1

CHAPTER 1

INTRODUCTION

1.1 PROPERTIES OF STEVIOL GLYCOSIDES

1.1.1 Physical properties of Steviol Glycosides

The herb Stevia rebaudiana (bertoni) accumulates several sweet

tasting diterpene glycosides in its leaves. The commercial importance of

Stevia rebaudiana stems from the use of these glycosides as sugar substitutes.

There are eight known glycosides in the plant (Figure 1.1) (Kennelly 2002;

Starrat et al 2002).

Steviol glycosides are high-intensity sweeteners ranging from 50-

300 times sweeter than sugar, with low water solubility and high melting

points (Table 1.1) (Crammer and Ikan 1987).

These molecules in solution are highly stable at broad pH and

temperatures. Prolonged heating of a stevioside solution results in a decrease

by 16.7 % after 12 hours at 100 °C in neutral condition and 46.0-53.8 % after

4 hours in acidic environment (Chang and Cook 1983). Significant decrease

in stevioside concentration at 80°C was found only at highly acidic conditions

(pH 1) in solution and at 140°C as a solid (Th.Kroyer 1999).

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Figure 1.1 The structure of the steviol glycoside backbonePosition 19 and 13 are marked on the backbone. R1, except in the case of

rebaudioside B is a glucose unit attached to the carboxyl group. R2 contains

various numbers of glucose residues that are attached to C13 depending

upon the specific glycoside.

Table 1.1 Physical properties of different steviol glycosides The molecular weight, melting point and solubility in water of the different

steviol glycosides are given. RS of the molecules is with respect to a 0.4% w/v

solution of sucrose.

Compound Molecular weight

Melting point (0C)

Solubility in water (%)

RS (Relative Sweetness)

Stevioside 804 196-198 0.13 143

Rebaudioside A 966 242-244 0.80 242

Rebaudioside B 804 193-195 0.10 300

Rebaudioside C 958 215-217 0.21 50

Rebaudioside D 1128 283-286 1.00 221

Rebaudioside E 966 205-207 1.70 174

Steviolbioside 642 188-192 0.03 125

Dulcoside A 788 193-195 0.58 50

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The advantages of stevioside as a dietary supplement for human

subjects are manifold: it is stable, it is non-calorific, it maintains good dental

health by reducing the intake of sugar and could be a good substitute for

diabetic and phenylketonuria (PKU) patients and obese persons (Jan 2003).

The omission of excessively added sugar in the food is beneficial to diabetics

by lowering the blood sugar content. Steviol glycosides are also safe for PKU

patients as no aromatic amino acids are involved. Obese persons might lose

weight by the fact that excessive sugar in the food is replaced by Stevia.

Omitting the added sucrose in foods increases the relative proportion of

polymeric carbohydrates. This has a beneficial effect for balanced food intake

and for human health.

1.1.2 Stevioside Biosynthesis pathway

Steviol glycosides have the ent-kaurene backbone similar to the

Gibberellic acids (GA). With glycosylation at C13 and C19, the different

steviol glycosides differ in the number of saccharide units added at C19

position. The saccharide units are added by various glycosyltransferases

(Shibata et al 1991; Richman et al 2005). The precursor of the backbone,

steviol, which was originally thought to be synthesized by the mevalonate

pathway, was later proved to be synthesized by the Methyl-Erythritol-

Phosphate (MEP) pathway (Totte et al 2000). The end product of the MEP

pathway, Iso-Pentenyl Di-Phosphate (IPP) is converted to Geranyl Geranyl

Di-Phosphate (GGDP) before being cyclised into Copalyl Di-Phosphate

(CDP). The CDP is then converted to kaurene by Kaurene synthase (KS).

Both CDP synthase (CDPS) and Kaurene synthase have been characterized in

stevia (Richman et al 1999). Kaurene oxidase (KO), a cyt-p450

Monoxygenase, then converts kaurene to Kaurenoic acid(Bennett et al 1967).

Both KS and KO are duplicated in the stevia genome (Humphrey et al 2006).

