Supercritical Fluid Extraction of Mogrosides from Siraitia Grosvenorii

Yan Xia

Department of Food Science and Agricultural Chemistry

McGill University

Montreal, Québec, Canada

September, 2006

A thesis submitted to McGill University in the partial

fulfillment of the requirement of the Degree of Master of Science

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Mogrosides, the main active components in S. grosvenorii SWINGLE, are considered to be sorne 250 times sweeter than sucrose and to possess several medicinal attributes. Previous isolation processes used large quantities of toxic solvent that resulted in toxic residues of organic solvent in this high value food. Supercritical fluids fulfill the requirements of non-toxicity, recycle ability, and useful solvent characteristics. The work presented in this thesis is directed to the extraction of mogrosides from the powdered S. . grosvenorii concentrate (SG) and the crude extract after resin treatment (MG) with sub critical water and supercritical CO2. Because no source of mogroside V reference materiaI is available commercially, the first objective of this research was to isolate mogroside V of sufficient purity that it could be crystallized. This objective was achieved by selecting suitable eluates from resin chromatography coupled with preparative thin layer chromatography (TLC). Crystalline white isolate was further characterized by 13C-NMR and by MS and determined to he mogroside V, which was suitable as a reference material for subsequent experiments.

The process variables for both sub critical water and supercritical carbon dioxide extraction were evaluated and optimized so that conclusions could be formulated regarding the relative merits of the two proposed extraction methods. The efficiency of extraction was determined spectrophotometrically based on the recovery of mogrosides from the starting material following the vanillin-HCI04 method.

When compared with Soxhlet solvent extraction, supercritical fluid extraction with either sub critical water or supercritical C02 provided improved recoveries and consumed less organic solvent. In addition, the purity of the extracts differed greatly. For identical SG samples, sub critical water extraction was demonstrated to be more efficient (62.4 % recovery) compared with 37.0 % recovery by EtOH modified SCC02 extraction or 5.1 % for Soxhlet extraction with hexane. Résumé

Mogrosides, le composant actif principal dans le grosvenorii SWINGLE de S., sont considérés comme environ 250 fois plus doux que le sucrose et posséder plusieurs attributs médicinaux.

L'isolement précédent traite les grandes quantités utilisées de dissolvant toxique qui ont eu comme conséquence les résidus toxiques du dissolvant organique en cette haute nourriture de valeur. Les fluides supercritiques remplis les conditions de la non-toxicité, du recyclability, et des caractéristiques dissolvantes utiles. Le travail a présenté dans cette thèse a été dirigé vers l'extraction des mogrosides du concentré en poudre de grosvenorii de S. (SG) et de l'extrait brut après le traitement de résine (magnésium) avec l'eau critique secondaire et le CO2 supercritique.

Puisqu'aucune source de documentation de référence du mogroside V n'est disponible commercialement, le premier objectif de cette recherche était d'isoler le mogroside V de la pureté suffisante qu'il pourrait être cristallisé. L'isolat blanc cristallin a été encore caractérisé par

I3C_NMR et par la MS et déterminé pour être le mogroside V, qui convenait comme documentation de référence pour des expériences suivantes. Les variables de processus pour l'eau critique de sous-marin et l'extraction supercritique de CO2 ont été évaluées et optimisées de sorte que des conclusions aient pu être formulées concernant les mérites relatifs des deux méthodes proposées d'extraction. L'efficacité de l'extraction a été déterminée spectrophotométriquement a basé sur le rétablissement des mogrosides à partir du produit de départ après la méthode vanillin-HCI04 en comparaison avec l'extraction par solvants de Soxhlet, l'extraction liquide supercritique avec l'eau critique de sous-marin ou le CO2 supercritique a

fourni des rétablissements améliorés et a consommé le dissolvant moins organique. En outre, la

pureté des extraits a différé considérablement. Pour les échantillons identiques de SG;

l'extraction critique de l'eau de sous-marin a été démontrée pour être plus efficace (62.4 % de

rétablissement) comparée à 37.0 % de rétablissement par l'extraction scC02 modifiée EtOH ou à

5.1 % pour l'extraction de Soxhlet avec de l'hexane. Acknowledgements

It is pleasant that 1 have now the opportunity to express my gratitude for many people who supported me.

The flfst person 1 would like to thank is my supervisor William D. Marshall. 1 have been in his project since 2004 when 1 started my MSc assignment. During the se years, his gentlemanly personality and integral view on research has made a deep impression on me. 1 owe him lots of gratitude for gui ding my work and providing me with valuable comments on this thesis. 1 am really glad that 1 have come to know Dr. Marshall in my life.

1 would like to thank all the colleagues in our lab and department. 1 thank Kebba Sabally, for providing me brotherly advice that helped me a lot in staying at the right track. Tao Yuan has shared instruments and many experiences with me. Shiyi Gong has provided me valuable comments. Mario Rivero-Huguet has helped me translate my abstract in French. 1 thank Sadia Ehsan and Liang Yu, for being helpful in the labo 1 would also thank Muhammad Alu Datt for helping me in preparing my defense presentation.

1 also thank a nice couple, Changying Shi and Juntao Luo, for giving me lots of academic advice and emotional support. A special thanks goes to Ting Xie, whom 1 have known for more than ten years now and who showed to be a mostly helpful and trustful friend.

1 am very grateful for my husband, Xue Feng Ren, for his love and patience. He has always accompanied me and supported me since we met. Sorne of the greatest help came from my mother-in-law, Cuihua Lou, who visited us in the first term and had taken really good care ofmy daughter and my family.

Finally, please allow me to devote my deepest love to my father, Zhenzhong Xia. Although he passed away last year, 1 know he's blessing me forever.

III Table of Contents

Abstract- ,------,-1 Résumé ------, ------II Acknowledgements --- ,------III

Table of Contents ------,------~------rv List of Figures ------VII List of Charts ,------VII List of Tables------, ,------VIII List of Abbreviations ------x

Chapter 1 Introduction ...... 1 1.1 SeIected components of Siraitia grosvenom fruit ...... 1 1.1.1 Studies of S. grosvenorii ...... , ...... 1 1.1.2 Production ...... 2 1.1.3 Main components of interest: Cucurbitane-glycosides ...... 3 1.1.4 Other components ...... 6 1.2 Attributes of mogrosides ...... , ...... , ...... 10 1.2.1 Chemical and organoleptic properties ...... 10 1.2.2 Toxicity studies ...... 10 1.2.3 Characteristics of mogrosides ...... Il 1.2.3.1 Sweet property ...... Il 1.2.3.2 Health considerations ...... 12 1.2.4 Use and antioxidant potential ...... 13 1.3 Isolation ofmogrosides ...... 14 1.3.1 Solvent extraction...... 14 1.3.2 Column chromatography ...... 15 1.3.3 High Performance Liquid Chromatography (HPLC) ...... 16 1.4 Supercritical f1uid extraction ...... 17

IV 1.4.1 Principles of supercritical fluids ...... 17 1.4.2 Supercritical fluid extraction (SFE) ...... 19 1.4.3 Selected applications of SFE for natural products isolation ...... 22 1.5 Objectives ...... 24

Chapter 2 Isolation of mogroside V reference material...... 27 2.1 Introduction ...... 27 2.2 ,Materials and Methods ...... , ...... 29 2.2.1 Materials ...... 29 2.2.2 Isolation ofmogroside V reference material ...... 29 2.2.2.1 Preparation of resin columns ...... 31 2.2.2.2 Procedure for the removal of impurities in the crude S. grosvenorii extract by Amberlite XAD-2 resin ...... 32 2.2.2.3 Discoloration of the solution by aion exchange resin ...... 33 2.2.2.4 Purification of Mogroside V by preparative Thin Layer Chromatography (TLC) ...... 34 2.2.3 Identification of mogrosides ...... 34 2.2.3.1 Characterization by TLC ...... 34 2.2.3.2 Spectroscopie method ...... 34 2.2.3.3 MS and NMR spectra ...... 36 2.3 Results and discussion ...... 36 2.3.1 Isolation ofreference material by resin chromatography ...... 36 2.3.1.1 Standard curve ofmogroside V ...... 37 2.3.1.2 Optimal extraction conditions ofresin column chromatography .... 38 2.3.2 Characterization by Thin Layer Chromatography ...... 41 2.3.3 Mass spectrometry ...... 44 2.3.4 Nuclear Magnetic Resonance Spectroscopy (NMR) ...... 48

Chapter 3 Supercritical Fluid Extraction of Mogrosides from S. grovenorii ...... 52 3.1 Introduction ...... 52 3.2 Materials and methods ...... :...... 54

v 3.2.1 Materials ...... 54 3.2.2 Sub critical water extraction (SWE) ...... 55 3.2.2.1 Equipment ...... 55 3.2.2.2 General SWE extraction procedure ...... 57 3.2.3 Supercritical CO2 extraction ...... 59 3.2.3.1 Equipment ...... 59 3.2.3.2 General extraction procedure ...... 60 33 Results and discussion ...... ;...... 63 3.3.1 Sub critical water extraction (SWE) ...... 63 3.3.1.1 EfIect of extraction temperature in SWE ...... 63 3.3 .1.2 EfIect of packing material in SWE ...... 64 3.3.1.3 EfIect of extraction time in SWE ...... 65 3.3 .1.4 Effect of sub critical solvent ...... 67 3.3.1.5 EfIect of extraction pressure in SWE ...... 67 3.3.1.6 Optimized extraction conditions for SWE ofmogrosides from SG 68 3.3.2 Supercritical C02 extraction ...... 68 3.3 .2.1 EfIect of pressure on SCC02 extraction ...... 68 3.3 .2.2 EfIect of scC02 extraction temperature on mogrosides recovery .... 70 3.3.2.3 EfIect of scC02 extraction time on mogrosides recovery ...... 71 3.3.2.4 EfIect of packing material in SCC02 extraction ...... 72 3.3.2.5 EfIect of modifier in SCC02 extraction ...... 73 3.3.2.6 Optimized SCC02 extraction conditions ...... 75 3.3.3 Comparasion of Soxhlet extraction, sub critical water extraction and supercritical CO2 extraction of mogrosides ...... 75 3.4 Conclusion ...... 77

References ...... 78

VI List of Figures

Figure 1.1 Structures of sweet glycosides isolated from the fruit of S. grosvenorii ...... 5 Figure 1.2 Chemical structure of mogroside V ...... 6 Figure 1.3 Structures of flavones isolated from the fruits of S. grosvenorii ...... 7 Figure 1.4 Phase diagram for water and carbon dioxide ...... 18 Figure 1.5 Solvent Polarity ofwater/Organic Solvents ...... 22 Figure 2.1 Structure of Amberlite XAD-2 resin ...... 28 Figure 2.2 Standard curve of mogroside V generated relative to a control sample that did not contain any analyte ...... 38 Figure 2.3 Sorption capacity of Amberlite XAD-2 resin ...... :...... 39 Figure 2.4 Effect of eluting solvent on the elution ofmogroside ...... 39 Figure 2.5 The discolouring effect of selected anion exchange resins ...... 41 Figure 2.6 TLC separations ofvarious fractions ...... 43 Figure 2.7 ESI-MS spectra of fmal crystallized eluate in positive and negative mode . .46 Figure 3.1 Sub critical water extraction unit...... 56 Figure 3.2 Supercritical C02 extraction system ...... 62 Figure 3.3 Effect of temperature on the sub critical water extraction efficiency of mogrosides from SG ...... 64 Figure 3.4 Effect of extraction time on the recovery of mogrosides from SG with sub critical water ...... 66 Figure 3.5 Comparison of sub critical solvent ...... 67

List of Charts

Chart 2.1 Isolation Procedure of mogroside V reference material ...... 30

VII List of Tables Table 1.1 Mogrosides components in S. grosvenorii ...... 4 Table 1.2 The content of the amino acid hydrolysate ...... 8 Table 1.3 Protein, sugar and vitamin contents of the fruit of S. grosvenorii ...... 9 Table 1.4 Inorganic content (ppm) in the fruit of S. grosvenorii ...... 9 Table 1.5 Comparison ofphysical and transport properties of gases, liquids and SCFs.18 Table 1.6 Sorne commonly used solvents in supercritical fluid extraction processes ..... 20 Table 2.1 Effect of acidity on adsorption to the XAD resin ...... 40 Table 2.2 Proposed fragmentation pattern (upper pane) ...... 47 Table 2.3 Proposed fragmentation pattern (middle pane) ...... 47 Table 2.4 Proposed fragmentation pattern for ESI in the negative ion mode (lower pane) ...... 48 Table 2.5 Experimental l3C NMR chemical shifts for the aglycone moiety of the TLC isolate compared with published data for mogroside V and mogrol (0 / ppm) ...... 49 Table 3.1 Conditions for SWE runs during optimization ...... 58 Table 3.2 Effect of packing material on sub critical water extraction of mogrosides from SG ...... 65 Table 3.3 Effect of extraction time on the cumulative recovery of mogrosides for the extraction of SG with sub critical water...... 66 Table 3.4 Optimized Parameters for SWE of mogrosides from SG ...... 68 Table 3.5 Effect of pressure on the SCC02 extraction efficiency of mogrosides from SG69 Table 3.6 Effect of pressure on the SCC02 extraction efficiency of mogrosides from MG ...... 69 Table 3.7 Effect of scC02 extraction temperature on the recovery of mogrosides from SG ...... 70 Table 3.8 Effect of SCC02 extraction temperature on the recovery of mogrosides from MG ...... 70 Table 3.9 Effect of SCC02 extraction time on the recovery of mogrosides from SG ...... 71 Table 3.10 Effect of scC02 extraction time on the recovery of mogrosides from MG .72 Table 3.11 Effect of packing material on the SCC02 mediated recovery of mogrosides

VIII from SG ...... 73 Table 3.12 Effect of padang material on the scCOz mediated recovery of mogrosides fromMG ...... 73 Table 3.13 Effect of modifier addition for the scCOz mediated recovery of mogrosides fromMG ...... 74 Table 3.14 Established Parameters for scCOz extraction ofmogrosides from SG and MG ...... 75 Table 3.15 Conditions used to compare .soxhlet, SWE and scCOz extractions ...... 76

IX List of Abbreviations ESI electrospray ionization GC gas chromatography HPLC high performance liquid chromatography LC automated liquid chromatography MG crude S. grosvenorii extract after resin treatment which contained mainly mogrosides MS mass spectrometry MW. molecular weight m/z mass-to-charge ratio NMR nuclear magnetic resonance spectroscopy Tc critical temperature TLC thin layer chromatography

PARs Polycyclic aromatic ~ydrocarbons Pc critical pressure PCBs polychlorinated biphenyl compounds scC02 supercritical carbon dioxide SCF supercritical fluid SFE supercritical fluid extraction SG s. grosvenorii powdered plant concentrate SWE sub critical water extraction ss stainless steel TPEV temperature and pressure equilibration vessel pSI unit of pressure (pounds per square inch) cm centimeter hr hour Ld. inner diameter mg milligram mm minute mL milliliter MPa megaPascal

x wt% weight percent w/w weight per weight. oc degrees Celsius

XI Chapter 1 Introduction

Chapter 1

Introduction

1.1 Selected components of Siraitia grosvenom fruit

The fruit of S. grosvenorii SWINGLE (also known as Momordica grosvenori, Fructus momordicae, Luo Han Guo), a perennial vine grown in a restricted area of southem China, has been used as a natural sweet food as well as a medicinal herb for treating cough and sore throat (Kinghom and Soejarto, 1986). Research concerning the fruit of S. grosvenorii and its components has been reported since the 1980s. The extract from this fruit has been reported to be about 150 times sweeter than sucrose despite having a minimal caloric content (Lee, 1975). BeneficiaI characteristics of the extract to health have also been the object of several studies in recent years. As examples, mogrosides, the principle sweet components, have antioxidant properties and are valuable as cancer-chemopreventive agents (Konoshima and Takasaki, 2002).

