration and separation of

the constituents of herbal materials. Another interesting recent development is the on-

line coupling of SFE to an uterotonic bioassay by Sewram, et al (2000). In the on-line

SFE uterotonic bioassay system SFE extracts from four local medicinal plants were

transferred directly to a uterus muscle chamber to identify the active fractions (i.e. the

fractions capable of inducing muscle contraction can be determined rapidly, safely, and

sensitively). This novel on-line SFE bioassay method could also be adapted for

screening plants with other therapeutic properties, e.g. those used for treatment of

diabetes mellitus and hypertension. Another novel approach involving the off-line

coupling of SC-CO2 to gas chromatography (GC) in such a way that the glass liner of a

programmed temperature vaporizer is placed after the separation vessels of the SC-CO2

extraction module has been recently demonstrated by Blanch, et al. (1999) to be

effective for the sensitive and selective analysis of complex plant matrices.

1.6. Cost and safety

From what has been discussed so far, it is apparent that SC-CO2 offers attractive

technological alternatives to many of the existing industrial processes and unit

operations. Even though these processes usually offer clear advantages over traditional

ones, a few intrinsic limitations, such as limited solubilities of reactants for specific

reactions, the lack of realistic economical studies are the main drawback for industrial

scale. The cost of reactor design for SCF involving processes must be carefully

considered, as otherwise the alternative technology becomes commercially unviable.

Minimizing the size of the reactor for high-value products may minimize the cost, as the

Chapter 1

20

relative cost of a SFE process is estimated to scale as (V*Q)1/4 (V is the column

volume, Q the flow rate) (Perrut, 2000).

As high pressures are involved, the safety aspects of SCF based processes must

also be carefully considered, especially while planning for scale-ups (Lucas, et al.,

2003). A parameter used for measuring the safety limit of any solvent is the „threshold

limit value time weighted average‟ (TLV–TWA). Basically for a 40 h work-week, it is

the concentration of the solvent vapor to which all healthy workers may be repeatedly

exposed without any adverse effect. Such data for SCF must be generated before it is

widely adopted by the industry. These issues have received enough attention and it is

reasonable to expect a healthy growth of SCF-based industries (Mckenzie, et al., 2004).

The possibility of accidental leakage of CO2 during processing can be minimized by the

implementation of CO2 detectors, which are regarded as reliable, accurate and are

largely used at the industrial scale.

Chapter 1

21

Fig. 1.1. Phase diagram of SC-CO2. The sublimation, melting and saturation lines are

equilibrium curves, i.e. two phases are present in the lines except at the triple point

where three phases coexist and at the critical point (T = 31.3 oC, P = 73.8 bar) where

liquid and vapor phases become one phase.

Chapter 1

22

Fig. 1.2. P–T diagram of CO2 demark interest at densities from 100 to 1200 g/L

(Brogle, 1982).

Chapter 1

23

Fig. 1.3. Critical points in ternary mixtures (Brunner, 1994).

Chapter 1

24

Table 1.1

Selected physicochemical properties of liquids, gases and supercritical fluids (McHugh,

et al., 1994)

Properties

Liquid

Supercritical fluid

Gas

Density (kg/m3)

1000

200−800

1

Viscosity (mPas) 0.5−1.0 0.05−0.1 0.01

Diffusivity (cm2/s) 10

-5 10

-4−10

-3 0.1

Chapter 1

25

Table 1.2

Critical properties of selected solvents (Klesper, 1980; McHugh, et al., 1994)

Solvent

TC

(oC)

PC

(bar)

PC

(g/ml)

Ammonia

132.5

113.5

0.24

Benzene 289.0 48.9 0.30

n-Butane 152.0 38.0 0.23

Carbon dioxide 31.1 73.8 0.45

Chlorotrifluoromethane 28.8 39.5 0.58

Dichlorodifluoremethane 111.7 39.9 0.56

Ethane 32.2 48.9 0.20

Ethanol 243.3 63.8 0.28

Ethylene 9.3 50.4 0.22

Isopropanol 235.3 47.6 0.27

Methanol 240.5 79.9 0.27

Nitrous oxide 36.5 72.3 0.46

n-Propane 96.8 42.6 0.22

Propylene 91.9 46.2 0.23

Toluene 318.6 41.1 0.29

Water 374.2 221.2 0.34

Chapter 1

26

Table 1.3

Summary of the extraction of bioactive compounds from plants by SC-CO2

Plant

material

Compounds

Of interest

Related

functional

activities

Extraction

conditions

Analytical

technique

Reference

Cynanchum

paniculatum

Paeonol

Anti-

inflammatory,

antidiabetic,

cardiovascular

protective

CO2+methanol,

150 bar, 55 oC,

20 min (static) +

90 min (dynamic)