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This overexpression of the KS and KO genes should cause an increase in flux

through the GA biosynthesis pathway, but stevia does not have increased

concentrations of either GA or Kaurenoic acid in its tissues.

Spatial and temporal separation of the key enzymes in steviol

biosynthesis from the GA biosynthetic pathway is one reason for keeping the

GA concentrations under check (Davidson et al 2005). Work on duplicating

the CPS and KS genes in Arabidopsis thaliana also proved that regulation of

GA concentrations in tissues was further downstream from these enzymes, at

the dioxygenase, which is the first committed step to GA biosynthesis (Fleet

et al 2003). Arabidopsis was able to accumulate surprisingly high

concentrations of the Kaurenoic acid without any ill effects.

In stevia, increased flux of Kaurenoic acid could be handled by the

presence of the sink pathway leading to steviol glycoside biosynthesis.

Kaurenoic acid -13-hydroxylase is the first committed step to steviol

glycoside biosynthesis (Figure1.2). At this point GA precursors are

hydroxylated at C7. This enzyme has immense applications in biotechnology

and there is some controversy regarding reported sequences of this enzyme

(Kim et al 1996; Brandle and Telmer 2007).

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Figure 1.2 Stevioside biosynthesis pathway CDPS - Copalyl Di Phosphate Synthase, KS - Kaurene Synthase, KO

- Kaurene Oxidase, KAH - Kaurenoic acid Hydroxylase, UGT -

UDP-Glycosyl Transerase. (Adapted from Brandle and Telmer,

2007)

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1.1.3 Phytochemical constituents of Stevia leaves

The interest in use of the plant for sweetening purposes has lead to

extensive investigation of the constituents of the plant material. The leaves of

the plant accumulate the diterpene glycosides responsible for the sweet taste

and other phytochemicals. The nutritional composition of the leaves was

analyzed and reported by many workers (Savita et al 2004; Dr.Duke’s

phytochemical and ethnobotanical databases, accessed on 22.07.2008;

Tadhani and Subhash 2006).

Stevia leaves have been reported to contain tannins. Tannins are

synthesized in a wide variety of plants and trees (Haslam 1981; Okuda et al

1990; Kumar and Vaithiyanathan 1990) which can accumulate in the wood,

bark, leaves or galls of the plants. Gallo and ellagitannins or mixtures of both,

classified as hydrolysable tannins, are found in stevia leaves. Hydrolysable

tannins consist of central glucose attached to many gallic or ellagic acid units.

Complex reaction products of this core structure leads to many

uncharacterized tannins in plants (Nonaka 1989). There are many methods

available for analysis of tannins (Irene 2001). Analytical methods for tannins

are dependant on sample preparation, storage and extraction techniques.

Mixtures of tannins may be analyzed by general tannin assays such as

precipitation with metals or proteins, and, by colorimetric assays for total

phenols. While colorimetric assays are widely used for tannin analysis, TLC

and HPLC provide more sensitive methods for differentiating and quantifying

tannins. The high polyphenolic concentrations in stevia leaves are significant

because of their sensory properties and anti-oxidant activity (Zhang and Lin

2008; Beninger and Hosfield 2003; Amarowicz et al 2000). Antioxidants are

free radical scavengers, inhibitors of lipid peroxidation and chelating agents.

Phenolic compounds like gallic acid, ellagic acid and their derivatives,

flavonoids and even vitamins are classified as primary anti-oxidants or chain-

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breakers. These compounds act by oxidizing the free radicals which cause

oxidative stress.

Pigmented tannin-like polyphenols cause astringency of wines

(Vidal et al 2004). Tannins also have anti-protein activity but, the major factor

acting against their presence in food is their contribution to taste. Astringency

and bitterness of tannins are a function of their molecular weights, with low

molecular weight tannins being astringent and high molecular weight tannins

being bitter.

1.1.4 Sweetness perception of Steviol glycosides

The sensory attributes of stevia extracts are, therefore, due to a

complex interaction between the various constituents of the leaf. Sweet and

bitter taste properties are found in most chemical classes of sweeteners, and a

close relationship between sweet and bitter tastes is found in many structural

categories (Schiffman et al 1991). A single compound can have both sweet

and bitter qualities. The interdependence of sweet and bitter tastes comes

from psychophysical taste experiments using mixtures containing varying

amounts of sweet and bitter components. This phenomenon called mixture

suppression, suggests a possible relationship between the sweet and bitter

components in a mixture.