1.1.1 Studies of S. grosvenorii

Initial research into the sweet component of S. grosvenorii has been attributed to C.H. Lee, who published an English report in 1975. He reported that an extract from S. grosvenorii with 50% ethanol-water, possessed an excellent sweetness that resulted from the presence of a triterpene glycoside, but he neither isolated nor determined the precise chemical structure of the active component(s) (Lee, 1975). Takemoto and colleagues performed several studies on the dry fruit of S. grosvenerii in Japan in the early 1980s (Takemoto et al., 1983b, 1983c). For example, they reported that S. grosvenorii contains appreciable quantities of fructose in 1983. They also described the tirst three major sweet cucurbitane-glycosides and called them mogroside IV, mogroside V and Chapter 1 Introduction 2 mogroside VI. A further four minor glycosides, siamenoside 1 (organoleptically sweet), ll-ixo-mogroside V (organoleptically sweet), and mogrosides lIE and IllE (both tasteless), had been isolated previously from this fruit by Kasai et al. in 1989 (Kasai et al., 1989). At approximately the same time, Matsumoto et al. reported the isolation and identification of another minor cucurbitane-glycoside, mogroside III (tasteless) (Matsumoto et al., 1990). Subsequently, Si et al. isolated a new cucurbitane-glycoside from the fresh fruits of S. grosvenorii which they named neomogroside (Si et al., 1996) .. Researchers have also examined the health characteristics of the extract as weIl as their components. By 2002, eighteen triterpenoid from the ethanol extract of the fruit had been reported and demonstrated to be potent anti-cancer agents (Ukiya et al., 2002). The structure of a latest mogroside from S. grosvenorii to he identified, Grosmomoside l, was determined by detailed spectral analysis (Yang et al., 2005).

1.1.2 Production

S. grosvenorii is a member of the family Cucurbitaceae. W.T. Swingle who named the plant Momordica grosvenorii, fust pub li shed the botanical description in 1941. After exarnining more plant specimens, Charles Jeffrey moved this species from the genus Momordica to Thladiantha in 1979 and to Siraitia in 1980 (Kinghorn and Compadre, 2001). The plant is found only rarely in the wild and has hence been cultivated for hundreds of years. Records as early as 1813 mention the cultivation of this plant in the Guangxi province of China (Wikipedia, 2006).

The most extensively cultivated areas of S. grosvenorii are in China. The plant may climb 2-5 meters as a cultivated dioecious vine with bifid tendrils and grows mainly in the mountainsides in Guangxi and Guangdong province of China. The region surrounding Guilin in Guangxi province by itself now has 1533 hectares of S. grosvenorii under active cultivation that produce over 9,800 fruits annually with a value Chapter 1 Introduction 3 of over 60 million RMB (Anonymous, 2006). The commercialization of S. grosvenorii fruits for food and medicinal purposes has been very rapid since its introduction to other countries (Anonymous, 1996). Thus, in 1987, a total of two metric tons of S. grosvenorii fruit containing mogroside V was utilized in Japan, representing a value of 40 million yen (Kinghorn and Compadre, 2001). Although millions of S. grosvenorii fruit are consumed fresh each year, oruy dried fruits or extract were exported to Southeast Asia, Japan, and North America. S. grosvenorii fruits in Europe and North America are available mostly in Chinese grocery and herb stores.

1.1.3 Main components of interest: Cucurbitane-glycosides

The sweet taste of S. grosvenorii primarily results from the content of mogrosides, a group of cucurbitane-type triterpene glucosides that are present at the leve1 of about 1 % of the flesh of the fruit (Kinghorn and Soejarto, 1986). To date, 12 glucosides have been isolated from this fruit and identified as summarized in Table 1.1. The non-sugar component (aglycone moiety) is mogrol, the glycosidic bonds are formed at C-3 and C-24 positions and each has a Pconfiguration. The structures of the main mogrosides in the fruit of S. grosvenerii are presented in Figurel.1 (Si et al., 1996). Chapter 1 Introduction 4

Table 1.1 Mogrosides components in S. grosvenorii

Component Formula

mogroside V ~601l102()29-2112()

mogrosides IV ~54fl92()24-1I2()

mogroside VI ~6611112()34

siamenoside 1 ~541192()24 -7/2112()

Il - ()X() - mogroside V ~6oll100()29-71211 2()

mogroside II E ~421182()19

mogroside III ~481182()19

Mogroside IllE ~481182()19

MogrosideA ~421192()14-3112()

neomogroside ~~ll2()34-5112()

mogroester ~441156()4

Grosmomoside 1 ~541192() 24 Chapter 1 Introduction 5

RI R2

Gle Gle

2 Gle GI~Gle

6-1 3 Gle (I)--Glc (fi)

~Gle(V) 6-1 4 Gle (I)--Gle (II) Gle(IV)~ 2-1 Gle (VI)

. 6-1 Gle (I)-.-Glr (U) ~Glc(V) s 2-1 Gle(IV)~ Gle (DI) 2-1 Gle (VI)

6 H H

1. mogroside lIE 2. mogroside IllE 3. mogroside III 4. mgroside IV 5. mogroside V 6. mogrolGlc: P-D-glucopyranosyl

Figure 1.1 Structures of sweet glycosides isolated from the fruit of S. grosvenorii (Si et al., 1996)

The principal sweet triterpene glycoside is called mogroside V. A previous investigation on commercially available samples of dried sweet S. grosvenorii fruit indicated that the content of mogroside V varies in the range of 0.81-1.29 % (w/w) and highest levels of Chapter 1 Introduction 6 mogroside V occurred in the endocarp of the fruits rather than in the peel or in the seeds (Makapugay et al., 1985). The structure of mogroside V (Figure 1.2) has been established by Takemoto and colleagues with chemical and spectroscopie evidence (Takemoto et al., 1983c). Carbon 13 (BC) and proton eH) NMR spectra demonstrated that the C-3 position of aglycone was connected to a diglucoside and the C-24 position was connected to a triglucoside (Si et al., 1996).

Figure 1.2 Chemical structure of mogroside V (Takemoto et al., 1983c)

1.1.4 Other components

Two types of flavones, Grosvenorine 1 and Grosvenorine II (Figure 1.3), were isolated from the fresh fruit and their structures were determined by means of UV, FAB-MS, IH)H COSy, BC)H COSY and NOE difference spectra (Si et al., 1994). The contents of the hydrolysis to liberate amino acids are presented in Table 1.2 (Moore, 1999). Chen performed research on the second chemical composition. He was able to isolate protein material, a low molecular weight sugar, selected vitamins and fatty acids as summarized in Table 1.3. Twenty six inorganic elements that were determined in the fruit, are listed Chapter 1 Introduction 7 in Table 1.4, sixteen of which are essential for humans (Meng et al., 1989). It should be noted that the content of selenium (Se) is 2 - 4 times greater than the levels in typical grains. It has been reported that Se possesses anti-cardiac, anti aging and anticancer properties.

OH o RI Grosvenorine (1) Rha Rha 2-1 Glc Il Rha Rha

Figure 1.3 Structures of flavones isolated from the fruits of S. grosvenorii (Si et al., 1994) Chapter 1 Introduction 8

Table 1.2 The content of the atnino acid hydrolysate (Moore, 1999) Chapter 1 Introduction 9

Table 1.3 Protein, sugar and vitamin contents of the fruit of S. grosvenorii

Content % of the total fresh weight

Protein 7.1 -7.8 % of the total dried fruit

Vitamin BI 3.38 % (mglg)

VitaminB2 1.23 % (mg/g)

VitaminC 3.39 - 4.87% (mglg)

Glucose 0.8%

Fructose 1.5%

Oil 0.8%

Table 1.4 Inorganic content (ppm) in the fruit of S. grosvenorii

Element Content Element Content Element Content

Mn 22.7 Na 16.5 Co 0.1

Fe 29.2 Cd 0.02 Se 0.2

Ni 1.8 Sr 1.7 Sn 0.2

Zn 12.8 Ba 3.3 As 0.1

Mg 550.0 Cr 0.5 1 1.0

Ca 667.5 Al 7.7 Si 645

Pb 0.07 Be 0.01 F 0.9

Cu 0.5 Ti 0.3 Mo 004

K 12290.8 V 0.2 Chapter 1 Introduction 10

1.2 Attributes of mogrosides

1.2.1 Chemical and organoleptic properties

In its pure fonn, mogroside V is a white, crystalline material with a melting point of 194-201 oC, an optical rotation of -16.3° in water, and an elemental composition of C6oH10202ge2H20 and a mok~cular weight of 1286 dalton (Takemoto et al., 1983c). Mogroside V is a polar compound containing five glucose residues and is readily soluble in either water, methanol, ethanol or pyridine but is insoluble in petroleum ether, diethyl ether or ethyl acetate (Kasai et al., 1989).

Mogrosides are relatively thennostable. It has been reported that mogrosides do not decompose under continuous heating at 120°C for 12 hr (Zhao, 1993). Mogroside V can be hydrolysed with mineral acid or enzymes such as maltase or cellulase to produce 'mogrol. Other mogrosides (IV, IllE and IlE) behave similarly (Kasai et al., 1989; Takemoto et al., 1983b).

1.2.2 Toxicity studies

S. grosvenorii fruit and extracts have been used for more than 300 years in China for both their sweet flavor and their medicinal properties. No account of adverse reactions due to the ingestion of these materials has appeared in the scientific literature and, on this basis, S. grosvenorii does not seem to present a potential toxic risk to humans.

Previous acute and subacute toxicity studies with S. grosvenorii extracts administered to rats have not demonstrated of any significant toxic effects (Takemoto et al., 1983a). Chapter 1 Introduction 11

Mortalities among mogroside-treated dogs (at doses up to 3 g/kg body weight for 4 weeks) did not differ significantly when compared with control animals (Su et al., 2005). An aqueous extract of S. grosvenorii fruit exhibited an LDso (lethal dose required to kill 50 % of a test population) to mice was greater than 10 g/kg (Kinghom and Soejarto, 1989; 1986; Su et al., 2005).

The compound has been observed to be non-mutagenic when tested in a forward mutation assay using Salmonella typhimurium strain TM677 (Kinghom and Soejarto, 1989; 1986). Recent studies have demonstrated that partially purified extract of S. grosvenorii fruits is not mutagenic, when evaluated with Salmonella typhimurium strains TA97, TA98, TAI 00, and TA 102, either in the presence or absence of metabolic activating systems (Su et al., 2005).

1.2.3 Characteristics of mogrosides

1.2.3.1 Sweet property

Usually the sweetness ofhigh-intensity natural sweetener in plants has been traced to the e1evated content (greater than 5 % w/w of sugars and polyols) (Hussain et al., 1990). The yield of saccharides and polyols in the S. grosvenorii dried fruit is only 2.4 % (w/w). It is assumed that the sweetness is not merely the result of the sugar content; other sweetening components must also be present. As the result of further extensive studies, it has been observed that the most important sweetening components were sapomn glycosides that incIuded mogroside V, mogroside VI, siamenoside l, and II-oxomogroside V. Chapter 1 Introduction 12

A 0.02 % aqueous solution of mogroside V was rated as being about 260 times sweeter than fructose by a human taste panel (Takemoto et al., 1983a), although this compound was characterized by an undesirable aftertaste (Kinghom and Soejarto, 1986). In another test, the relative sweetnesses of mogroside IV, mogroside V, siamenoside l, and II-oxomogroside V were determined to be 392,425, 563, and 84 times higher than a 5% aqueous solution of sucrose, respectively. By contrast, another triterpene glycoside, mogroside III (M-III), was tasteless (Kasai et al., 1989; Matsumoto et al., 1990). The mixed mogrosides have been estimated to be about 300 times as sweet as sugàr by weight, so that the 80% extracts are nearly 250 times sweeter than sugar (Kasaï et al., 1989). These compounds represent sorne of the sweetest plant glycosides known (Kinghom et al., 1998).

ln studies of structure-taste relationships of compounds and derivatives, the number of glucose units and the oxygen functionality at the Il-position of the aglycone moiety seemingly IS responsible for the perception of taste; glycosides of lla-hydroxy-compounds taste sweet, whereas glycosides of llP-hydroxy-compounds are tasteless and ll-keto-glycosides taste bitter (Kaiser et al., 1987; Kaiser et al., 1988). Glycosides of 11a-hydroxy-aglycone and five glucosyl units in mogroside V confer a sweet taste (Matsumoto et al., 1990).

1.2.3.2 Bealth considerations

The health properties of S. grosvenorii have been recorded in all editions of Chinese Pharmacopoeia after 1977 (Zhou, 2003). Recent investigations have demonstrated that mogrosides can produce a significant reversal or suppression of the early stages of cancer development. Mogrosides have value as cancer chemopreventive agents (Kinghom et al., 1998; Konoshima and Takasaki, 2002; Takasaki et al., 2003). Ukiya and collegues reported that 18 mogrosides possess potent inhibitory effects on the Chapter 1 Introduction 13 induction of Epstein-Barr virus early antigen (EBV-EA) induced by tumor promoter ID Raji cells, a primary screening test for antitumor promoters (Ukiya et al., 2002). Additionally, mogroside V and II-oxo-mogroside V exhibited significant inhibitory effects on the two-stage carcinogenesis test of mouse skin tumors using 12-dimethylbenz[a]anthracene (DMBA) or peroxynitrite (ONOO) as an initiator and 12-o-tetradecanoylphorbol-13-acetate(TPA) as a promoter (Takasaki et al., 2003).

The crude extract from S. grosvenorii possesses anti-hyperglycemic effects in rats and suggests that its sweetening component can represent a remedy for diabetes. It is not only because it can substitute for caloric sugars normally consumed in the diet but also because sweet components can inhibit both oxidative modification of low-density lipoprotein (LDL) and the elevation of postprandial blood glucose levels after a single oral administration of maltose in rats (Suzuki et al., 2005; Takeo et al., 2002). LDL plays an important role in the initiation and progression of atherosclerosis in patients with diabetes mellitus.

Mogrosides have also been found to possess sorne microbial inhibiting activity (Zhou, 2000). Moreover, even small concentrations of the triterpene glycoside (0.7 mg/mL) combined with 0.1 mg/ml licorice extract, can inhibit oral bacteria which are known to causes of oral malodor, gum disease and tooth decay (Zhou and James, 2000).

1.2.4 Use and antioxidant potential

For centuries, the fruit of S. grosvenorii have heen employed 'in foods and beverages in south China. In 1987, S. grosvenorii was listed among the fust group of items that can be used as a dietary supplement by the Administration of Chinese Medicine of the Ministry of Hygiene in China. To date, mogrosides can he used as a food additive in Chapter 1 Introduction 14

China, Japan, South Korea, Thailand, Singapore, and UK (Zhou, 2003). Generally, mogrosides are not utilized as sweeteners in pure form. Rather, they have been used in Japan predominantly as S. grosvenorii extracts with varying degrees of purity (Anonymous, 1985; Kim and Kinghom, 2002).

The fruit has been used for centuries in Traditional Chinese Medicine as a remedy for colds, coughs, sore throats, diabetes and blood pressure control (Ichikawa et al., 1997; Lee, 1975; Tsunematsu et al., 1978); historically, it has been known as the "longevity fruit". It has been suggested that mogrosides could be used as cancer-chemopreventive agents because of the research reports on the medicinal application of mogrosides that inc1ude antioxidant activity against free radicals and lipid peroxidation (Shi et al., 1996).

1.3 Isolation of mogrosides

A number of laboratory extraction, purification and determination procedures, which were developed originally for fresh or dried fruits of S. grosvenorii or their active component mogrosides, have been reported in the two decades that followed the mid 1980s. The majority of these procedures for the purification of mogrosides have involve initial extraction into an aqueous solvent, followed by purification involving one or more of the following: selective extraction into a polar organic solvent, decolourization, precipitation, coagulation, adsorption, ion exchange, and crystallization (Zhang and Qi, 2005; Zhou, 2003). Much ofthis type of information has appeared in the patent literature (Tsunematsu et al., 1978; Zhou, 2000; Zhou and He, 2000; Zhou and James, 2000).

1.3.1 Solvent extraction

1 Traditional extraction of mogrosides from fresh or dried fruits was usually performed Chapter 1 Introduction 15 with water or an organic solvent such as methanol, ethanol, or pyridine. These laboratory extraction processes have proved to be rather complicated, provided low recoveries and have been applied, most frequently, as a first step prior to further purification by chromatography. More recent research has demonstrated that either ultrasonic energy or microwave energy can improve extraction yield (Li et al., 2003; Li et al., 2004).

1.3.2 Column chromatography

Most extractions procedures have been completed by filtration using a macroporous synthetic pol ymer resin such as Diaion HP-20 (Mitsubishi Chemical Industries, Ltd., Tokyo, Japan) or AB-8 (The Chemical Plant of Nankai University, Tianjin, China) combined with column chromatography over silica gel or alumina.