HSCCC,

HPLC-DAD

Sun,

et al., 2008

Cassia

tora L. seeds

Volatile oil

Antioxidant

CO2 + ethyl acetate

(10%), 250 bar,

45 oC

GC-MS

Zhang,

et al., 2007

Elettaria

cardamomum

Volatile oil,

fatty acids,

tocopherol

Antioxidant

CO2, 300 bar,

35 °C

GC-MS;

HPLC-FD

(ex;295 nm,

em:330 nm),

HPLC-DAD

Hamdam,

et al., 2008

Coriandrum

sativum L.

Isocoumarins

-----

CO2, 80 bar, 35 °C,

2h (dynamic)

HSCCC

Grosso,

et al., 2008

Garcinia

mangostana

L.

Xanthones

Antioxidant

CO2 + ethanol (4%),

200 bar, 40 ºC

HPLC-

ESIMS

Zarena and

Udaya

Sankar,

2009

Zingiber

Corallinum

Hance

Essential oil

Antipyretic

CO2, + methanol,

100 bar, 30 °C,

40 min

GC-MS

Zhannan,

et al., 2009

Hibiscus

cannabinus

Oil

Antioxidant

CO2, 200 bar,

80 °C, 150 min

_

Chan, et

al., 2009

Pinus sp.

Flavanoids

Antioxidant

CO2 + ethanol (3%),

200 bar, 40 °C

HPLC-UV

(280 nm)

Yesil-

Celiktas,

et al., 2009

Rhodiola

rosea roots

Rosavin

Antioxidant,

anti-stress

CO2 + water (10%),

200 bar,

80 °C, 3 h

HPLC-UV

(UV 254 nm)

Iheozor-

Ejiofor and

Szwajcer

Dey, 2009

Valiriana

officinalis L.

Valerenic

acid

Tranquilizing

CO2 + ethanol, 360

bar, 37 °C, 20 min

GC-MS

Salimi

et al; 2008

Viscum album

L.

Cytotoxic

compounds

Anti-cancer

drugs

CO2, 350 bar, 35 °C

GC-MS

Cebovic, et

al., 2008

Chapter 1

27

Scope of the present work

Objectives

The main objective of this thesis was

1. Isolation and screening of mangosteen pericarp extract for their bioactive

components obtained by SC-CO2 and by conventional method like Soxtec™

extract.

2. Characterization and evaluation of the extracts for their anti-oxidant properties

in- vitro assay.

3. Formulation of bioactive compounds as deliverable form.

Specific objectives for the individual studies were:

The present work focuses on the SC-CO2 extraction of bioactive components

from mangosteen pericarp with and without entrainer along with Soxtec™

method.

To study the antioxidant capacity using different assay methods. To compare

the yield and xanthone content of the SC-CO2 extracts with Soxtec™ extracts.

To develop a validated HPLC/LC-ESI-MS method to qualitatively and

quantitatively analyze the extracts in SC-CO2 and Soxtec™ extracted

mangosteen pericarp. Structural elucidation of some of the xanthone by NMR.

Chapter 1

28

Optimization of SC-CO2 parameters under various temperatures, pressures,

solvent to material ratio and with respect to time by response surface

methodology.

Enzymatic glucosylation of α-mangostin with glucose to prepare water soluble

α-mangostin-D-glycosides using amyloglucosidase. A central composite

rotatable design (CCRD) was employed with five parameters namely, pressure,

temperature, enzyme concentration, pH and buffer volume to arrive at optimum

glucosylation conditions.

To identify and quantify phenolic acid and anthocyanin-based color as a natural

food colorant from mangosteen pericarp.

Formulation of emulsion and to study their rheological property and storage

stability.