Although sweet taste is usually regarded as pleasant and bitter taste

as unpleasant, there is much evidence to suggest that the two tastes are quite

closely related with respect to their transduction mechanisms (DuBios et al

1991). Biochemical studies suggest that the degree of interdependence of

bitter and sweet ratings may be compound specific and related to the

transduction mechanisms involved in inducing the taste sensations (Walters

et al 1991). Generally, with increasing concentration, high-potency

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sweeteners including acesulfame-K, neohesperidin dihydrochalcone, sodium

saccharin, rebaudioside-A and stevioside tend to become bitter. Low-potency

sweeteners including fructose, sucrose, and lactitol tend to become less bitter

with increasing concentration (Schiffman et al 1995).

Sweetness enhancement by odour and colour associations plays a

major role in taste perception (Joseph and Harry 1998). In fruits, ripening is

often associated with a transition from green to red colour, and this often

parallels a major increase in the sugar content of fruits. If green colour in

fruits represents unripeness or a preponderance of acid (sour) taste over

sweetness, then an association of lighter green colour with less acid and

sweeter taste seems reasonable as well. An association of certain acceptable

colours with foods is thought to begin early in our cognitive development and

to stay with us as we age. It is also possible due to a cohort effect that depends

on their specific experiences with some commercial products. For example,

orange coloured soft drinks are associated with acidic taste of orange fruit.

Statistically, colour and odour have a stronger influence on sensory

perception in adults than children. A control over colour and odour thereby

influences sensory perception of specific attributes (Clydesdale 1993).

It is due to the significant contributions of colour and odour on

sweetness perception of stevia, that purification becomes necessary.

Phytochemical synergy is important in determining the health benefits of any

herbal food ingredient. Although the main use of stevia is the sweet taste, the

associated free radical scavenging activity is also important. Furthermore,

processing is an expensive step towards producing a commercially acceptable

sweetener product. When minimal processing is sought to be introduced, the

product must have commercially acceptable organoleptic properties.

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The sweetness of the steviol glycosides varies significantly with

small variations in structure. The magnitude of sweetness and quality of taste

improves according to the number of glucosyl units attached to the C13 to

some extent. Cyclomaltodextrin glucanotransferases (CGTases) were used to

treat stevioside in the presence of starch as a donor. Significant improvements

in the taste profile were observed in the products which were mono and di-

glycosylated at the C13 position (Fukunaga et al 1989). Other factors

affecting taste of the glycosides need to be studied.

1.2 LITERATURE REVIEW

1.2.1 Analytical Methods for Steviol Glycosides

The glycosides of the plant, Stevia rebaudiana, accumulate in all

the photosynthesizing parts of the plant. In wild cultivars, stevioside

accumulates between 8-10% on a dry weight basis in the leaves and

rebaudioside A between 1 and 3%. Selective breeding has developed many

varieties with altered steviol glycoside composition. Many analytical

techniques have also been developed for detection and quantification of these

glycosides in plant and food samples.

1.2.1.1 Thin Layer Chromatography

TLC-colorimetric methods were among the earliest to be

standardized and reported for steviol glycosides (Metivier and Viana 1979).

Silica gel (SG60) chromatoplates were used for TLC analyses of Stevia

sweeteners with isopropanol: ethyl acetate: acetone: water (30:53:2:15, v/v) as

mobile phase and orcinol 0.5 g% in methanol: sulphuric acid (95:5) as

revelator following heating at 10–58 ºC for 2–4 min (Antonio et al, 2005).