Researchers have evaluated changes in the polarity of the mobile phase and have restricted their studies to only one or two stationary phases for the purification of mogrosides. For example, a United States Patent quoted by Zhou and colleagues, described a purification process with a macroporous resin that was eluted with an alcohol (Zhou and He, 2000). Another separation procedure used Diaion HP-20 e1uted, in succession, with H20, 50 % MeOH, 80 % MeOH, MeOH and (CH3)2CO. The fractions that were e1uted with 50 % and 80 % methanol were separated further by chromatography on silica gel with CHCh-MeOH-H20 of different polarities to result in the separation of seven glycosides. Glycoside G was identified as mogroside V (Matsumoto et al., 1990).

The component mogrosides all contain varying proportions of sugar groups that are bound to the same aglycone. The structural similarities of these compounds make them very difficult to separate. Therefore, it has been necessary to use three or more columns Chapter 1 Introduction 16 to isolate individual mogroside. For example, one process to separate mogroside V, mogroside IV and mogroside VI from S. grosvenorii was patented in 1978 by Takemoto (Tsunematsu et al., 1978). He used activated carbon, an activated alumina column and then Celite 535 to separate aIl three mogroside. Chromatography over silica gel was then repeated to isolate the desired components.

Further, Makapugay and coIleagues have reported a detailed analytical method for the isolation/purification of mogroside V (Makapugay et al., 1985). A silica gel column was eluted successively with mixtures of CHCh-MeOH-H20 of increasing polarity. The purity of the isolate was increased relative to other methods; however, toxic organic solvents inc1uding chloroform (which has been listed as a carcinogen) were used extensively in this procedure.

In a study in which mogroside III, IV and V were recovered from fresh fruit of S. grosvenorii, Si reported the application of sequential stages involving a high porosity polymer resin, silica gel, an RP2 reverse-phase column and a Sephadex LH20 column (Si et al., 1996).

1.3.3 High Performance Liquid Chromatography (HPLC)

Reversed phase HPLC methods have been used extensively after pre-column treatment by open column chromatography. Mogroisde V are first separated with a 25 cm (4.6 mm i.d) Zorbax NH2 column, eluted with acetonitrile-water (3:1, adjusted to pH 5 with H3P04) at the flow rate 2 mL/min (pressure 8276 KPa and ambient temperature) (Makapugay et al., 1985). Five triterpene glycosides were isolated by HPLC separation on a Pegasil ODS II column at 25°C, eluted with MeOH-H20 (Ukiya et al., 2002). Yu recovered mogroside V from fresh fruits of S. grosvenorii by semi-preparative HPLC Chapter 1 Introduction 17 after pretreatment by an AB-8 polymer adsorption column and a D-280 ion exchange column (Yu et al., 2003). She employed an Econosphere NH2 column (Alltech Associates) at 40°C eluted with acetonitrile -water (2:1, v/v) as mobile phase at a flow rate of 5 mL/min. Detection was by ultra violet (UV) at 203 nm. The purity of the product was reported to be greater than 98 %.

1.4 Supercritical f1uid extraction

Supercritical fluid extraction is a relatively newer method based on the unique physical properties of a substance in the supercritical state that favour the extraction of selected components within a matrix. The supercritical state is achieved when temperature and pressure exceed a substance's critical point. In this phase, the substance retains much of the solvating power of a liquid phase while retaining much of the lower viscosity of the gaseous phase. The supercritical fluid can readily penetrate a range of porous sample matrices, enabling it to possess a high solvating power. Supercritical fluid extraction processes offer an environmentally benign alternative to other extraction methods, which commonly use relatively large volumes of noxious organic solvents. Supercritical carbon dioxide has been described as a "green" solvent for its usefulness in replacing traditional solvents in certain chemistry-related applications (Hauthal, 2001).

1.4.1 Principles of supercritical f1uids

The phase diagram for carbon dioxide or water is presented in Figure 1.4 (Steel, 2006). Four main phases exist, the solid, liquid, gas, and supercritical; they are all governed by the conditions of temperature and pressure. A fluid within the shaded region of Figure 1.4 is referred to as a supercritical fluid and is neither a true liquid nor a true gas. In this state, the fluid possesses a gas-like viscosity. A comparison of typical values of density, viscosity and diffusivity of gases, liquids and supercritical fluids is presented in Table 1.5 (Kang, 2006). Chapter 1 Introduction 18

Figure 1.4 Phase diagram for water and carbon dioxide (Steel, 2006)

Table 1.5 Comparison of physical and transport properties of gases, liquids and SCFs (Kang, 2006)

Property Density (kg/ml) Viscosity (cP)

Gas 1 0.01 1-10

SCF 100-800 0.05-0.1 0.01-0.1

Liquid 1000 0.5-1.0 0.001

The density of a SCF is 0.1 - 0.8 times the values that are typical for the liquid phase, the viscosity is 10 - 100 times lower than for the liquid phase whereas the diffusivity is 10- Chapter 1 Introduction 19

100 times higher than for the liquid phase. These parameters result in greater selectivity, a more rapid mass transfer, and a higher flow rate of seF when compared to the liquid phase. As a result, their use as solvents can make SFE a much more efficient separation technique when compared to conventional extraction.

The properties (especially solvating power) of SF's can be finely turned by quite small changes in temperature and/or pressure (especially in the region of the critical point). By controlling or regulating pressure and temperature, the density, or solvent strength, of supercritical fluids can be changed to approximate organic solvents ranging from chloroform to methylene chloride to hexane (McHugh and Kruk:onis, 1994). This dissolving power can be applied to purify, extract, fractionate, infuse, and recrystallize a wide array of materials.

1.4.2 Supercritical fluid extraction (SFE)

The strong solvent capabilities of supercritical fluids was first reported more than a century ago (Hogarth and Hannay, 1879), but the rapid development of SFE technology occurred in the era after 1969 (Gere et al., 1997). Supercritical fluid technology has now found many analytical and industrial-scale applications in various fields that include the production of polymers, aromatics and essential oils, fats, natural products, the decontamination of soil, as weIl as in the agricultural and pharmaceutical industries (Bowadt and Hawthorne, 1995; Anklam et al., 1998; Kaiser et al., 2001; Lang and Wai, 2001; Radcliffe et al., 2000; Rudzinski and Aminabhavi, 2000).

The extraction process can be considered to occur in four steps: penetration of the supercritical fluid into the porous sample matrix, release of solutes within the matrix, diffusion of solutes out of the matrix, and final removal of the solutes from the sample. Chapter 1 Introduction 20

Subsequently, the solutes can be recovered from the supercritical solvent when it is brought back to ambient conditions. The supercritical solvent is returned to a gas (if that is its normal state at ambient conditions) and either released as waste, or recyded for further use (King, 1995; Turner et al., 2001).

A wide range of supercritical fluids have been used for many SFE applications; the critical properties of certain commonly used supercritical fluids are listed in Table 1.6 (Rozzi and Singh, 2002).

Table 1.6 Sorne commonly used solvents in supercritical fluid extraction processes

Fluid Critical Temperature Tc: (K) Critical Pressure Pc: (bar)

Carbon dioxide 304.1 73.8

Ethanol 514.15 61.8

Ethane 305.4 48.8

Ethylene 282.4 50.4

Propane 369.8 42.5

Propylene 364.9 46.0

Ammonia 405.5 113.5

Water 647.3 221.2

Cyc10hexane 553.5 40.7

Toluene 591.8 41.0 Chapter 1 Introduction 21

Of these, carbon dioxide has been the solvent of choice for more than 90 % of aH analytical SFEs (Bowadt and Hawthorne, 1995). Apart from its low critical temperature and pressure parameters (31.1 oC, 73.8 bar), carbon dioxide is available in high purity at relatively low cost; it is readily separated from the extract, is non-toxic, chemically inert, and non- flammable. It can be used with or without the addition of a polar modifier (such as alcohols or other organic chemicals) to obtain a desired polarity, enhance extraction efficiency, and to manipulate the range of solvated materials in the supercritical fluid (Boselli and Caboni., 2000·; Rudzinski and Aminabhavi, 2000; King, 1995; Zougagh et al., 2004). Without modification by polar cosolvents, carbon dioxide can only extract non-polar materials or certain polyaromatic hydrocarbons of low polarity (PARs), aldehydes, esters, and low molecular weight alcohols.

Water has been another choice as an extraction fluid for analytical SFE in recent years.

Water has desirable properties similar to CO2 such as a lack of toxicity and expense. Although ambient water is too polar to dissolve many organic species, water at elevated temperature and pressure behaves like a polar organic solvent. Thus, sub-critical water has been demonstrated to be an effective extraction fluid for several classes of organic compounds. Water extractions often use sub-critical water as extraction solvent at temperatures between its boiling point (lOO°C) and its critical temperature (374.15°C) and under sufficient pressure to maintain water in the liquid phase (Li et al., 2000; McGowin et al., 2001). Extractions can be effective using water without the need to go to the supercritical state.

The dielectric constant is the key parameter in controlling the analyte solubility in water (Hawthorne and Kubatova, 2002). Under sub-critical conditions, the dielectric constant

of water is greatly reduced from that at ambient conditions (E = 80), thereby decreasing its polarity. The curve in Figure 1.5 summarizes the effect of temperature and pressure on solvent polarity (as measured by dielectric constant) of water compared to that of Chapter 1 Introduction 22 some representative organic compounds. For example, by raising the temperature to 300°C at a pressure that can maintain the liquid state (3448 KPa), the dielectric constant

(e) of water drops to be approximately 20, making it similar to ethanol (e = 24) and methanol (e = 33) (Hawthorne and Kubatova, 2002). This change results in an increase in its solvating power for organic compounds. By changing the temperature, the solvating properties of water can be more finely tuned to match the polarity of the target analytes without the necessity to mix solvents of different polarities.

100 r------~--~------~ 90

1$ 60 ~ d -1:! so ] ~ 40 _.. metbanol 30 -o-akrot.oluene -ethallol 20 -butaao- benzIildebyde -meth)lamlne 10 phenol ::t-PCBs ~ne ·~,PAu. 0 25 50 75 100 125 150· 175· 200 225250'27$ 300 325 350 375 400 450500 SSO ... Température ( C) ...... 100 bar:~ 200 bar ...... 300 bar -e- 400 bar

Figure 1.5 Solvent Polarity of water/Organic Solvents (Hawthorne and Kubatova, 2002)

1.4.3 Selected applications of SFE for natural products isolation

With the increasing interest in herbaI and naturai products as pharmaceuticaIs, food additives or as natural pesticides, a variety of active compounds from herbs and other

plants have been processed with supercritical fluids. Numerous SF CO2-based methods have been developed for the isolation of natural products, including the extraction of lipid soluble vitamins (A, D, E, and K), various seed, nut, bean, and wood oils, essential Chapter 1 Introduction 23 oils, meat fats, phospholipids, pesticides, cholesterol, and pharmaceutical components (Kaiser et al., 2001; Lang and Wai, 2001; Modey et al., 1996).

Successful applications of sub-critical water to extract natural products have been reported for essential oils and other bioactive compounds from plants (Femândez-Pérez et al., 2000; Ibanez et al., 2003; Jimenez-Carmona et al., 1999; Ju and Howard, 2005; Kubatova et al., 2001; Ozel et al., 2003; Rovio et al., 1999; Shotipruk et al., 2004; Soto Ayala and Luque de Castro, 2001).

In SFE, the solvating power of the fluid can be modified readily by changing pressure and/or temperature; therefore, it may achieve a remarkably high selectivity, which is particularly useful for the extraction of complex mixtures from plant materials (Ellington et al., 2003; Reverchon et al., 1993). One good example is the selective extraction of a vindoline component from among more than 100 alkaloid compounds from the leaves of Catharanthus rose us (Lang and Wai, 2001).

SFE, especially with CO2 that has mild critical parameters, provides conditions of stability for thermally labile materials because extraction at low temperatures reduces the risk of analyte degradation during processing. As an example, if supercritical C02 was used to extract Australian-grown ginger, many undesirable reactions including hydrolysis, oxidation, degradation and rearrangement were effectively prevented (Bartley, 1994).

For many of these methods, the use of SFE was preferred to conventional extraction techniques because of the comparatively rapid reaction kinetics and reduced use of Chapter 1 Introduction 24 environmentally hostile organic solvent(s). The extraction of polychlorinated biphenyl (PCB) compounds from sediment and mussel tissue standard reference materials are examples. The efficiencies of extraction were comparable but SFE reduced extraction time (50 min compared to 18-24 hr) and organic solvent usage (7.5 ml as compared to 250 ml) (Zougagh et al., 2004)

One of the attractive features of supercritical fluid extraction is that it is relatively simple to couple the extraction technique directly with chromatographie techniques, which reduces potential loss of extract and eliminates sample pre-treatment. Additional advantages inc1ude a short sample extraction time and high sensitivity, as 100 % of the extract is transferred into the chromatographie system. SFE has been coupled successfully to thin layer chromatography (TLC), automated liquid chromatography (LC), gas chromatography (GC), packed and capillary column SFC (Radcliffe et al., 2000). Subcritical water can be used as an e1uent in reversed-phase high-performance liquid chromatography as an alternative to methanol-water or acetonitrile-water mixtures (Smith and Burgess, 1997).

In addition, SFE can be applied to systems of different scales. For instance, extraction can be scaled up from an analytical scale (less than a gram to a few grams of samples) to a preparative scale (several hundred grams of samples), to a pilot plant scale (many kilograms of samples) and up to large industrial scale (tons of raw materials, such as SFE of coffee beans) (Lang and Wai, 2001).

1.5 Objectives

People have realized that the consumption of sucrose as a sweetener can entail several nutritional and possible medical complications inc1uding dental caries, obesity and high

blood pressure. New alternative "low calorie" sweetener~ for dietetic and diabetic applications have been in increased demand worldwide. For certain synthetic sweetners Chapter 1 Introduction 25 such as duicin, saccharine and sodium cyclamate, toxicity has been evaluated and has resulted in limitations or bans in certain countries (Kim and Kinghom, 2002)

Comparable sweetening products from natural sources possess the advantage of the toxicological safety. As of mid-2002, over 100 plant-derived sweet compounds of 20 major structural types had been isolated from more than 25 different families of green plants (Kim and Kinghom, 2002). Nevertheless, orny a few of the se compounds (including the mogroside group) have been exploited commercially. For example, a major corporation in the United States has a patent on the use of extracts of S. grosvenori to enhance the taste of sweet juices (Fischer et al., 1994).

As mentioned previously, mogroside V has been demonstrated to be non-mutagenic. It may fmd application for diabetes victims or as a cancer chemopreventive agent. These attributes, coupled with the high sweetness compared with other natural products, could serve to stimulate further study of extracts of the fruit of this plant and its wide market prospect.

Although laboratory extraction, purification and determination processes for fresh or dried fruit of S. grosvenorii and mogrosides have been reported since the 1980s, many isolation processes used large quantities of toxic solvent, that resulted in the possibility of toxic residue of organic solvent in this high value food. Supercritical fluids offer opportunities in this area to replace and lor elimination of hazardous solvents due to the ease of solvent removal, recyclability and suitability of solvent parameters. Carbon dioxide and water fulfil the requirements of non-toxicity, ease of use and are inexpensive. The extraction of mogrosides in sub-critical water or supercritical C02 was anticipated to satisfy these requirements. Chapter 1 Introduction 26

The overall objective of this research was to identify a "green" extraction method t9 extract mogrosides from a dried powdered S. grosvenorii. The specifie objectives of this studywere: a. ro purify and characterize an isolate ofmogroside V to act as a reference material. b. To investigate and optimize extraction conditions for the mobilization ofmogroside V from the powdered plant concentrate using sub-critical water.

c. To investigate and optimize the process variables for the supercritical CO2 mobilization of mogroside V from the powdered plant concentrate. d. ro formulate conclusions regarding the relative merits of the two proposed extraction methods Chapter 2 Isolation of mogroside V reference material 27

Chapter2

Isolation of mogroside V reference material

2.1 Introduction

At present, no source of mogroside V reference material is available commercially. An objective of this research was to isolate mogroside V of sufficient purity that it could he crystallized. This objective was achieved by selecting suitable eluates from column chromatography coupled with preparative thin layer chromatography (TLC) and favorable conditions for isolation were identified.