Chapter 2

Extraction and characterization of

bioactive compounds in mangosteen

pericarp obtained by SC-CO2

Chapter 2

30

Part 2A

Preliminary study on the influence of SC-

CO2 extraction of bioactive compounds

Chapter 2

31

2A.1. Introduction

Garcinia mangostana Linn. belonging to the Guttiferae family is one of the most widely

recognized tropical fruits and is commonly known as mangosteen. The fruit has a

universal appeal because of its quality in color, shape and flavor. Since the fruit is

eloquently delicious it is nicknamed as ‘Queen of fruits’. Mangosteen is presumed to

have originated in Southeast Asia or Indonesia and has largely remained indigenous to

Malay Peninsula, Myanmar, Thailand, Cambodia, India, Vietnam and the Moluccas

(Akao, et al., 2008). Worldwide production of mangosteen is about 150000 tons per

annum (Loo and Huang, 2007). This tree can reach 6–25m in height and it has leathery,

glabrous leaves and is slow growing (Morton, 1987). The entire fruit is typically 2.5–

7.5 cm in diameter (Fig. 2A.1.a–e). The edible fruit aril is white, soft and juicy with a

sweet, slightly acid taste and a pleasant aroma (Martin, 1980). The edible portion of the

fruit comprises only about 25% of the total volume, whereas the remainder is tough,

bitter pericarp, 6–10 mm in diameter which exudes a yellow resin (hence the term

xanthones or yellow in Greek). The pericarp is dark red to purplish and composes about

two-thirds of the whole fruit weight as agricultural waste. The pericarp is rich in

bioactive compounds with potential applications as therapeutic agents or as functional

food additives. In fact, the nonedible pericarp has been used for treating diarrhea,

wounds and skin infections in traditional Thai medicine and has potential impact for

lowering risk of human diseases (Pedraza-Chaveri, et al., 2008).

The mangosteen pericarp comprises an array of polyphenolic acids that assure

astringency to discourage infestation by insects, fungi, plant viruses, bacteria and

Chapter 2

32

animal predation while the fruit is immature. Color changes and softening of the

pericarp are natural processes of ripening, which indicates that the fruit can be eaten and

the seeds finish developing. Phytochemical studies have shown the pericarp of the fruit

contains various secondary metabolites, such as tannins, triterpenes, anthocyanins,

xanthones, polysaccharides, phenolic compounds, vitamins B1, B2, C and other

bioactive substances (Kondo, et al., 2009). Among the constituents of the pericarp,

xanthones are the most characteristic secondary metabolite. They are biologically active

phenols that naturally occur in a restricted group of plants and some fungi and lichens

(Jung, et al., 2006). Over 200 xanthones are currently known to exist in nature and

approximately 50 of them are found in the mangosteen.

Chemically xanthones (9H-xanthen-9-ones) are heterocyclic compounds with

the dibenzo-6-pyrone framework (Fig. 2A.1f). Xanthones are represented as C6-C1-C6

system with molecular formula C13H8O2. The xanthones possess a six-carbon

conjugated ring structure with multiple double carbon bonds. The xanthone nucleus is

numbered according to a biosynthetic convention with carbons 1-4 being assigned to

acetate-derived ring A and carbons 5–8 to the shikimate-derived ring B. The other

carbons are indicated as 4a, 10a, 8a, 9 and 9a for structure elucidation purposes (Pedro,

2002). The great diversity and pharmacological activities due to their unique structure

and its substitutions has made its use widely as a botanical dietary supplement in several

countries.

Very few reports appear in the literature concerning the extraction of xanthones

by supercritical fluid extraction (SFE). Da Costa, et al. (1999) have demonstrated the

Chapter 2

33

feasibility of using SFE for extracting xanthones from Maclura pomifera tree bark,

Cocks, et al. (1995) have applied SFE to extract xanthones from Aspergilus fumigates.

The objective of this chapter was to screen the most appropriate solvent and

method to extract active constituents (preferably xanthones) from mangosteen pericarp.

Based on the extraction method this section is divided in three parts namely:

Part A deals with the preliminary study on the extraction of xanthones using SC-

CO2 and by Soxtec™ method using methanol, ethyl acetate, hexane and ethanol

as solvents.

Part B deals with SCF extraction with pure CO2 (or CO2 alone).

Part C deals with SCF extraction performed with CO2 with added modifier such

as methanol, ethyl acetate and ethanol.

The effect of the main operating parameters namely, extraction pressure,

temperature, time, solvent to material ratio and effects of modifiers or co-solvents on

the extraction yields were investigated. A HPLC/LC-ESI-MS method to qualitatively

and quantitatively analyze the extracts was developed and structural elucidation of

xanthones was carried by NMR.