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A HPTLC method for steviol glycosides was most recently

described (Jaitak et al 2008). This method utilized pre-coated silica gel

HPTLC 60 F254 plates with a mobile phase consisting of ethyl acetate:

ethanol: water (80:20:12 v/v). Bands were visualized by spraying with acetic

anhydride: sulfuric acid: ethanol (01:01:10, v/v/v) followed by heating on

Camag HPTLC plate heater at 110°C for 2 min. This method provided a

lower detection limit of 80 ng/spot with linearity for quantification between

1-

1.2.1.2 Enzymatic method

An enzymatic method for quantification of stevioside was

standardized (Mizukami et al 1982). This method was based on the hydrolysis

of stevioside by a crude hesperidinase, which releases glucose. The released

glucose was followed by an enzyme based glucose detection system. The

enzyme was able to hydrolyze stevioside efficiently within two hours at 50°C,

but Rebaudioside A took more than 48 hours for complete hydrolysis. Hence,

the assay could be considered specific for stevioside under the conditions

prescribed. The specificity of the enzyme was questioned by the authors,

indicating that the presence of interfering oligosaccharides may give false

results. The advantage of the method was its simplicity and ability to use it in

analyzing large volumes of Stevia rebaudiana leaves.

1.2.1.3 High Performance Liquid Chromatography

High-Performance Liquid Chromatography has been used for

analysis of steviol glycosides. Hutapea et al (1999) developed a method using

a C-18 column with water: acetonitrile linear gradient and measurement at

210 nm. This method was capable of separation and quantitation of steviol

glcosides and their metabolite

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and steviol, found in blood, feces and urine. Kolb et al (2001) used an NH2

column with isocratic acetonitrile/water elution and detection at 210 nm for

separation and quantitation of these glycosides in leaf extracts. Vanek et al

(2001) used a C-18 reverse phase column and a linear gradient of

water/acetonitrile ending in acetonitrile/water in 30 minutes. Detection was at

205 nm with a Photodiode array UV detector. Samples analyzed included leaf

and fruit teas commercially available. The method proposed by Hutapea et al

mL mL. Isocratic elutions are

faster than gradient elutions while providing similar efficiency of separation.

NH2-bonded silica is the most common packing material in the columns used.

The sensitivity and robustness of HPLC methods are their advantage.

Single column HPLC combined with ESI-MS was employed for

optimizing conditions for supercritical extraction of steviosides from the leaf

(Young et al 2002). Negative ion method was found to be more reliable for

stevioside when compared to positive ion and UV mode. Detection limits for

positive and negative ionization modes were 10 ng and 1 ng respectively.

Stevioside also lacks any chromophores hence UV absorption was not

considered a sensitive method for detection.

A two dimensional Liquid Chromatography method combined with

Time of Flight Mass Spectrometry (TOF-MS) was employed for analysis of

leaf extracts (Jaroslav et al 2007). Two dimensional LC provides a high

separation power because of the use of two columns with varying separation

mechanisms. LCxLC combined with ESI-TOF-MS was capable of resolving

the mixture of glycosides and characterizing them. A combination of C-18

and NH2 bonded columns in that order gave the best possible separation.

Single column HPLC was not capable of separating all the eight known

glycosides as reported by these workers.

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1.2.1.4 Capillary Electrophoresis

Capillary electrophoresis (CE) or capillary zone electrophoresis

(CZE) can be used to separate ionic species by their charge and frictional

forces. CE was designed to separate species based on their size to charge ratio

in the interior of a capillary filled with electrolyte. CE offers unparalleled

sensitivity to resolution of analytes with little physical differences. CE in the

micellar mode is a valuable alternative to HPLC. It allows separation of non-

ionic analytes, including plant secondary metabolites. CE was used for the

separation and analysis of steviol glycosides from the plant material (Mauri et

al, 1996). The system used has a linear response to concentrations between

0.2 and 5.0 mg/mL. Although CE is a viable alternative to HPLC in analysis

of steviol glycosides, it is not widely used.

1.2.1.5 Near Infra Red Reflectance Spectroscopy

Near Infrared spectroscopic analysis of stevia leaves for estimating

the content of stevioside in the leaves was proposed (Nishiyama et al 1992).

Near Infrared radiations are capable of analyzing bulk material with little or

no sample preparation due to their ability to penetrate the material deeper. But

analytical techniques based on NIR spectroscopy lack sensitivity. In the

analysis of stevioside, there was an overestimation of the stevioside content.