A suitable c1ass of stationary phase for column chromatography is the macroporous resin. These resins, which are composed of durable nonpolar and slightly hydrophilic polymers, are capable of efficient adsorption with the possible recovery of the adsorbed molecules, due to their porous polymeric matrix (Weber and van Vliet, 1981). Compared with conventional organic solvent extraction resins have the advantages of simplicity, physical and chemical stability, speed, relative1y modest cost, favorable environmental impact, and facile regeneration in the presence of normal organic solvents.

Macroporous resins have been used extensively for water treatment. It has been reported that more than 25 organic species can be removed efficiently from waste water by macroporous resins, including ketones, alcohols, benzenes, phenols, aniline, indenes, alkyl benzothiophenes and alkyl naphthalenes (Kunin, 1976). They have also been used extensive1y in natural product extractions from citrus and grapefruit (Ericson, 1990; Di et al, 1999; Wang et al, 2005). Chapter 2 Isolation of mogroside V reference material 28

Macroporous resins have been applied successfully for the separation of glycosides from plant materials. A process for extracting triterpene glycosides from botanical sources has been patented by Zhou and colleagues (Zhou et al., 2000). Other successes have included extractions of glycosides from Stevia rebaudiana and Ginseng (Bian et al, 1986; Cai et al, 2001). Mogroside separations have also succeeded with macroporous resin such as AB-8 resin, DIOl resin and Diaion HP-20 from fresh or dried S. grosvenorrii fruit (Matsumoto et al., 1990; Li et al., 1995; Zhang and Qi, 2005; Qi et al, 2005).

Mindful of these previous studies, it was decided to evaluate the removal of impurities from crude isolates by means of certain resins that are available commercially in North America. The resin of choice was Amberlite XAD-2 resin. This non polar resin is a hydrophobie polyaromatic polymer manufactured by the Rohm and Haas Company. The main structure, a polystyrene-divinylbenzene, is illustrated in Figure 2.1. This resin has been used extensively to remove hydrophobie compounds.

--~C-CH---~C-CH ---H~-CH--

--~-CH ---H2C-CH---H~-CH--

Figure 2.1 Structure of Amberlite XAD-2 resin (Kunin, 1976) Chapter 2 Isolation of mogroside V reference material 29

2.2 Materials and Methods

2.2.1 Materials

This study was based on the use of a powdered plant concentrate of S. grosvenorii, made available by the China Natural Products Group, Inc. (V.S.A).

Adsorbent resin, Amberlite XAD-2 and strongly basic anion exchange resin IRA 410, IRA 400, Dowex 1 and Cellex D were obtained from Sigma-Aldrich Co., Oakville, ON, Canada. Perchloric acid (70 %, w/v), acetic acid (glacial) and ethanol were purchased from Fisher Scientific, Ottawa, ON, Canada. Vanillin (purity, 99 %) and I-butanol were purchased from Sigma-Aldrich Co., Oakville, ON, Canada. Para-anisaldehyde (purity, 98 %) was obtained from Alfa-Aesar, Ward Hill, MA, USA. Analytical TLC plastic sheets (Silica gel 60 F-254) and preparative TLC glass plates (Vniplates Silica Gel GF 20 x 20 cm) were purchased from Analtech Inc., Newark, DE, USA. AIl reagents were ACS reagent grade or better.

2.2.2 Isolation of mogroside V reference material

This study optimized the use of the macroporous resin, Amberlite XAD-2 to obtain preparative quantities of mogrosides and selected an ion exchange material from among four resins to discolour crude aqueous isolates. Additional purification of mogroside V was accompli shed with preparative thin layer chromatography (TLC). The detailed procedure is swnmarized in Chart 2.1. Chapter 2 Isolation of mogroside V reference material 30

S. grosvenorii aqueous concentrate

Amberlite XAD-2 resin column

1 Adsorbate 1 Aqueous Eluate Elution with EtOH-H20

1 Eluate

Ion exchange resin column

Aqueous. Fraction 1

Preparative TLC

~ Second band 1

MeOH filtration

1 Crystalization 1

Chart 2.1 Isolation Procedure of mogroside V reference material Chapter 2 Isolation of mogroside V reference material 31

2.2.2.1 Preparation of resin columns

Methods of resin adsorption for the isolation of mogroside are simple and rapid for large volumes. However, a disadvantage of these methods is the possible incorporation of impurities into the sample from the stationary phase; therefore, it is paramount that the following rinsing procedure be applied prior to adding the crude sample to the column.

The resin was washed with water then, sequentially during 24 hr, with methanol, diethyl ether, acetonitrile, and ethanol and stored in ethanol until use. Before column packing, ethanol was rinsed from the resin with distilled water until the complete elimination of excess reagent occurred. To test this point, the column eluate had to remain c1ear when it was mixed with the equal volume ofwater.

To activate the strongly basic ion-exchange resin, Amberlite IRA-410, IRA 400, Dowex 1 or Cellex D, were equilibrated with dilute lM NaOH then eluted with water until the pH was decreased to approximately 7. The colour of resin changed from light yellow to brown after pretreatment.

A slurry of pretreated resin was added to a glass column (30 x 2 cm i.d.) that had been fitted with a filter and nylon end plug to a final packed length of about 15 cm. The resin packing was added while eluting excess water from the base but always maintaining sufficient water in the column to coyer the resin. This procedure minimized the presence air bubbles or channels within the resulting resin bed.

These resins could be regenerated. If adsorption proved to be inefficient, the column was treated, in succession, with dilute NaOH (lM) followed by water until the pH of the Chapter 2 Isolation of mogroside V reference material 32 eluate had decreased to about 7 and then dilute HCI (lM) followed by water until the eluate became neutral again.

2.2.2.2 Procedure for the removal of impurities in the crude S. grosvenorii extract by Amberlite XAD-2 resin

Powdered plant concentrate of S. grosvenorii was dissolved in water (8 mg/mL, pH iO) then added to the head of a previously washed column of Amberlite XAD-2 resin and eluted at ~2 mL! min. Elution with water was continued until the eluate was no longer coloured. The adsorbed mogrosides were displaced from the column by elution with 5 resin bed volumes of 50 % ethanol. The combined aqueous ethanol extract was then evaporated under reduced pressure at 45°C to obtain a brownish residue. In preliminary trials ofmogroside recovery, the sorption capacity of Amberlite XAD-2 resin, the choice of the solvent and acidity were examined.

Sorption capacity

If the capacity of the resin was exceeded, then no more mogrosides would be adsorbed and the concentration of the liquid phase eluate would remain stable. Following this procedure, 47 mL of aqueous crude extract (equal to one time of resin bed volume) was added to the column and the concentration of mogroside in the eluate was estimated spectrophotometrically by comparison with standards using the vanillin-perchloric acid method. Equal quantities of sample solution was loaded successively until the resin had become saturated, which was signaled when the concentration in the eluate no longer increased.

Effeet of eluting solvent mixture Chapter 2 Isolation of mogroside V reference material 33

With a view to economic and toxicological factors, it was proposed to limit the solvents to water and ethanol. In order to test the effect of various concentrations of ethanol in the eluting solvent mixture, aqueous crude extract that had been adsorbed to the head of the column, was eluted with 20 %, 50 %, 70 % or 90 % ethanol (v/v). The evaluation of mogroside in the eluate was performed with the vanillin-perchloric acid method.

Effect of acidity on adsorption

The same concentration and volume of sample solution, before and after passage through the resin, was studied in the subsequent experiments. The pH of the eluting solvent was varied by adding either IN HCI or IN NaOH.

2.2.2.3 Discoloration of the solution by aion exchange resin

Previous studies had reported that a strongly basic anion exchange resin could remove pigments after an initial clean-up step. The resins tested in this study were Amberlite IRA-400, Amberlite IRA-4l0, Cellex D and Dowex-l.

The residue, after Amberlite XAD-2 treatment, was diluted to 1000 ml. Four aliquots, (30mL) of the residue solution were added to separate 4 g aliquots of resin. Spectrophotometric analyses were completed each 30 min at a wavelength (Â.) of 590 nm. After choosing the most suitable anion exchange resin, the solution was added to the head of a column packed with this resin and eluted with water. The solvent was subsequently concentrated to dryness in a fume hood. Chapter 2 Isolation of mogroside V reference material 34

2.2.2.4 Purification of Mogroside V by preparative Thin Layer Chromatography (TLC)

Mogrosides were purified further by preparative TLC on glass plates (20cmx20cm) with 1-butanol-acetic acid-water (4:1:1) as eluting solvent. The colourless band with Rf 0.23 was recovered from the plate and recrystallized 3 times from methanol (5m1).

2.2.3 Identification of mogrosides

Materials were identified by TLC and spectrosphotocopic methods. The rmal crysta1lized eluate was characterized further by mass spectrometry (MS) and nuc1ear magnetic resonance spectroscopy (NMR).

2.2.3.1 Characterization by TLC

Extracts for TLC analysis were concentrated to dryness, dissolved in 10J,lL methanol, and analyzed by TLC on silica gel. Purity of the isolate was indicated by its appearance as a single spot after chromatography, using 1-butanol:acetic acid:water (4:1:1) (Takemoto et al, 1983c). Visualization was achieved by spraying the developed TLC plate with p-anisaldehyde:sulfuric acid:acetic acid (1 : 1:48) followed by heating at 130°C for 5 min (Papadopoulou et al, 1999) or with 30 % v/v sulfuric acid followed by heating at 110°C for 10 min (Makapugay et al, 1985).

2.2.3.2 Spectroscopic method

The deterinination of mogrosides can be carried out with a spectrophotometric method. Mogrosides were observed to develop an intense blue colour at 590 nm in the presence of an oxidant, although the mechanism of colour formation remained unc1ear. Two Chapter 2 Isolation of mogroside V reference material 35 methods based on vanillin colour development, the vanillin-perchloric acid method and the vanillin- sulfuric acid method, have been reported in the literature (Li et al, 1993; Li et al, 2003). Both methods provide a linear relationship between the mogrosides concentration and the adsorbance. However, it was observed that a control sample was more intensely coloured in the vanillin-H2S04 procedure than in the vanillin-HCI04 method, which might cause increased measurementerrors. The vaniHin-HCI04 method was applied to aH subsequent experiments.

Establishment of a standard curve

Approximately 30 mg of mogroside V reference material was weighed accurate1y, dissolved in water and transferred quantitative1y to a 5 mL volumetric flask. The solution was diluted to the mark and mixed thoroughly. Aliquots, 20, 30, 40, 50, 60, or 70 ilL were transferred to separate 5 mL test tubes each fitted with a screw cap. After the solution had been evaporated to dryness, the residue was combined with 0.2 mL of freshly prepared vanillin - glacial acetic acid (5 % w/v) solution and 0.8 mL of perchloric acid. After thorough mixing, the solution was heated at 60°C for 15 min then cooled immediately in ice bath. Glacial acetic acid, 5 mL, was added and the absorbance of the solution at 590 nm was recorded after 10 min of reaction. Control solution that contained no analyte was treated analogously. With solvent as the blank, a curve of mogrocide V concentration vs. absorbance was generated using a Model Ultrospec-100 spectrophotometer (Biochrom Ltd, Cambridge, England). The solution was characterized by a maximum adsorbances at 590 nm. AH tests were completed within one hour.

Measurement of mogrosides

A 100 ilL aliquot of unknown sample was added to a 10 mL test tube fitted with a screw cap. The content of mogrosides was estimated foHowing the procedure outlined above Chapter 2 Isolation of mogroside V reference material 36 for the development of a standard curve. The curve was then used to estimate the content of mogrosides in the extract.

The influence of extraction parameters was determined by comparing recoveries which in turn, was defined as the ratio of mogrosides content in the extract relative to the content in the original crude powder.

2.2.3.3 MS and NMR spectra

The characterization of the isolate as mogroside V was corroborated further by mass spectrometry (MS) and nuc1ear magnetic resonance spectroscopy (NMR). The experimental spectra were compared with data reported in the literature (Takemoto et al., 1983c; Si et al., 1996; Kasaï et al, 1989; Chang et al, 1994). Electrospray ionization mass spectrometry (ESI-MS) spectra were recorded by the mass spectrometry laboratory of the Chemistry Department of McGill University. Mass spectra were recorded in the positive and negative ion modes in the mass range 200-2000 m/z (mass-to-charge ratio). l3C NMR experiments were condcted by the spectrometry laboratory of the Chemistry

Department of McGill University. Chemical shifts were given in ~ relative to TMS as an internaI standard.

2.3 Results and discussion

2.3.1 Isolation of reference material by resin chromatography

Macroporous resins are highly porous structures with internal surfaces that can adsorb and then desorb a wide variety of difIerent species depending on the environment in Chapter 2 Isolation of mogroside V reference material 37 which they are used. According to the literature, such a resin can reject most of small molecular weight impurities and part of the pigments from a crude plant extract. AB-8 resin has been exploited frequently to recover mogrosides by several Chine se researchers and has properties that are similar to Amberlite XAD-2 resin. In consequence, Amberlite XAD-2 resin was evaluated to determine optimal conditions for the isolation of these compounds.

An aqueous extract containing mogrosides was added to the head of an Amberlite XAD-2 resin column and adsorbed by the resin. Polar impurities were washed from the column by eluting with water. At the same time, most of coloured pigments were washed from the XAD resin as weIl. The resin was then washed with an alcohol-water solvent mixture to recover a fraction that contained mogrosides. Subsequently, an ion exchange resin was to be used to remove other coloured impurities.

2.3.1.1 Standard curve of mogroside V

A coloured addition product formed by reaction of mogrosides with o-vanillin provided a sensitive means of estimating the quantity of these compounds. A standard curve generated under specifie conditions of reaction time and temperature was used to mlmmlze variation between assays and improve precision. A standard curve for mogroside V reference material was generated from five concentrations assayed on the Same day. Figure 2.2 provides an example of a standard curve. It's clear that there was a linear relationship between absorbance and concentration ofmogrosides. Chapter 2 Isolation of mogroside V reference material 38

1.2 y =1.9556x + 0.0187 1 R2 = 0.9985

II) CI)u 0.8 c ca .c ~ 0.6 0 .cII)

0 0 0.1 0.2 0.3 0.4 0.5 0.6 Weight of mogroside (mg)

Figure 2.2 Standard curve of mogroside V generated relative to a control sample

that did not contain any analyte

2.3.1.2 Optimal extraction conditions of resin column chromatography

The sorption capacity of Amberlite XAD-2 resin was examined with the vinallin-perchloric acid method. As suggested by Figure2.3, the adsorption capacity of the resin was determined to be 7 times the resin bed volume (BV), which is equivalent to approximately 48 mg sample per mL wet resin.

Mogrosides were not detected in the aqueous column eluate by either TLC or by the vanillin spectrophotometric method. However, they were eluted efficiently with a concentration of 50% ethanol or greater as summarized in Figure 2.4. A concentration of 50% ethanol was chosen for further studies. In addition, it was observed that the crude Luohanguo extract dissolve more readily in ethanol water solution than in pure water or pure ethanol. Chapter 2 Isolation of mogroside V reference material 39

20.0 ...J -E 18.0 CD 16.0 -E -c::: 14.0 0 12.0 ~ ..c::: 10.0 CI) () c::: 8.0 0 0 6.0 CI) "0 4.0 "ie 2.0 CD 0 0.0 :::riE 2 3 4 5 6 7 8 Elutate Volume (BV)

Figure 2.3 Sorption capacity of Amberlite XAD-2 resin

:::r 1. 20 ~ 1. 00 .§. c 0 +1 0.80 ...l'CI ..c CIl () 0.60 C 0 0 CIl 0.40 "C in ...0 CI 0.20 0 :E 0.00 0 10 20 30 40 50 60 70

Percent Ethanol (v/v) Figure 2.4 Effect of eluting solvent on the elution of mogrosides Chapter 2 Isolation of mogroside V reference material 40

Acidity also had an effect on the adsorption by the Amberlite XAD-2 resin. As recorded in Table 2.1, the recovery ofmogroside V from the column was quantitative at pH 9-10. Amberlite XAD-2 resin was characterized by increased selectivity athigher pH value as weIl as an obvious discoloring effect. Coloured materials were converted to their corresponding salts in alkaline media and their solubilities in water were increased.