2A.2. Materials and methods

The fresh mangosteen fruits were procured from Coimbatore (Tamil Nadu,

India). The pericarp of the fruit was separated and dried at 40 °C in a cabinet tray drier

Chapter 2

34

(Precision products, Ahmedabad, India). The dried pericarp was ground in a hammer

mill with a weight mean particle diameter 506.06±29.15µm as measured using

computerized particle inspection system (Model: CIS-100, M/s Galai Production,

Israel). Carbon dioxide (purity 99.9%) was supplied by M/s Kiran Corporation, Mysore

(India).

Rota vapor (model RE-111 Buchi, Switzerland), Soxtec™ system (HT2 1045,

Foss Tecator, Sweden). Formic acid, methanol, acetonitrile, isopropanol, water and

acetic acid were of HPLC grade. All the other chemicals used were of analytical grade.

2A.2.1. Supercritical carbon dioxide extraction

The samples were extracted on a pilot scale unit, with a high-pressure equipment

(Nova Swiss, Nova Werke AG, EX 1000-1.4-1.2 v, Switzerland) designed for working

pressures of 1000 bar and temperature of 100 °C. Fig. 2A.2 depicts a schematic flow

diagram of the SC-CO2 extraction equipment used in the present study. The dried and

powdered pericarp was loaded into a steel cylinder of 1L capacity equipped with

sintered metal plate on both ends. The loaded cylinder was then introduced into the

extraction vessel (E) and two Julabo thermostat water circulators were set to the desired

extractor and separator temperature, respectively. A flexible electrical heating tape with

a regulator is wound around the pipe connecting the separator to the expansion valve, to

prevent the blocking of the separator pipe with the extracted material. CO2 supplied

from a gas cylinder (G) was compressed by two diaphragm compressor (C1 and C2) to

the desired pressure by adjusting the pressure controller and heated to the specified

temperature by means of a heat exchanger (TC1) to reach the supercritical state. At the

Chapter 2

35

extractor exit; the gaseous solution leaving the extractor passes through the pressure-

reduction valve (PCR), here the pressure of the CO2 is reduced causing the extracted

components to precipitate in the separator (S). Thus, CO2 along with the extracted

material is depressurized to separate the extract; and vapor CO2 is recycled through a

micro filter (MF). The temperature, pressure and frequency of the pre-calibrated flow

meter (FM) were recorded to give solvent loading (amount dissolved in the solvent) as a

function of solvent usage. Once the scheduled time was achieved, the extraction vessel

was depressurized and the extracts were collected from the separation vessel. In

experiments where co-solvent was used, the solvent was removed using a rotary

evaporator (Buchi Model RE 111, Switzerland) under vacuum at 40 °C and weighed in

an analytical balance.

2A.2.2. Soxtec™ extraction

The dried and powdered pericarp (5g) was extracted using Soxtec™ apparatus

with different solvents. The solvent systems used were ethyl acetate (EtAc), hexane

(Hx) and acetone:water (80:20) (AW), methanol (MeOH) and ethanol (EtOH).

Extractions were carried out for 2h that include initial boiling for 30 min. After filtering

the extract through Whatman No. 1 paper, each of the filtrates was concentrated using

rota vapor at 40 oC, the weight of the each extract was noted and the final volume was

made up to 25 mL in a volumetric flask. The extract were kept in airtight amber bottles

after flushing with nitrogen gas for 30s and stored in freezer at -20 oC until they were

analyzed.

Chapter 2

36

2A.2.3. HPLC analysis

HPLC analysis was performed using Shimadzu LC 10A (Japan) with PDA

detector (SPD–M 10 Avp, Shimadzu, Japan). Class 10 software was utilized for

instrument control, data collection and data processing. The column was RP C–18, 150

X 4.6 mm, SS, Exsil ODS 5 μm particle size (SGE, Analytical science). The mobile

phase 1 consisted of A: 0.1% acetic acid in water and B: methanol, separations were

achieved using a gradient of 75 to 90% B over 0–40 min at the flow rate of 0.8 mL/min.

Injection volume was 5 μL of the extract concentration of 1 mg/mL. Absorbance was

recorded at 254 nm.

Mobile phase 2

The gradient mobile phase used for the analysis was solvent A: 0.03%

orthophosphoric acid in water and solvent B: acetonitrile:methanol (75:25). A flow rate

of 0.8 mL/min under the initial condition of 0 min 75% B, 15 min 90% B, 30 min 100%

B, 35 min 75% B. The temperature was held constant at 40 °C. The contents of the

compounds were expressed as % (w/w) on the basis of the calibration curve of standard

–mangosteen isolated in our laboratory. The square of the correlation coefficient for

–mangosteen was R2=0.994 and the results were calculated, as averages of triplicate

injection.