The proposed method was also sensitive only to the stevioside concentration.

Since NIR spectroscopic analysis was a rapid, high-throughput, non-

destructive technique, it has immense applications in continuous monitoring

during storage and for development of better varieties of the plant. But NIR

spectroscopy has not been widely reported to be used in stevioside analysis.

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1.2.1.6 Advantages of targeting glucose

The proposed assay method targets glucose released from the

glycosides by alkaline hydrolysis. There are multiple methods available for

analysis of glucose in clinical samples. Glucose monitoring is important for

diabetic patients. Hence, methods of assay of glucose have been developed

for increased accuracy, sensitivity, range of sample conditions and robustness.

High throughput automated methods for glucose analysis in plasma and urine

samples have become fairly routine in clinical labs. This ubiquitousness made

glucose an ideal target for assaying when trying to determine total level of

steviosides in the sample. The glucose oxidase based method for assay of

glucose was proposed in 1956 (in Fales 1963). But the peroxide coupling

reaction was reported to be prone to much interference from components

present in biological samples. A hexokinase coupled to Glucose-6-phosphate

hydrogenase system where the reduction of NAD was followed at 340nm was

later proposed and evaluated (Walter et al 1971). This method although

validated to be accurate and reliable is not quite as popular as the Glucose

oxidase based method for clinical samples.

The advantages of the glucose oxidase method are readily available

chemicals, stable solutions, specificity and the ability of the user to adjust the

sensitivity of the reaction over a wide range of glucose concentrations (John

and Kathie 1975). The reagents are not hazardous and are capable of being

adapted to the auto analyzer.

The glucose oxidase based system has been adapted to a sequential

injection system with spectrophotometric detection, where the enzyme is

immobilized in glass beads packed in a micro-column. This method has been

successfully applied to real world samples (Kanchana et al 2007). Sequential

injection analysis coupled with chemiluminscent detection was found to be

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advantageous since the enzyme need not be immobilized (Anastasias et al,

2006) and similarly glucose oxidase also formed the basis of a Lab-on-Chip

system for glucose detection (Vijay et al 2004) The ability to immobilize the

enzyme and couple it to a sensor was used to develop a needle type sensor for

monitoring blood glucose in fish (Hideaki et al 2006). Auto analyzers utilize

an oxygen electrode instead of the coupled dye method in the manual method.

Both methods are linear until 25mM glucose concentration after which it

becomes necessary to dilute the samples. (Violet and John 2000) The

increased sensitivity offered by the enzymatic method ensured that highly

turbid plasma samples could also be analyzed since small volumes of samples

were required. When turbidity is present, it is necessary to appropriately

dilute the samples. Triple enzyme electrodes for simultaneous detection of

glucose, L-Lactate and pyruvate were constructed by immobilizing the

respective enzyme detection systems on platinum disks (Toshio et al 2004).

It is not necessary for pure enzyme to be available. Microbial biosensors

working on the same principle of glucose oxidation have been developed

(Min et al 1997). Some adaptations of the glucose oxidase based method

include amperometric sensing using bilayer electrodes (Naruhide and

Hirokazu 2006).

Many more sensitive methods for analysis of glucose are available.

Post-column derivatisation of glucose by iodine and subsequent LC/MS based

detection have been proposed (Eduard et al 2007). The limit of quantitation

was approximately 50pg on the LC column. Non-enzymatic electrochemical

sensors are popular despite a number of problems including poisoning of the

electrode and irreproducible current responses. Boron doped diamond

electrodes were successfully applied to the detection of glucose at different

concentrations. These electrodes provide long term reliability and are easily

regenerated (Joowook and Su-Moon 2005). Capillary electrophoresis which

was successfully used for stevioside detection has also been applied for

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glucose detection and quantitation (Gang et al 2005). But among all the

systems proposed, enzymatic biosensors have been studied extensively and

adapted for a wide variety of applications. The review of literature suggested

an abundance of glucose assay systems which were sensitive, robust and

widely used.