Table 2.1 Effect of acidity on adsorption to the XAD resin

Volume Final Colourof Absorbance Colorof50% pH ofresin mogroside water ofEluate ethanol eluate ~cml weight~mg} eluate 3-5 15 0.206 232 Light yellow yellow

7 15 0.268. 301 Light yellow yellow

9-10 15 0.353 396 Dark yellow Light yellow

In conclusion, the first stage of purification of crude extract consisted of a partial purification on a column of Amberlite XAD-2 with water followed by 50% ethanol as eluting agent that had been rendered alkaline (pH: 9-10). The recommended flow rate was 2 mL / min and sample concentration was 8 mg/ml. The wet resin was capable of treating 150 mg of sample per unit (cm) of packed wet resin column.

The solution, after treatment with the macroporous resin, appeared to be light yellow indicating that coloured impurities remained. The separation of pigments from glycosides remained difficult to achieve because of similarities in their polarities (Qi et al, 2005). A strongly basic anion exchange resin IRA-410, had been demonstrated to he suitable to remove pigments during a mogrosides extraction procedure (Makapugay et al, 1985). The IRA-41O resin was compared with three other anion exchange resins. The Chapter 2 Isolation of mogroside V reference material 41 discolouring effects of the various resins are presented in Figure 2.5. Both IRA 410 and Dowex 1 resin possessed similar discolouring effects. Cellex D was less suitable and the eluted solution remained turbid. IRA 410 was chosen for subsequent studies.

1.6 1.4 1.2 II) CI) u 1 ---dowex 1 c ---lr- cellex D !... 0.8 0 II) ~IRA400 oC ct 0.6 -e- IRA410 0.4 0.2 0 0 30 60 90 120 150 180 Time (min)

Figure 2.5 The discolouring effect of selected anion exchange resins

2.3.2 Characterization by Thin Layer Chromatography

Thin layer chromatography (TLC) is a very convenient method to characterize and to assess the purity of a fraction. The TLC separations, performed on the original S. grosvenorii concentrate, thè extracts purified partially by XAD-2 resin treatment, by subsequent IR-410 resin treatment and finally crystallized material are presented in Figure 2.6.

The purity of the final crystallized material was indicated by its appearance as a single zone after TLC on silica gel GHLF (Analtech, Inc., Newark, DE), using I-butanol-acetic Chapter 2 Isolation of mogroside V reference material 42 acid-water (4: 1: l, Rf 0.23) as eluting solvent. Visualization of the eluted plate was effected with 30% w/v sulfuric acid (llOoC, 10 min). Further characterization was perfonned by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Chapter 2 Isolation of mogroside V reference material 43

1. Original S. grosvenorii concentrate (lanes 1 and 2);

2. Eluate after XAD-2 treatment (lanes 3 and 4);

3. Eluate after subsequent resin treatment (lanes 5 and 6);

4. mogroside V reference material (lanes 7 and 8).

Figure 2.6 TLC separations of various fractions Chapter 2 Isolation of mogroside V reference material 44

2.3.3 Mass spectrometry

MS has superior selectivity and it can readily discriminate against interfe.rence from background or other eluted materials. In a mass spectrometer, compounds are vaporized, ionized, then separated according to their mass-to-charge ratio (mlz), and detected. In this study, mass spectra were recorded using an electrospray ionization (ESI) technique. Since the ESI process involves the formation of high charged liquid droplets, the possibility of multiple charging is implicit.

The ESI-MS spectra in both positive and negative modes of the final crystallized eluate are presented in Figure 2.7. The positive-ion ESI mass spectra reproduced in the upper and middle panes were characterized by ions corresponding to cation attachment to the molecular specie, and to several of the lower mie fragment ions. These fragment ions are consistent with the sequentialloss of sugar substituents as summarized in Table 2.2 and Table 2.3. The fragmentation pattern for the negative-ion mass spectrum in the bottom pane is presented in Table 2.4.

In the upper pane of Figure 2.7, which is the full mass spectrum (labelled as c+), a prominent peak with mie 1309.6 is consistent with mogroside V (M.W. 1287.447) minus H (1.008) plus Na (M.W. 22.9292), so that it is considered to represent mogroside V that has lost a hydrogen atom and captured a sodium atom [M - H + Nat. The prominent fragment peak with mie 1310.6 represents mogroside V that has captured a sodium atom [M+Nat·

In the middle pane, there is a pattern that results from the fragmentation of the base peak (1309.6 mlz) in the upper spectrum. This fragmentation of the ion [M - H + Nat resulted in a prominent fragment with mie 1147.5, by the loss of 162 mass units, Chapter 2 Isolation ofmogroside V reference material 45 corresponding to the loss of a glucoside fragment ([M - H + Nat - Glu). Multiple losses of glucoside fragments were also observed. For example, the prominent fragment with mie 985.5 correspond to lose two glucoside fragments ([M - H + Nat - 2glu) and the fragment with mie 823.3 was considered to result from the loss of three glucoside molecules ([M - H + Nat - 3g1u). The sodium ions presumably came from the solvent salt solution.

Negative-ion ESI mass spectra of this compound were relatively simple when compared to their positive-ion ESI mass spectra. The negative-ion mass spectrum in the bottom pane yielded strong fragment with mie 1285.6/1286.7 associated with Mt, which is the negative ion fragmentation of mogroside V and a fragment at 1127.7 that corresponded to the loss of a glucoside fragment from the molecular ion.

In conclusion, the major mass fragmentation information appeared to support the structural assignment as mogroside V. The compound had the molecule weight of 1286 and possessed at least 4 glucose moieties. Chapter 2 Isolation of mogroside V reference material 46

U~1:aU_Ul"LI-'~UL1Wh1t•• :Sl·:AI HI: U.llf.t~ AV: Il Nl: 1.UUt;( -t:;l T: .0 Fullms [150.00.2000.00) 1309.6 100 i 1310.6 J61 1308.9 ! 40 663.3 Jj 1305.9 ~ 20 666.4 750.2 11325.4 1949.8 154.7 268.9 303.2 324.9 423.3 492.2 582.3 655.3 b674.4 1 807.3 821.3 960.6 983.4 1025.21131.5 114!.6 1228.3 1299.4, 1 • 1386.1 147t4 1515.51563.5 1708.4 1740.11799.9 1868.4

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 mIz

051220_01ELP~uUwhft•• 51·57 RT: l32·l42 AV:7 NL:8.21E8 -t:;l T: .0 [email protected] [360.00.1320.00) 1147.5 100

1148.5

r 1027.5 j dl 833.4 '1028.5 1309.5 Q: 1149.5 805.4 '834.5 865.4 898.3 1291.5 464.2 509.2 5~7.1.539.4 583.4 664.8 6713 703.5 753.4 1057.51088.8 1129.7J 1164.8 11,89.5 1219.4 i i 1 i i i i i \ '9 il' i i , 1 , 1 i .,. , "1 .• , 1 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 mlz

051220_01ELP~uUwhlt•• 20·24 RT: 0.58·0.67 AV:5 Nl: 2.61E7 -t:;l T: ·0 FuUms [ 150.00·2000.00] 1285.6 10 1

~ ! " 1287.6 rl 20 1283.6,

o~ 160.9 235.0 263.1 3411 383.5 454.1 514.0 635.4 660.6 718.0 800.7 844.2 91lS 961.6 985.11105.41~.7 1139,5 1269.6 13~t51384.4 1447.7 1476.3 1584.3 1669.317082 1796.3 1977.8

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 mlz Figure 2.7 ESI-MS spectra of final crystallized eluate in positive and negative mode Chapter 2 Isolation of mogroside V reference material 47

Table 2.2 Proposed fragmentation pattern (upper pane)

+ESI (Observed) Calculated Estimated Formula Relative Intensity

1309.6 1309.4 M-H+Na 100.0

1310.6 1310.4 M+Na 60.5

1306.9 1306.5 M+H2O+H 52.7

1307.8 1307.5 M+H20 + 2H 48.9

663.3 662.4 M + Na - 4Glu frag 37.7

1311.6 1311.4 M+Na+H 24.4

666.4 662.4 M + Na - 4Glu frag 12.5

1325.4 1325.4 M-H+Na+O 10.8

1147.6 1147.2 M - H + Na - Glu frag 5.3

Table 2.3 Proposed fragmentation pattern (middle pane)

+ESI Relative Calculated Estimated Formula (Observed) Intensity

1147.5 1147.4 M-H + Na-Glufrag 100

1148.5 1148.4 M + Na-Glufrag 55

1027.5 1027.5 M + H20+ 2Na -2Glu frag 31

967.5 967.2 M-H + Na-2Glu-H2O 23

1309.5 1309.4 M-H+Na 19

985.5 985.2 M - H + Na- 2Glu frag 18.5

1291.5 1291.4 M-H-H2O+Na 10

805.4 804.9 M - H + Na - 3Glu - H20 9.5 Chapter 2 Isolation of mogroside V reference material 48

Table 2.4 Proposed fragmentation pattern for ESI in the negative ion mode (lower pane)

- ESI (Obsen"ed) Calculated Estimated Formula Relative Intensity

1285.6 (base peak) 1286.4 M-H 100

1287.6 1287.4 M 22

1283.6 1283.6 M-4H 16

1123.7 1124.3 M-H-Glufrag 8

2.3.4 Nuclear Magnetic Resonance Spectroscopy (NMR)

Nuclear Magnetic Resonance Spectroscopy (NMR), which is based in quantum mechanical properties of nuclei, has become the most important analytical tool available for natural products chemistry. Carbon 13 (l3C) and proton eH) NMR spectra have been reported for the identification of the structure of mogroside V (Takemoto et al., 1983c; Si et al., 1996). Chang and colleagues determined the structure of mogroside V by lH NMR ,BC NMR, lH _lH COSY, BC _lH COSY and NOE differential spectroscopie techniques (Chang et al, 1994).

l3C NMR signaIs of the final crystallized compound were assigned as summarized in Table 2.5. The signals of the aglycone (mogrol), were assigned in Reference 3 (Chang et al, 1994). Assignments of the NMR signaIs of mogroside V were reported by Kasai et al. in 1989 and by Chang et al. in 1994. Table 2.5 indicates that the observed BC NMR chernical shifts for this compound are in accord with published data for mogroside V. Comparations of the experimental BC NMR spectra with reported chernical shifts for mogrol, this chromatographie isolate has the same molecule structure except the downfield shifts of signal due to C3 and C24 (87.1, 93.7) compared with those of mogrol (C3:76.2; C24:79.0). These observations suggest that the sites of glucosylation Chapter 2 Isolation of mogroside V reference material 49 are the C-3 position and the C-24 position.

Table 2.5 Experimental BC NMR chemical shifts for the aglycone moiety of the TLC isolate compared with published data for mogroside V and mogrol (0 / ppm)

Reference 2 Reference 3 Reference 1 Obseved mogrol mogrosideV Carbon Chemical mogrosideV (Kasaï et al, Shift (Chang et al, (Chang et al, 1989) 1994) 1994)

1 26.9 26.8 26.8 25.8

2 29.7 29.4 29.4 30.8

3 87.1 87.4 87.4 76.2

4 41.8 42.2 42.3 42.2

5 144.5 144.3 144.5 144.3

6 119.1 118.4 118.2 119.1

7 24.2 24.6 24.7 24.5

8 43.1 43.5 43.7 43.6

9 39.6 40.1 40.3 40.2

10 37.2 36.7 36.8 36.9

11 78.5 77.9 78.0 77.8

12 39.6 41.0 41.2 41.2

13 47.1 47.4 47.6 47.4

14 49.4 49.7 49.8 49.8 Chapter 2 Isolation of mogroside V reference material 50

Reference 2 Reference 3 Reference 1 Obseved mogrol mogrosideV Carbon Chemical mogrosideV (Kasaï et al, Shift (Chang et al, (Chang et al, 1989) 1994) 1994)

15 35.9 34.5 34.7 34.5

16 28.3 28.5 28.6 28.4

17 50.9 51.0 51.2 51.0

18 16.5 17.1 17.2 17.0

19 26.9 27.0 27.1 26.7

20 35.9 36.3 36.5 36.3

21 19.2 19.1 19.2 18.9

22 33.2 33.2 33.4 34.2

23 29.7 29.4 29.6 29.0

24 93.7 91.9 91.7 79.0

25 73.2 72.7 72.8 72.0

26 24.2 24.6 24.7 25.8

27 26.1 26.2 26.3 26.2

28 19.2 19.4 19.5 19.3

29 26.9 27.6 27.8 27.3

30 26.1 26.2 26.3 26.2 Chapter 2 Isolation of mogroside V reference material 51

Furthennore, the sugar moieties in glycosides typically result in chemical shifts between o 99.0""109.5 ppm in l3C NMR spectra (Wu, 2003). The five resonances that were observed at 0 102.1, 102.3, 102.6, 102.9, 104.4 in the NMR spectrum, were considered to be characteristic of the five glucose units of this compound. Further assignment of the linkage positions of the sugar moieties would require more advanced NMR technique.

On the basis of the above spectroscopic analyses, the structure of this final compound was concluded to be mogroside V. The aglycone moiety was mogrol and the five glucose units were connected with the aglycone at the C-3 and C-24 positions. In conclusion, NMR spectra provided more detailed structural information of this compound. l3C NMR spectra coupled with the MS data corroborated the conclusion that the final crystallized eluate was mogroside V, which is suitable as a reference material for subsequent experiments. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 52

Chapter3

Supercritical Fluid Extraction of Mogrosides

from S. grovenorii

3.1 Introduction s. grosvenorii SWINGLE (also known as Momordica grosvenori, Fructus momordicae, Luo Han Guo), a perennial vine grown in a restricted area of southem China, has been used as a natural sweet food as well as a medicinal herb for treating cough and sore throat (Kinghom and Soejarto, 1986). The extract from this fruit has been reported to be about 150 times sweeter than sucrose (Lee, 1975). BeneficiaI characteristics of the extract to health have also been the object of several studies in recent years. As examples, mogrosides, the principle sweet components, have showed promising results for diabetes victims and valuable antioxidant properties as cancer-chemopreventive agents (Konoshima and Takasaki, 2002).

Since mogrosides in S. grosvenorii are in low concentrations (about 1% of the flesh of the fruit) (Kinghom and Soejarto, 1986), a great deal of research has been done to develop more effective methods for the selective extraction to recover these compounds from fresh or dried S. grosvenorii fruit. Studies using solvent extraction, TLC, HPLC and resin chromatography have been reported. However, many isolation procedures used large quantities of toxic solvent that resulted in the possibility of toxic residues in this high value food. Therefore, developing- altemate extraction techniques with increased efficiency and environmental innocuity is highly desirable. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 53

Supercritical fluids provide opportunities in this area due to the ease of solvent removal, their recyclability and useful solvent characteristics; they can replace and/or eliminate hazardous solvents. In addition, the absence of light and oxygen in the extraction process minimizes/eliminates oxidation reactions (Diaz-Reinoso et al., 2006). This latter point is of special interest for the extraction of mogrosides that are prone to oxidative decomposition.

Supercritical fluid extraction (SFE) is based on the unique physical properties of a substance in the supercritical state that favour the solubilization of selected components within a matrix. In comparison with liquid organic solvents, supercritical fluids have a higher diffusivity but a lower density, viscosity, and surface tension. In addition to these desirable features, the properties of supercritical fluids can be modified over a wide range (changing the operating pressure) to achieve a remarkably high selectivity, which is particularly useful for the extraction of complex mixtures from plant materials (Ellington et al., 2003; Reverchon et al., 1993). SFE of active natural products from plant materials has become one of the most important areas of application for supercritical fluids (McHugh and Krukonis, 1994).

Carbon dioxide has been the solvent of choice for more than 90 % of all analytical SFEs (Bowadt and Hawthorne, 1995). Apart from its low critical temperature and pressure parameters (31.1 oC, 73.8 bar), carbon dioxide is available in high purity at relatively low cost; it is readily separated from the extract, is non-toxic, chemically inert, and non flammable. Numerous scC02-based methods have been developed for the isolation of natural products, including the extraction of lipid soluble vitamins (A, D, E, and K), various seed, nut, bean, and wood oils, essential oils, meat fats, phospholipids, pesticides, cholesterol, and pharmaceutical components (Kaiser et al., 2001; Lang and Wai, 2001; Modey et al., 1996). Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 54

Water has been another choice as an extraction fluid for analytical SFE in recent years. Water as SF solvent has been reported by BroU et al. and used mainly as a reaction medium (BroU et al., 1999). Sub critical water (water under pressure and above 100°C but be10w its critical point of 374°C) has been used for extraction of herbal samples (Femândez-Pérez et al., 2000; Ibanez et al., 2003; limenez-Carmona et al., 1999; lu and Howard, 2005; Kubatova et al., 2001; Ozel et al., 2003; Rovio et al., 1999; Shotipruk et al., 2004; Soto Ayala and Luque de Castro, 2001).