2A.2.4. LC-MS analysis

LC analysis were performed using Waters Alliance system 2695 separation

module with auto sampler and Waters 2996 Photo diode array detector (USA). For LC

Chapter 2

37

separation was accomplished on a 150 x 4.6 mm, 5 μm ODS, RP C-18 column

(Phenomenex, Torrance, CA) with the following elution condition: flow rate of 0.8

mL/min, temperature 40 C. The mobile phase consisted of (A) water with 0.5% acetic

acid, (B) acetonitrile with 0.5% acetic acid and (C) isopropanol, which were applied in

the following gradient elution: 40% A/ 40% B/ 20% C (v/v/v) in 10 min to 5% A/ 70%

B/ 25% C (v/v/v) in 35 min; finally in the next 10 min to 100% B. For the MS, mass

spectra were acquired using Ultima ESI-Q-TOF (Microssmass Limited, UK). Drying

gas: N2, drying gas temperature was 325 °C. For negative ESI analysis the parameters

were, capillary voltage: -3.00 KV; Cone: 100, source temperature: 120 °C, desolvation

temperature: 300 °C, cone gas flow 50 L/hr, desolvation gas: 500 L/hr. The mass scan

range was from 200 to 900 m/z, scan speed 1000 amu/sec. Data acquisition and

processing was done with the software Masslynx 4.0 and held at that composition for 5

min. Each run was followed by equilibration time of 10 min.

2A.2.5. Structural identification using preparative HPLC, 1H &

13C NMR

For preparative HPLC, the same condition was used as mentioned in section

2.2.3 (mobile phase 2) with the difference being that fractions were collected for several

cycles. HPLC analyses were carried on LC-8A (Shimadzu, Singapore) UV-visible

detector. The column used was RP C-18, Phenomex 250 x 50 mm. Flow rate was 25

mL/min, injection volume was 5 mL. Each of the peaks were individually collected and

evaporated at 40 ºC. Two-dimensional heteronuclear single quantum coherence transfer

spectra (2D HSQCT) were recorded on a Brüker Avance 500 MHz spectrometer

operating at 500.13 MHz for 1H and 125 MHz for

13C at 35

oC. Proton and carbon 90

o

Chapter 2

38

pulse widths were 12.25 and10.5 μs, respectively. Spectra of the samples were recorded

in DMSO-D6 solvent, chemical shifts were expressed in ppm relative to internal

tetramethylsilane standard.

2A.2.6. Determination of moisture content and extract yield

The moisture content (ASTA, 1985) of the mangosteen pericarp powder was

determined by toluene distillation method. The pericarp powder (5g) was co-distilled

with toluene and the amount of water collected was noted and expressed as percentage

of moisture using the formula.

(g)takensampleofWeight

(mL)collectedwaterofVolume(%)Moisture

The yield of the evaporated dried extracts based on dry weight basis (% db) was

calculated from the equation:

Yield (%) = 1002

1x

W

W

where W1 was the weight of extract after evaporation of solvent and W2 was the dry

weight of the pericarp powder.

2A.3. Results and discussion

2A.3.1. Effect of SC-CO2 parameter on the extraction yield

A preliminary supercritical extraction of mangosteen pericarp powder was

performed with and without entrainer. The ground powder (300–500 g) was packed in

the extraction vessel and extraction was carried out at 300 bar and 50 ºC with carbon

dioxide alone and along with ethanol at 2–3% using dosing pump (Milton Roy, USA).

Chapter 2

39

To monitor the extraction rate, the extracts were collected at different intervals and the

percentage recovery is plotted against solvent to material ratio to compare the extraction

yield. The extract was further analyzed for compound characterization by HPLC, and

LC–ESI–MS analysis was undertaken to confirm the identity of the xanthones.

The limitation of CO2 is that polar organic compounds are often difficult to

extract from plant materials though they are soluble in SC-CO2. The extraction of polar

molecule requires addition of modifiers like methanol and ethanol. Xanthones being

polar compounds, ethanol was used as modifier. Fig. 2A.3.a. provides the extraction of

mangosteen powder with carbon dioxide without entrainer, while Fig. 2A.3.b provides

the extraction with alcohol as entrainer at 2% by weight to carbon dioxide; the entrainer

increased the average extract concentration by 200-300 times in case of the polar

compounds. Fig. 2A.3.c depicts the recovery of the extracts with solvent to material

ratio with and without entrainer from the individual rate extraction curves that follow

logarithmic model