1.2.1.7 Outline of work

There are both quantitative and qualitative methods available for

analysis of steviol glycosides. TLC provides an efficient method of separating

and identifying the glycosides quickly. The other chromatographic methods,

HPLC, HPTLC, GC and CE provide rapid and sensitive methods for detection

and quantification of the glycosides in plant material and food matrices.

All methods rely on the separation of the glycosides before

individual quantification. When expressing quality of leaf material or

concentration of a mixture of these glycosides in food, the quantities of

individual glycosides are usually added together. The above mentioned

methods utilize external standards of individual glycosides. While trying to

find the total glycoside concentrations in the plant material, all the known

glycosides have to be procured and used as standards. A new method to

overcome the cumbersome method of quantifying using individual standards

was felt.

In addition, leaf material had to be analyzed at source for

determining quality and commercial value. Laboratories equipped with the

expensive and sensitive instrumentation required for analysis of the leaf are

not commonly found in rural areas. Hence, a robust method that would work

on a ubiquitous assay platform would become widely accepted.

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The method outlined in this work addresses both these concerns by

targeting glucose, released from the glycosides by an alkaline hydrolysis step.

The hydrolysis step can be easily performed by using a hot plate and glucose

assay kits which are widely used for routine blood analysis. The ability to

work with interference from other components of the leaf material was

evaluated.

1.2.2 Purification Strategies for Steviol Glycosides

Aqueous and alcoholic extracts of stevia are associated with a

number of impurities, including plant pigments and phenolics which

contribute to colour, odour and taste that are not intrinsic to the molecule.

Brown to yellow colour is contributed by phenolics which also affect the

stability of any formulation made out of a semi-pure extract, since phenolics

are easily oxidisable. Green and yellow colour is contributed by chlorophylls

and xanthophylls which impart a leafy odor and taste to the extract. Odour is

contributed by volatile components which again contribute to herbal or leafy

sensations. Taste is affected by the presence of polyphenolic components.

Low molecular weight phenolics are astringent and high molecular weight

phenolics are bitter. Aqueous extracts of stevia typically contain polymeric

hydrolysable polyphenols contributing to a bitter taste, which acts to amplify

the inherent bitterness of the molecule. Purification was felt to be necessary

due to the above reasons.

1.2.2.1 Fractionation/Ion-exchange/Crystallization

Even though it has low solubility in water, stevioside is soluble in

dioxane, methanol and alcohols up to butanol, insoluble in chloroform,

hexane and other non-polar solvents. Choice of solvent, sequence of usage,

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temperature and pH in the case of aqueous extraction, influence the extraction

efficiency as well as the amount of impurities eluted out.

One of the earlier patents for purification of stevioside (US Patent

No: 3723410) involved defatting of coarsely ground leaves with chloroform

and subsequent extraction with dioxane or water in the presence of an alkali.

In this case the alkali served to precipitate out the phenolic acids by salting

them out of the organic phase. Ion-exchange resins were already being used

for purification of the molecule, the resin of choice being Amberlite IR-120

(Harry et al 1955). In both processes the final step was crystallizing out of the

molecule from cold methanol before recovery. Strong acidic and weak basic

ion exchange resins have been used on a decoction of stevia leaves, followed

by filtration to obtain pure products (US Patent No.: 4892938). This was also

one of the first patents to completely eliminate the use of organic solvents in

the processing.

One major modification in later methods involving solvent

extraction was the use of chelating agents which perform the function of the

ion-exchange resin in removing charged impurities (US Patent No:4599403).

The chelatant of choice was organic di or tricarboxylic acids. Phosphoric acid

was the only mineral acid capable of producing the desired reduction in color.

Phosphoric acid has the added advantage of contributing phosphate ions

which are nutritive when the residual sludge is used as animal feed. The acids

could remove metal ions, proteins and some phenolic acids from the extract.

Calcium oxide and hydroxide served to precipitate out impurities that are not

chelated by acidic precipitation. These include plant pigments contributing to

green and yellow colour. Subsequent fractionation of the molecule into a

water immiscible alkanol gave further purification. Addition of acids and

alkaline salts for clarification provided the advantage of cost, easy waste

handling and simple unit operations.