The work presented in this chapter, deals with the extraction of mogrosides from S. grosvenorii with sub critical water and supercritical C02. To date, no report on supercritical fluid processing to extract mogrosides has been published. Soxhlet extractions were also performed for comparison. The process variables for both sub critical water and supercritical CO2 were evaluated and conclusions were formulated regarding the relative merits of the two proposed extraction methods.

3.2 Materials and methods

3.2.1 Materials

This study was based on the use of powdered plant concentrate of S. grosvenorii (SG), supplied by the China Natural Products Group, Inc. (U.S.A) and crude extract after resin treatment by our lab (MG). Mogroside V reference material was isolated and characterized by NMR and MS as reported in Chapter 2.

Perchloric acid (70 %, w/v), acetic acid (glacial) and ethanol were purchased from Fisher Scientific, Ottawa, ON, Canada. Carbon dioxide and N2 gas were obtained from MEGS, St-Laurent, QC, Canada. Vanillin (purity, 99 %) and Aluminum Oxide neutral (150 mesh) were purchased from Sigma-Aldrich Co., Oakville, ON, Canada. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 55

Para-anisaldehyde (purity, 98 %) was obtained from Alfa-Aesar, Ward Hill, MA, USA. AIl reagents were ACS reagent grade or better.

3.2.2 8ub critical water extraction (8WE)

3.2.2.1 Equipment

Sub critical water extraction (SWE) was perfonned in a laboratory built apparatus described in Fig. 3.1. The extraction system consisted of an HPLC pump and an extraction cell. An HPLC pump (Beckman, model 100A) was used to deliver water at a constant flow rate. The I-mL (4 mm i.d., 75 mm long) extraction cell was obtained from Supelco Canada (Mississauga, ON, Canada) and contained a stainless steel filter at both ends. Both the extraction cell and a I-m preheating coil [fabricated from 1/16 in. (1.6 mm) stainless steel tubing] were mounted within a gas chromatographic oyen (Hewlett-Packard, 5890 II). The extraction cell was mounted vertically in the oyen with water flowing from top to bottom. A I-m section oftubing (1/16 in.) exited the GC oyen and transferred the eluate from the extraction cell to a beaker cooled in an ice bath. The outlet pressure was maintained at 11724 KPa with a tenninal regulator made of capillary quartz (25 cm x 0.050 mm) tubing (Chromatographic Specialties, Brockville, ON), ensuring that the water remained in the liquid state at all of the temperatures that were tested. High pressure needle valves (SSI model 02-0120, U.S.A.) were placed between the pump and the preheating coil and between the cell outlet and the outlet back pressure regulator. Chapter 3 Supercritical Fluid Extraction ofMogrosides trom S. grovenorii S6

r··v~~~..··l

---(---~h // // ...._;; ...._ ..._.- r····p~~h~~~i~~··c~ù·········l ~...... :

,...... : .// / L!'~tr:"'ti~~_~"!I_J / l'-" rvaÏ;';l i i l - .,/,,,,- : ...... :

...••...•••.

r········HPLC··P~~········] I··········c~ù~~~i~~··vi~ï"·········l r"'w~~~~""l : ! [::.:::::.::?~::?~~~:::::::::.:] !...... J L...... ~...... :

Figure 3.1 Sub critical water extraction unit Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 57

3.2.2.2 General SWE extraction procedure

As no previous information conceming extraction of mogrosides by SFE was available, preliminary assays were carried out to establish appropriate protocols. These experiments were performed using 50 mg of S. grosvenorii powdered plant concentrate (SG). On the basis of the initial observations, it was considered necessary to extract mogrosides by placing the sample between two layers of packing material within the extraction cell.

The water was degassed with ultrasound to remove dissolved oxygen prior to the extraction. The extraction procedure was initiated by heating the oyen to the required temperature and pressurizing the system with water to 11724 KPa with a flow of 0.7 mL / min. When the oyen temperature had stabilized (5 min), the needle valve was opened and the collection of elutes was commenced. Six-mL fractions of eluate were collected, diluted to 10 mL with water then stored to await analysis.

In between runs, the extraction cell was cleaned thoroughly with warm soapy water and the extaction assemly was washed by water to prevent cross contamination. At lease one trial of any series were performed in triplicate; it was presumed the variability observed among replicates was characteristic of the other experiments.

To determine the optimal conditions for extraction, the temperature, extraction time and packing materials for each run were varied during the optimization studies. Table 3.1 summarizes the settings for the conditions of sub critical water extractions. Additionally, ethanol as sub critical solvent modifier was also assessed at various temperatures (70 - 170°C). In all these experiments, the pressure (11724 KPa) and flow rate (0.7 mL/min) were maintained constant as these conditions was found to be the most efficient • Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 58 operating parameters in the preliminary assays.

Table 3.1 Conditions for SWE runs during optimization

Run Packing material Temperature \C) Time(min)

Ah03 neutral 70 10

Effect of extraction Ah03 neutral 100 10 temperature Ah03 neutral 120 10

Ah03 neutral 150 10

Ah03 neutral 170 10

Ah03 neutral 150 10

Effect of packing Ah03 basic 150 10

material Celite 545 150 10

Silica gel 150 10

Ah03 neutral 150 5

Ah03 neutral 150 7

Effect of extraction Ah03 neutral 150 10 time Ah03 neutral 150 15

Ah03 neutral 150 20

Ah03 neutral 150 30

The efficiency of an extraction was determined relative on the recovery of mogrosides Chapter 3 SupercriticaI Fluid Extraction ofMogrosides frOID S. grovenorii 59 from the starting material. Mogroside yield (%) was calculated as follows:

Content of mogroside in final product

Mogroside Recovery (%) = x 100%

Content of mogroside in SG

The starting material, and post-extraction isolate were analyzed spectrophotometrically (Model Ultrospec-l00, Biochrom Ltd, Cambridge, England) following the vanillin-HCI04 methods (Li et al., 1993).

3.2.3 Supercritical CO2 extraction

3.2.3.1 Equipment

The supercritical C02 extraction system was assembled in the laboratory as summarized in Figure 3.2. The unit consisted of a compressed C02 cylinder, a modifier mixing system and an extractor unit. As received, the cylinder of C02 had been pressurized to 12414 KPa (MEGS, Ville St-Laurent, QC, Canada); the gas was further pressurized with a diaphragm compressor (Newport Scientific, Jessup, MD, USA). A Varian 9010 solvent delivery system (Varian Associates Inc., Walnut Creek, USA) was used in the constant flow mode to deliver modifier to a mixing tee [1/16 in, inner diameter (id)] where it was merged with a SCC02 stream (about 1 mL/min). The supercritical fluid was then passed through a I-m preheating coil [1/16 in. stainless steel (ss) tubing] that was connected to an empty ss HPLC column assembly (10 mm id x 25 cm), which acted as a temperature and pressure equilibration vessel (TPEV). The TPEV was connected in series with a ss high pressure cylindrical extraction cell that was of the same dimensions as the cell for Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 60 sub-critical water extraction (4 mm id, x 75 mm long HPLC column, Supelco). The I-m preheating coil, TPEV and extraction cell were positioned within a gas chromatography oven (Hewlett-Packard, 5890A) to provide repeatable temperature control. Pressure within the assembly was maintained with a terminal restrictor made of capillary quartz (25 cm x 0.05 mm) tubing (Chromatographie Specialties, Brockville, ON, Canada). High pressure needle valves valve (SSI model 02-0120, AIltech, USA.) mounded at the entrance and exit of the oven completed the assembly. The exit tip of the capillary restrictor was immersed in ethanol (5 mL) to trap products from the reactor eftluent.

A pressure release valve (Tyco Valves & Controles, Montreal, QC, Canada) that incorporated a rupture disk (rated to 27586 KPa) was positioned upstream from the extractor column and was configured to vent the system if the pressure within the reactor column became excessive.

3.2.3.2 General extraction procedure

Samples were prepared for SFE by mixing packing material with the powdered plant material (50 mg SG or 50 mg MG). The extraction cell was filled with a small quantity of sand at the outlet first (to prevent plugging of the ceIl), overlayered with the sample-packing material and completed with sand.

In operation, a short delay served to purge residues of air from the system (during which time only scC02 was fed to the extractor). During the extraction, ethanol was added continuously via the HPLC pump to the SCC02 stream as the polar modifier and transported to the TPEV. It was presumed that the ethanol was weIl mixed with

supercritical CO2 before they entered the extraction cell. The SCC02 extraction was carried out at a tempe rature range of 40-80°C, pressure range of 13793-20690 PKa and Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 61 and stored to await analysis.

The efficiency of an extraction was detennined based on the recovery of mogrosides from the starting material. Extracts from both the starting plant powder and post-extraction material were analyzed spectrophotometrically (Model Ultrospec-lOO, Biochrom Ltd; Cambridge, England) following the vanillin-HCI04 method (Li et al., 1993).

In between runs the extraction cell was cleaned thoroughly with wann soapy water and the extraction system was washed with ethanol to prevent cross contamination. An trials were perfonned in triplicate so that each entry in the Tables represents the mean recovery (%) ± one relative standard deviation. Chapter3 SupercriticalFluid ExtractionofMogrosides from S. grovenorii 62

,...... ":

r.. ··~~·co~ ...... J

l...... ! r------,L/--//~~~~:--:~~~~:~:~s~;~~~~~

..._~ 1./, ExtractIon Cell 1 ...... 1"...... ' l ...... !

---~ ......

[.~~?~:::.J . [:~~:~~~:~~~~~:~~~::::::l

, ......

z ...... [:y.~y.~:::] !..... ~~~.~.~.~~ ..~.~~~~.~ ..~~~~~ ...... !

Figure 3.2 Supercritical C02 extraction system Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 63

3.3 Results and discussion

3.3.1 Sub critical water extraction (SWE)

3.3.1.1 Effect of extraction temperature in SWE

Temperature is the most important physical parameters in SWE. Previous studies have demonstrated water's ability to selectively extract different classes of compounds depends primarily on the temperature used. The more polar analytes with high solubility in ambient water are extracted most readily at lower temperatures, whereas moderately and nonpolar organics with low water solubility at ambient temperature (e.g., PAHs) require a less polar medium (water at temperatures up to 250 or 300°C) (Yang et al., 1998; Hawthorne et al., 2000). The main reason for this change is that water's dielectric constant is reduced at elevated temperature, thereby decreasing its polarity.

The influence of temperature on the efficiency of extraction of mogrosides from powdered S. grosvenorii concentrate (SG) with sub critical water is presented in Figure 3.3. It was concluded that sub critical water can provide differing extraction efficiencies for the same compound. Increasing the temperature of water from 70°C to 150°C resulted in extraction enhancements ranging from 22.9 % to 47.1 %.

On the other hand, increasing temperature above 150°C did not increase of recovery further. It was observed that the recovery of mogrosides at 170°C was decreased compared to the efficiency at 150·C presumably because of degradation of the sample. The decreased recovery was accompanied by a visible darkening of the extract and the smell became increasingly acrid. Since the highest extraction efficiency was obtained at Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 64

I50·C, this temperature was selected for use in the subsequent experiments.

60.0

50.0 ..&.. :.40.0 / - ~ '" ;... ~ 30.0 / o ./ ~20.0 ~ 10.0 0.0 1 1 1 1 70 100 120 150 170 Temprature(C)

Figure 3.3 Effect of temperature on the sub critical water extraction efficiency of mogrosides from·SG

3.3.1.2 Effect of packing material in SWE

The influence of four different packing matrices (neutral aluminum oxide, basic aluminum oxide, Celite 545, and silica gel) in eluting mogrosides from SG with sub critical water was evaluated. The effect of various packing materials is presented in Table 3.2. Although water at I50aC was sufficient to elute mogrosides regardless of the packing material studied, the packing type greatly influenced the elution efficiency under subcritical water conditions. The large st amounts of mogrosides were extracted at 150°C using either neutral aluminum oxide or silica gel as packing material, the percentage of recovery being 56 or 54 %, respectively. On the basis of this result, neutral aluminum oxide was selected in order to extract a maximum quantity of mogrosides. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 65

Since mogrosides are nonionic compounds,the results from two kinds of alumina oxide stationary phase would be expected to be similar. The neutral surfaces of the Ah03 can interact with the hydroxyls in mogrosides, which results a 10 % increase in recovery compared to using basic Ah03 packing material that is anticipated to display increased interactions with cationic compounds.

Table 3.2 Effect of packing material on sub critical water extraction of mogrosides from SG

Packing material Recovery (0/0)*

Ah03 neutral 55.9 ± 1.0

45.9

Silica Gel 53.9 ± 1.4

eelite 545 34.2

* Mean recovery ± one relative standard deviation based on triplicate trials.

3.3.1.3 Effect of extraction time in SWE

The SWE ofmogrosides from SG is a rapid process. One half of the mogrosides content was mobilized within 5 minutes as reported in Figure 3.4. At the same time, the efficiency of the SWE process tailed off dramatically after the majority of the compounds had been removed from the matrix surfaces. This is because the remaining solutes are held within the structures of the sample partic1es and can be mobilized only with prolonged extraction times. For instance, the cumulative extraction efficiency increased only slightly (about 4 %) when the extraction time was increase from 10 to 30 min (Table 3.3). It is presumed that increasing the extraction time to 1- 2 hr would not increase the cumulative extraction efficiency appreciably. Therefore, using a prolonged extraction time to gain a slightly higher recovery might not be economically worthwhile. An optimum extraction time for the recovery of mogrosides from SG is recornmended to Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 66 be 30 minutes.

Table 3.3 Effect of extraction time on the cumulative recovery of mogrosides for the extraction of SG with sub critical water.

Extraction Time(min) Recovery (%)*

0 0

5 49.8 ±2.0

7 55.7 ± 1.9

10 58.3 ± 0.97

15 59.7 ± 0.48

20 61.1 ± 0.50

30 62.4 ±.0.47

* Mean recovery ± one relative standard deviation based on triplicate trials.

70 60 - ?f?- 50 ~ 40 ! èo ;> 8 30 / o p::: 20 / 10 / o j o 5 7 10 15 20 30 Extraction time (min)

Figure 3.4 Effect of extraction time on the recovery of mogrosides from SG with sub critical water Chapter 3 SupercriticaI Fluid Extraction ofMogrosides from S. gravenorii 67

3.3.1.4 Effect of sub critical solvent

Ethanol as a sub critical solvent (78.3°C - 243°C and pressurized to maintain the liquid phase) was also evaluated to extract mogrosides from SG. The recovery was appreciably lower than for the sub critical water as presented in Figure 3.5. Ethanol in sub critical phase was inefficient for the recovery.

60.0

50.0

";!. 40.0 ~ o Subcritical Ethanol ~ 30.0 o u • Subcritical Water &. 20.0 10.0

0.0 70 100 120 150 170 Temprature

Figure 3.5 Comparison of sub critical solvent

3.3.1.5 Effeet of extraction pressure in SWE

Many researchers have demonstrated that increased pressure up to the critical point can be used to retain the solvent in the liquid state. The operating temperature has a little effect on the solvating characteristics of water as long as it remains in the liquid state (Miller and Hawthorne, 1998). For example, increasing the pressure from 0.1 to 10 MPa

yields an increase of E of only 0.3 7 (Vematsu and Franck, 1980). Therefore, the effect of extraction pressure was not considered in the current sub critical water extraction design. Considering that most literature studies have been conducted at 5 MPa, a pressure of 11724 KPa was applied in subsequent experiments. Chapter 3 SupercriticalFluid Extraction ofMogrosides from S. grovenorii 68

3.3.1.6 Optimized extraction conditions for SWE of mogrosides from SG

On the basis of the results reported above, the final process for the extraction of mogrosids from powdered S. grosvenorii concentrate (SG) was described in Table 3.4. The results obtained using this optimized SWE method were then compared with efficiencÏes obtained with the supercritical COz extraction method.

Table 3.4 Optimized Parameters for SWE of mogrosides from SG

Water flux 0.7mLlmin

Pressure 11724 KPa

Temperature 150°C

Pac1ang material Neutral A}z03

Extraction time 30 min

Recovery 62.4%

3.3.2 Supercritical COz extraction

Supercritical COz (scCOz) extraction was performed on two types of samples, the powdered S. grosvenorii concentrate (SG), and the crude extract after resÏn treatment which contained mainly mogrosides (MG).

3.3.2.1 Effect of pressure on scCOz extraction

The extraction pressure is an influential parameter for SFE based on COz smce both density and solvating capacity increase with increasing pressure. Conditions and extraction efficiencies with modified scCOz on SG and MG performed at various Chapter 3 Supercriticai Fluid Extraction ofMogrosides frorn S. gravenorii 69

pressures is presented in Table 3.5 and Table 3.6. 1t is concluded that modified scC02 can extract mogrosides from both kinds of sample. There is a modest increase in recovery with increased pressure· in extraction of SG When MG represented the substrate, the recovery of mogrosides was increased from 37.8 % to 66.8 % when the pressure was increased from 13793 to 20690 PKa (2000-3000 psi). Since the highest extraction efficiency was obtained at 20690 PKa for both sample types, this pressure was selected for use in the subsequent experiments unless stated otherwise.

Table 3.5 Effect of pressure on the scC02 extraction efficiency of mogrosides from SG

Pressure (KPa) 13793 17241 20690

Packing material sand sand sand

Temperature (OC) 60 60 60

EtOH modifier addition rate (mL/min) 0.5 0.5 0.5

Extraction time (min) 15 15 15

Recovery (%)* 20.9 ± 4.4 23.6 ± 3.4 27.6 ± 1.5

* Mean recovery ± one relative standard deviation based on triplicate trials.

Table 3.6 Effect of pressure on the scC02 extraction efficiency of mogrosides from MG

Pressure (KPa) 13793 17241 20690

Packing material sand sand sand

Temperature (OC) 60 60 60

EtOH modifier addition rate (mL/min) 0.3 0.3 0.3

Extraction time (min) 90 90 90

Recovery (%)* 37.8 ± 1.4 59.6±4.7 66.8 ± 2.4

* Mean recovery ± one relative standard deviation based on triplicate trials. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 70

3.3.2.2 Effect of scC02 extraction temperature on mogrosides recovery

The effect of extraction temperature on the recovery of mogrosides from SG and MG is summarized in Table 3.7 and Table 3.8, respectively. For the temperature range of 40-80°C, extraction of both SG and MG indicate that the recovery is increased at 60°C relative to either 40 or 80°C. On the basis of those data, the optimized temperature was chosen to be 60°C for the extraction on SG and MG.

Table 3.7 Effect of scC0 extraction temperature on the recovery of mogrosides from SG 2 . Temperature 40 C 60·C 80·C

Packing material sand sand sand

Pressure (PKa) 20690 20690 20690

EtOH modifier addition rate (mL/min) 0.5 0.5 0.5

Extraction time (min) 15 15 15

Recovery (%)* 18.9 ± 3.2 27.6 ± 1.5 21.7±4.6

* Mean recovery ± one relative standard deviation based on triplicate trials.

Table 3.8 Effect of scC02 extraction temperature on the recovery of mogrosides from MG . . Temperature 40 C 60 C 80·C

Packing material sand sand sand

Pressure (PKa) 20690 20690 20690

EtOH modifier addition rate (mL/min) 0.1 0.1 0.1

Extraction time (min) 30 30 30

Recovery (%)* 0.91 ± 0.62 22.8 ± 1.6 18.4 ± 1.0

* Mean recovery ± one relative standard deviation based on triplicate trials. Chapter 3 SupercriticaI Fluid Extraction ofMogrosides from S. gravenorii 71

3.3.2.3 EfJect of scC02 extraction time on mogrosides recovery

In order to evaluate the effect of extraction time, the experiments with SG and MG were continued for 3 hr and 2hr, respectively, while trapping successive fractions that corresponded to 30 min of cumulative extractor eluate. The results indicated that the longer the supercritical C02 was in contact with the matrix, the greater was the amount of mogrosides extracted (Table 3.9 and Table 3.10). However, it can also be observed that the recovery increased only slightly for the longer extraction times, which suggested that extraction time, beyond 1.5hr, did not influence mogroside recovery significantly.

Table 3.9 Effect of SCC02 extraction time on the recovery of mogrosides from SG 1

Extraction time Cumulative Recovery (%) 2

30 min 9.0 ± 1.1

Ihr 13.1 ±2.6

1.5 hr 18.0

2hr 20.9

2.5 hr 22.3

3hr 22.1

1: AB trials were performed with Ah03 packing material in admixture with the substrate at 80°C with 0.5 mL/min of EtOH and the pressure was maintained at 17241 PKa. 2: Mean recovery ± one relative standard deviation based on triplicate trials. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. gravenorii 72

Table 3.10 Effect of SCC02 extraction time on the recovery of mogrosides from MG 1

Extraction time Cumulative Recovery (%) 2

30 min 20.7 ± 1.3

1 h 21.0 ± 1.5

1.5h 22.8+ 1.6

2h 22.8+ 1.7

1: AlI trials were performed with Ah03 packing material in admixture with the substrate at 80°C, with 0.5 mL/min of EtOH and pressure was held at 17241 PKa. 2: Mean recovery ± one relative standard deviation based on triplicate trials.

3.3.2.4 Effect of packing material in SCC02 extraction

It has been demonstrated previously that extraction efficiency was increased by the addition of granular particles to the substrate (Berg et al., 1997). These particles were presumed to enhance the extraction by pro vi ding an increased contact between supercritical fluid and the substrate, reducing the dead volume effects, and increasing the penetration of the sample matrix. Packing materials such as sand, Ce1ite 545 and neutral Ah03 were chosen for this study because of their larger grain size, chemical inertness, non-toxicity, and availability. Improved SFE recoveries were observed when SG or MG samples were blended with sand (30-60 mesh) before loading the samples into the SFE extraction cell (Table 3.11 and Table 3.12). As an example, for the extraction of MG (Table 3.11), the recovery ofmogrosides was increased approximate1y 20-fold (0.5 % to 9.3 %) by changing the packing material from neutral Ah03 to sand while maintaining the other parameters constant. Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 73

Table 3.11 Effect of packing material on the scC02 mediated recovery of mogrosides from SG

AhOJ Packing material Sand Celite 545 neutral

Temperature ( OC) 60 60 60

Pressure (pKa) 20690 20690 20690

EtOH modifier addition rate (mL/min) 0.5 0.5 0.5

Extraction time (min) 30 30 30

Recovery (%)* 4.2 ± 0.98 37.0 ± 3.2 25.5 ±2.0

* Mean recovery ± one relative standard deviation based on triplicate trials.

Table 3.12 Effect of packing material on the scC02 mediated recovery of mogrosides fromMG

Packing material neutral A120J Sand

Temperature ( OC) 60 60

Pressure (PKa) 20690 20690

EtOH modifier addition rate (mL/min) 0.1 0.1

Extraction time (min) 30 30

Recovery (%)* 0.46 9.3

*: Mean recovery ± one relative standard deviation based on triplicate trials.

3.3.2.5 Effect of modifier in scC02 extraction

Due to its non-polar character, supercritical CO2 was not anticipated to efficiently Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 74 mobilize the moderately polar mogrosides fraction from plant materials. The addition of a modifier was observed to be beneficial for mogrosides extraction in preliminary assays. In addition, previous researchers have indicated that a facile yet effective procedure to deal with clogging, a very common mechanical problem in SCC02 extractions, was to incorporate a liquid modifier into the mobile phase. The addition of modifier increased the solubility of target organic compounds and transferred them efficiently to the collecting vials (Heaton et al, 1993).

Ethanol was chosen as modifier for the SCC02 extraction of mogrosides because of its toxicological innocuity. In the presence of 0.0-0.5 mL/min ethanol, mogrosides extraction efficiency increased appreciably (Table 3.13). The recovery was 66.8 % when ethanol was added at 0.3 mL/min as modifier, which is judged not to be significantly different from the recovery at 0.5 mL/min (67.3 %). Both recoveries are improved appreciably relative to the 22.8 % observed with the addition of 0.1 mL/min modifier and much better than no addition ofmodifer at all (0.63 %).

Table 3.13 Effect of modifier addition for the SCC02 mediated recovery of mogrosides fromMG EtOH modifier addition 0 0.1 0.3 0.5 rate (mUmin)

Temperature ( OC) 60 60 60 60

Packing material sand sand sand sand

Pressure (PKa) 20690 20690 20690 20690

Extraction time (h) 1.5 1.5 1.5 1.5

Recovery (%)* 0.63 ± 0.27 22.8 + 1.6 66.8 + 2.4 67.3 + 1.2

*: Mean recovery ± one relative standard deviation based on triplicate trials. Chapter 3 Supercriticai Fluid Extraction of Mogrosides from S. grovenorii 75

3.3.2.6 Optimized SCC02 extraction conditions

On the basis of the results reported ab ove, the final process for the extraction of mogrosids from SG and MG has been summarized in Table 3.14.

Table 3.14 Established Parameters for scC02 extraction ofmogrosides from SG and MG

Sample SG MG

CO2 flux (mL/min) 1 1

Pressure (pKa) 20690 20690

Temperature C'C) 60 60

Packing material sand sand

Extraction time (hr) 0.5 1.5

EtOH modifier addition rate (mL/min) 0.5 0.5

Recovery (%)* 37.0 ± 3.2 67.3 ± 1.2

* Mean recovery ± one relative standard deviation based on triplicate trials.

3.3.3 Comparasion of Soxhlet extraction, sub critical water extraction and supercritical C02 extraction of mogrosides

Extractions of mogrosides from powdered S. grosvenorii concentrate were performed with a Soxhlet apparatus, with sub critical water (SWE), or with EtOH modifiered supercritical C02 (scC02). The conditions used for each extraction method and the results are summarized in Table 3.15. With a proper understanding of the techniques, the extraction of mogrosides can be achieved using any of these methods. However, the different methods have varying degrees of ability to provide mogrosides free from large amounts of co-extracted matrix materials. Compared with Soxhlet solvent extraction, supercritical fluid extraction either using sub critical water or supercritical C02 provided Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 76 improved recoveries, and consumed less organic solvent. In addition, the purity of the extracts differed greatly. The organic solvent extracts (Soxhlet) were much darker and highly turbid, while the extracts from the subcritical water process were yellow and c1ear, and the extracts from SFE were light yellow and somewhat turbid.

Table 3.15 Conditions used to compare Soxhlet, SWE and SCC02 extractions

Soxhlet SWE sceoz

Sample size Ig 50mg/run 50mg/run

Extraction Solvent ethanol, hexane water EtOH modified CO2

Solvent Volume (mL) 300 18 180

Time (h) 24 0.12 1.5

Pressure (pKa) ambient 11724 20690

Temperature eC) b.p. of solvent 150 60

Labor Intensive Low Low High

37.0 for SG; Sample Recovery 5.1 % for SG 62.4 % forSG 67.3 for MG

Extract col or orange yellow Light yellow

Sub critical water extraction permits the efficient partial extraction of mogrosides in a short time. Subcritical water extraction yields a higher recovery of mogrosides in 30 min, compared to 24 h of Soxhlet extraction or 1.5 hr of SCC02 extraction. For identical powderous S. grosvenorii concentrate samples, sub critical water extraction was demonstrated to be more efficient (62.4 % recovery) compared with 37.0 % recovery by scC02 extraction. As relatively few parameters affect the SWE process, optimization is Chapter 3 Supercritical Fluid Extraction ofMogrosides from S. grovenorii 77 relatively facile. Temperature is the major parameter to be considered. More rapid extractions are achieved with higher water temperatures but increased temperature must be used with caution, since degradation of the mogroside compounds was clearly evident at temperatures above 150°C. The main drawback of the scCOz extraction process is the elevated pressure. By contrast, sub critical water extractions require much lower pressure to keep the water in liquid phase, so that SWEs are inexpensive and relatively easy to perform. The greatest recovery of mogrosides was obtained at 150°C for 10 minutes, using subcritical water as solvent.

SFE with pure CO2 yielded lower recoveries than either technique, but comparable recoveries when ethanol was added as modifier. Supercritical CO2 extraction is more complicated and optimization is relatively more difficult than sub critical water extraction because of the numerous parameters that affect SCC02 extraction procedure including modifier identity and rate of addition, pressure, temperature, extraction time, and packing material. The main parameters to be considered in the extraction of mogrosides are the extraction pressure and the extraction temperature. The greatest recovery ofmogrosides was 37.0 % for SG and 67.3 % for MG.

3.4 Conclusion

For the first time, the supercritical fluid techniques based on either supercritical COz or sub critical water, have been applied to the extraction of mogrosides from powdered S. grosvenorii concentrate. Relatively facile extraction conditions for both fluids have been optimized. It has been demonstrated that SFE technique produces less waste and generates no hazardous product. Although SFE with pure COz yielded low recoveries, recoveries comparable to the sub critical water process were achieved if ethanol was added as a modifier. Sub critical water extraction has more desirable features than

supercritical CO2 extraction that include a shorter extraction time, lower pressure requirement, fewer parameters that need to be controlled and a higher recovery. References 78

References

Anklam, E.; Berg, H.; Mathiasson, L.; Sharman, M.; Ulberth, F., Supercritical fluid extraction (SFE) in food analysis: Areview. FoodAdd. Contam. 1998, 15(6),829-750.

Anonymous Guangxi SCIence and technology information network/resource system/Guilin. (July 27, 2006), http://www.gxsti.net.cnlesti/2resourse.htm#guilin

Anonymous, High sweetening agents. Japan Food Chem., 1985, 10, 12-25.

Anonymous, Market of Chinese herb medicine: analysis of the production and sales of Lo-Han-Kuo. J. Chinese Med. Inform. 1996,3(9), 13-13.

Bartley, J.P., Supercritical fluid extraction of Australian-grown gmger (Zingiber officinale). J. Sei. Food Agrie. 1994,66(3),365-371.

Berg, H.; Magard, M.; Johansson, G; Mathiasson, L., Development of a supercritical fluid extraction method for determination of lipid classes and total fat in meats and its comparison with conventional methods. Journal ofChromatography A 1997, 785(1-2), 345-352.

Bian, S.Z.; Sun, H.F.; Liu, L.L, Extraction of steviosides by macroporous resin. J. Chinese Tradit. Med. 1986, 17 (6), 12 - 13.

Boselli, E.; Caboni., M.F., Supercritical carbon dioxide extraction of phospholipids from dried egg yolk without organic modifier. J. Supererit. Fluid. 2000, 19(1),45-50.

Bowadt, S.; Hawthorne, S.B., Supercritical fluid extraction in environmental analysis. J. Chromatog. A, 1995,703(1-2),549.

BroU, D.; Kaul, C.; Kramer, A.; Krammer, P.; Richter, T.; Jung, M.; Vogel, H.; Zehner, P., Chemistry in Supercritical Water. Angewandte Chemie International Edition 1999, 38(20),2998-3014. References 79

Cai, X.; Liu, S.Q.; Wang, P.X., Purification glycosides from Ginseng by macroporous resin. J. Chinese Herb 2001, 32(9), 631-634

Chang, Q.; Chen, D.; Si, j.; Shen, L., The studies on the NMR spectra of mogroside V. Chin. J. Magnetic Resonanace 1994, 11(1),43-48

Di Mauro, A; Fallico, B; Passerini, A.; Rapisarda, P.; Maccarone, E, Recovery of hesperidin from orange peel by concentration of extracts on styrenedivinylbenzene resin. J Agric Food Chem. 1999,47(10),4391-4397.

Di Mauro A.; Fallico B.; Passerini A.; Maccarone, E., Waste water from citrus processing as a source of hesperidin by concentration on styrenedivinylbenzene resin. J Agric Food Chem. 2000, 48(6), 2291-2295.

Diaz-Reinoso, B.; Moure, A.; Dominguez, H.; Parajo, J. C., Supercritical C02 Extraction and Purification of Compounds with Antioxidant Activity. J. Agric. Food Chem. 2006,54(7),2441-2469.

Ellington, E.; Bastida, J.; Vïladomat, F.; Codina, C., Supercritical carbon dioxide extraction of colchicine and related alkaloids from seeds of Colchicum autumnale. Phytochem. Anal. 2003, 14(3), 164-169.

Ericson, A.P.; Matthews, RF.; Teixeria, A.A.; Moye, H.A., Recovery of grapefruit oïl from processing waste water using SDVB resins. Proc. Fla. State Hart. Soc. 1990,103, 280--282.

Femândez-Pérez, V.; Jiménez-Carmona, M.M.; Dolores Luque de Castro, M., An approach to the static-dynamic subcritical water extraction of laurel essential oil: comparison with conventional techniques. Analyst 2000,125(3),481-485.

Fischer, C.M.; Harper, H.J.; Jr., H.; J., W.; Jr., M.; J., M.; Romer, K.; Swaine Jr., RL., Sweet beverages and sweetener compositions. 1994; US Patent W094-US 1690.

Gere, D.R; Randall, L.G.; Callahan, D., Supercritical fluid extraction: principles and References 80 applications. 1997; Il,421-484.

Hauthal, W.H., Advances with supercritical fluids [review]. Chemosphere 2001, 43(1), 123-l35.

Hawthorne, S.B.; Kubatova, A., Extraction technology fundamentals-chapter 18-Hot (subcritical) water extraction. Comp. Anal. Chem. 2002,37(18),587-608.

Heaton, D.; Bartle,' K.; Rayner, C.; Clifford, A., Application of supercritica1 fluid extraction and supercritical fluid chromatography to the production of taxanes as anti-cancer drugs. Journal ofHigh Resolution Chromatography 1993, 16(11), 666-670.

Hogarth, J.; Hannay, J.B., On the solubility of solids in gases. Proc. Roy. Soc. 1879, A(29),324.

Hussain, R.A.; Lin, Y.M.; Poveda, L.J.; Bordas, E.; Chung, B.S.; Pezzuto, J.M.; Soejarto, D.D.; Kinghorn, A.D., Plant-derived sweetening agents: saccharide and polyol constituents of sorne sweet-tasting plants. J. Ethnopharmacol. 1990,28(1), 103-15.

Ibanez, E.; Kubatova, A.; Senorans, F.J.; Cavero, S.; Reglero, G; Hawthorne, S.B., Subcritical water extraction of antioxidant compounds from Rosemary plants. J. Agric. Food Chem. 2003,51(2),375-382.

Ichikawa, T.; Matsunra, T.; Uchida, M.; Murakami, A.; Shi, H., Blood pressure and lipid peroxides in tissues of SHR fed with hot water extracts of 10 han kuo fruits. J. Jap. Soc. Food Sei. Technol. 1997,44(2),140-143.

Jimenez-Carmona, M.M.; Ubera, J.L.; Luque de Castro, M.D., Comparison of continuous subcritical water extraction and hydrodistillation of marjoram essential oil. J. Chromatog. A 1999, 855(2), 625-632.

Ju, Z.Y.; Howard, L.R., Subcritical water and sulfured water extraction of anthocyanins and other phenolics from dried red grape skin. J. Food Sci., 2005, 70(4), S270-S276. References 81

Kaiser, C.S.; Rompp, H.; Schmidt, P.C., Pharmaceutical applications of supercritical carbon dioxide. Pharmazie 2001, 56(12), 907-926.

Kaiser, R.; Matsumoto, K.; Nie, R.L.; Morita, T.; Awazu, A.; Zhou, J.; Tanaka, O., Sweet and bitter cucurbitane glycosides from Hemsleya carnosiflora. Phytochem. 1987,26(5), 1371-1376.

Kaiser, R.; Matsumoto, K.; Nie, R.L.; Zhou, J.; Tanaka, O., Glycosides from Chinese medical plant, Hemsleya panacis-scandens, and structure-taste relationship of cucurbitane glycosides. Chem. Pharm. Bull. 1988,36(1),234-243.

Kang; J., Comparison of physical and transport properties of gases, liquids and SCFs. (July 27,2006), http://www.andrew.cmu.edu/user/jitkangl/SFE.htm

Kasai, R.; Nie, R.L.; Nashi, K.; Ohtani, K.; Zhou, J.; Tao, G-d.; Tanaka, O., Sweet cucurbitane glycosides from fruits of Siraitia siamensis(chi-zi luo-han-guo), a Chine se folk medicine. Agric. Biol. Chem. 1989,53(12),3347-3349.

Kim, N. C.; Kinghom, A.D., Highly sweet compounds of plant origin. Arch. Pharmacol. Res. 2002,25(6), 725-746.

King, J.W., Analytical-Process Supercritical Fluid Extraction: A synergestic combination for solving analytical and laboratory scale problems. Trend. Anal. Chem. 1995, 14(10), 474-481.

Kinghom, A.D.; Kaneda, N.; Baek, N.L.; Kennelly, EJ.; Soejarto, D.D., Noncariogenic intense natural sweeteners. Med. Res. Rev. 1998, 18(5),347-360.

Kinghom, A.D.; Soejarto, D.D., Sweetening agents of plant origin. Crit. Rev. Plant Sei. 1986,4(2), 79-120.

Kinghom, A.D.; Soejarto, D.D., Intensely sweet compounds ofnatural origin. Med Res. Rev. 1989,9(1),91-115. References 82

Kinghorn D.A.; Compadre, C.M., Less common high-potency sweeteners. Third Edition, Marcel Dekker, inc.: New York, 2001, 209-233.

Konoshima, T.; Takasaki, M., Cancer-chemopreventive effects of natural sweeteners and related compounds. Pure Appl. Chem. 2002, 74(7), 1309-1316.

Kubatova, A.; Miller, DJ.; Hawthorne, S.B., Comparison of subcritical water and organic sol vents for extracting kava lactones from kava root. J. Chromatog. A 2001, 923(1-2), 187-194.

Kunin R., The use of macroreticular polymeric adsorbents for the treatment of waste effluents. Pure Appl. Chem. 1976,46(2-4),205-211.

Lang, Q.; Wai, C.M., SupercriticaI fluid extraction in herbai and natural product studies -- a practicaI review. Talanta 2001,53(4), 771-782.

Lee, C., Intense sweetener from Lo Han Kuo. Experientia 1975, 31(5),533-534.

Li, B.; Yang, Y.; Gan, Y.; Eaton, C.D.; He, P.; Jones, A.D., On-line coupling of subcriticaI water extraction with high-performance liquid chromatography via solid-phase trapping. J. Chromatog A 2000, 873(2), 175-184.

Li, H.; Li, B.; Hu, S.; Yang, X.; Guo, S.; Cai, M., Microwave-assisted extraction of mogrosides. Food Sei. (Chinese) 2003, 24(2), 91-94.

Li, J.; He, R.; Hou, G.; Lu, C.; Danli, L., Effects ofultrasonic wave on the extraction of mogrosides. Food Ferment. Ind. (Chinses) 2004,30(10), 136-138.

Li, Y.Q.; Wang C.; Wang W.S., Adsorption of the polymetric adsorption AB-8 for mogroside. Ion Exchange and Adsorption, 1995, 11(4),363-367

Li, Y.; Wang, W.; Wang, C., Seperation and measurement of mogrosides from Luo Han Guo. Chinese Food Science 1993, 161(5),66-70.

Makapugay, H.C.; Nanayakkara, N.P.D.; Soejarto, D.D.; Kinghom, A.D., High References 83 performance liquid chromatographie analysis of the major sweet principle of Lo Han Kuo Fruits. J. Agric. Food Chem. 1985, 33(3), 348-350.

Matsumoto, K.; Kasai, R.; Ohtani, K.; Tanaka, O., Minor cucurbitane-glycosided from fruits of Siraitia grosvenori(Cucurbitaceae). Chem. Pharm. Bull. 1990, 38(7), 2030-2032.

McGowin, A.E.; Adom, K.K.; Obubuafo, A.K., Screening of compost for PAHs and pesticides using static subcritical water extraction. Chemosphere 2001, 45(6-7),857-864.

McHugh, M.A.; Krukonis, V. J., Supercritical fluid extraction: principles and practice, 2nd ed.; Butterworth-Heinemann: 1994.

Meng, X.; Zhou, Q.; Rong, X., Analysis of inorganic elements in Luohanguo. J. Guangxi Herb 1989, 12(6),42.

Modey, W.K.; Mulholl, D.A.; Raynor, M.W., Analytical supercritical fluid extraction of natural products. Phytochem. Anal. 1996, 7(1), 1-15.

Moore, R.J., 75-day premarket notification for new dietary ingredient. US Food and Drug Administration 1999; 22-23.

Ozel, M.Z.; Gogus, F.; Lewis, A.C., Subcritical water extraction of essential oils from Thymbra spicata. Food Chem. 2003, 82(3), 381.

Qi, X.; Zhang L.; Chen W., Method for isolation and purification of mogrosides with macroporous resin. J. Transactions o/the CSAE, 2005, 21 (9), 163- 166.

Radcliffe, C.; Maguire, K.; Lockwood, B., Applications of supercritical fluid extraction and chromatography in forensic science. J. Biochem. Biophys. Meth. 2000, 43(1-3), 261.

Reverchon, E.; Donsi, G; Osseo, L.S., Modeling of supercritical fluid extraction from herbaceous matrices. Ind. Eng. Chem. Res. 1993, 32(11), 2721. References 84

Rovio, S.; Hartonen, K.; Holm, Y.; Hiltunen, R; Riekkola, M.-L., Extraction of clove using pressurized hot water. Flavour Fragrance J. 1999, 14(6),399-404.

Rozzi, N. L.; Singh, R. K., Supercritical fluids and the food industry. Comp. Rev. Food Sei. Food Sa! 2002, 1,33-34.

Rudzinski, W.E.; Aminabhavi, T.M., A review on extraction and identification of crude oil and related products using supercritical fluid technology. Energy Fuels 2000, 14(2), 464-475.

Shi, H.; Hiramatsu, M.; Komatsu, M.; Kayama, T., Antioxidant property of fructus momordicae extract. Biochem. Mol. Biol. Int. 1996, 40(6), 111-112.

Shotipruk, A.; Kiatsongsenn, J.; Pavasant, P.; Goto, M.; Sasaki, M., Pressurized hot water extraction of anthraquinones from the roots of Morinda eitrifolia. Biotech. Prog. 2004,20(6), 1872-1875.

Si, J.; Chen, D.; Chang, Q.; Shen, L., Isolation and structure detennination of flavonol glycosides from the fresh fruits of Siriatia grosvenori. Acta Pharm. Sin. 1994, 29(2), 158-160.

Si, J.; Chen, D.; Chang, Q.; Shen, L., Isolation and detennination of cucurbitane-glycosides from fresh fruits of Siraitia grosvenorii. Acta Botan. Sin. 1996, 38(6),489-494.

Smith, RM.; Burgess, RJ., Superheated water as an eluent for reversed-phase high-perfonnance liquid chromatography. J. Chromatog. A 1997,785(1-2),49.

Soto Ayala, R; Luque' de Castro, M.D., Continuous subcritical water extraction as a useful tool for isolation ofedible essential oils. Food Chem. 2001,75(1), 109-113.

Steel, K., Phase diagram for water and carbon dioxide. (June 20, 2006), http://www.kobelco.co.jp/eneka/p14/sfeOl.htm#gaiyou References 85

Su, X.; XU, Q.; Liang, R.; Tang, Z; Wei, Y., Experiments studies on the non-toxicity action ofmogrosides. Food Sei. 2005,26(3),221-224.

Suzuki, Y. A.; Murata, Y.; Inui, H.; Sugiura, M.; Nakano, Y., Triterpene glycosides of Siraïtia grosvenori inhibit rat intestinal maltase and suppress the rise in blood glucose level after a single oral administration of maltose in rats. J. Agrie. Food Chem. 2005, 53(8), 2941-2946.

Takasaki, M.; Konoshima, T.; Murata, Y.; Sugiura, M.; Nishino, H.; Tokuda, H.; Matsumoto, K.; Kasaï, R.; Yamasaki, K., Anticarcinogenic activity of natural sweeteners, cucurbitane glycosides, from Momordica grosvenori. Cancer Lett 2003, 198(1),37-42.

Takemoto, T.; Arihara, S.; Nakajima, T.; Okuhira, M., Studies on the constituents of fructus Momordicae. 1. on the sweet princple. Yakugaku Zasshi 1983a, 103(11), 1151-1154.

Takemoto, T.; Arihara, S.; Nakajima, T.; Okuhira, M., Studies on the constituents of fructus Momordicae. II. structure of sapogenin. Yakugaku Zasshi 1983b, 103(11), 1155-1166.

Takemoto, T.; Arihara, S.; Nakajima, T.; Okuhira, M., Studies on the constituents of fructus Momordicae. III. structure of mogrosides. Yakugaku Zasshi 1983c, 103(11), 1167-1173.

Takeo, E.; Yoshida, H.; Tada, N.; Shingu, T.; Matsuura, H.; Murata, Y.; Yoshikawa, S.; Ishikawa, T.; Nakamura, H.; Ohsuzu, R; Kohda, H., Sweet elements of Siraïtia grosvenori inhibit oxidative modification of low-density lipoprotein. J Atheroscler. Thromb. 2002,9(2), 114-20.

Tsunematsu, T.; Tadashi, N.; Shigenobu, A.; Megumi, o. Glycosides having sweetness. 1978, US Patent 4,084,010.

Turner, C.; King, J.W.; Mathiasson, L., Supercritical fluid extraction and References 86 chromatography for fat-soluble vitamin analysis. J. Chromatog. A. 2001, 936(1-2), 215-237.

Ukiya, M.; Akihisa, T.; Tokuda, H.; Toriumi, M.; Mukainaka, T.; Banno, N.; Kimura, Y.; Hasegawa, 1.; Nishino, H., Inhibitory effects of cucurbitane glycosides and other triterpenoids from the fruit of Momordica grosvenori on epstein-barr virus early antigen induced by tumor promoter 12-0-tetradecanoylphorbol-13-acetate. J. Agric. Food Chem. 2002,50(23),6710-6715.

Wang, H.; Liu, y:, Application and development of macroporous adsorbing resins. J. Chinese Med. Mat. 2005,28(4),353-356

Weber, W. 1.; Van Vliet, B.M., Synthetic adsorbents and activated carbons for water treatment: overviewand experimental comparisons. J. Amer. Water Works Assoc., 1981, 73(8),420-426.

Wikipedia, Siraitia grosvenorii. (July 27, 2006), http://en.wikipedia.org/w/index.php?title=Siraitia~osvenorii&oldid=65859136

Wu, L., Natural products chemistry. People's medical publishing house 2003; Vol. 2, 146-148

Yang, x.; Zhang, J.; Qian, Z., Grosmomoside l, a new cucurbitane triterpenoid glycoside from fruits of Momordica grosvenori. Chinese Trad. Herbai Drugs 2005, 36(9), 1285-1290.

Yang, Y.; Belghazi, M.; Lagadec, A.; Miller, D. J.; Hawthorne, S. B., Elution of organic solutes from different polarity sorbents using subcritical water. Journal of Chromatography A 1998, 810(1-2), 149.

Yu, L.; Chen, Q.; Yi, X.; Yang, R.; Zhang, Y., Preparation of mogroside V from fresh fruits of Luohanguo by high performance liquid chromatography. J. Chromatog. (Chinese) 2003,21(4),397-399. References 87

Zhang, L.; Qi, X., Literature review of mogroside from S. Grosvenerii. Guangxi Tropical Agriculture (Chinese) 2005, 6(14), 23-25.

Zhao, J., Development and the utilization of Chine se medicine luohanguo. A corpus of the research (Beijing), 1993.

Zhou, J.H., HerbaI sweetening and preservative composition comprising licorice extract and mogrosides obtained from plants belonging to Cucurbitaceae and lor Momordica. . . . Patent 6103240. Chemical Abstracts 2000, 133(12), 168393u.

Zhou, J. H.; He, W., Process for extracting triterpene glycosides from botanical sources. 2000, US Patent 6,124,442.

Zhou, Q.; James H. Composition and method for inhibiting oral bacteria. 2000, US Patent 6,319,523.

Zhou, x., Components and application of Luo han guo. J. Traditional Med (Chinese) 2003,21(9), 1482-1483.

Zougagh, M.; Valcarcel, M.; Rios, A., Supercritical fluid extraction: a critical review of its analytical usefulness. Trend. Anal. Chem. 2004,23(5),399-405.