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Stevioside was reported to have a greater stability in methanol at

temperatures between 1200C and 1400C (Jaroslav et al 2007). In addition, the

authors also concluded that methanol is better for extraction in both static and

pressurized conditions. Water was concluded to provide a “green” alternative.

Supercritical fluid extraction has been proposed as a method increasing the

efficiency of extraction as well as affecting the composition of the final

product by adjusting the ratio of Stevioside and Rebaudioside A (Pasquel

et al 2000). A Japanese patent held by Tan (Japan Patent No.: JP-A-

62-000496) involves the use of acetone along with the aforementioned

solvents and further purification was achieved by adsorption. US Patent No.

5112610 simply described a method for using supercritical CO2 to remove

taste-impairing components without describing them. The gas was employed

in leaves, crude extracts, purified extracts in solid form as well as in liquid

extracts.

1.2.2.2 Adsorption Chromatography

Separation by column chromatography on a silica stationary phase

was easily accomplished. HPLC provided the advantage of greater separation

efficiency and faster flow rates. Derivatised silica, either Octadecylsilane

(Vanek et al 2001), glycerylpropyl (US Patent No: 4361697) or aminopropyl

(Kolb et al 2001), are commonly used. Cross linked starch and styrene gels

have also been used on HPLC systems (US Patent No.: 4171430). HPLC

though has the disadvantage of higher capital and operational costs.

Highly cross linked copolymer of ethyl styrene and divinylbenzene

with about 3% methyl-methacrylate has a high adsorption and desorption

capacity for steviol glycosides. Macroporous strongly basic anion exchange

resins with –N+(CH3)3 has a high decolourisation capacity. Shi et al (2002)

were able to synthesize a bifunctional polymeric adsorbent that combined

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both these activities by introducing the –N+(CH3)3 groups into the AB-8

adsorbent. Steviol glycosides could be desorbed from this polymer by

aqueous ethanol, whereas pigments required that the eluting solvent contain

sodium chloride or hydroxide. Based on the adsorption pattern, the authors

were able to conclude that steviol glycosides interact with the polymer based

on hydrophobic interactions, whereas the plant pigments had much more

complex mechanisms of interaction.

1.2.2.3 Filtration Techniques

Ultra and Nano filtration techniques provide means of separating

the steviol glycosides from the extract of leaves, with minimal use of solvents

and chemicals. There is also less generation of waste. Zhang et al (2000)

described a scheme for separation of the glycosides based on the use of ultra

and nanofiltration units. While pH did not affect the elution of the steviol

glycosides in the first stage of water extraction, lower pH reduced the amount

of coloured impurities. This was the basis of a patent (US Patent

No.:5972120). Suspended particles from the extraction step was removed by a

ceramic micro filter with a mean pore size of 0.3-

membrane with a cut-off size of 2.5-3.0KDa allowed the glycosides to pass

through while retaining the higher molecular weight impurities. Finally

nanofiltration allowed for concentration and removal of impurities which had

lower molecular weights. One additional step of ion-exchange resin based

clarification could allow for the production of high purity steviol glycosides.

A recent permutation of unit operations has allowed for supercritical

extraction to be used in conjunction with nanofiltration for increased

efficiency of separation (Sarrade et al 1998). This could find useful

applications in stevioside purification given our knowledge of supercritical

extraction of the glycosides.

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1.2.2.4 Evaluation of Process Choice

Optimum economic design of a process involves choosing the

process with least cost among many, provided the end results are the same. In

comparing different processes for phytochemical purification, the following

factors have to be considered (Peters and Timmerhaus 1991)

a. Technical – flexibility of process, yields, energy and

equipment requirement, health and safety hazards.

b. Raw materials – present and future availability, handling and

processing requirements.

c. Waste products – amount and value, method of disposal.

d. Location, time and cost evaluations

e. Technology availability.

In the case of extraction of stevia leaves, the end-use of the product

determines the processing method employed. In Japan, stevia based

formulations are mainly used in soy sauce, pickles, dried seafood and miso.

The sweetness of stevia is offset by its residual bitterness. Hence, in such

foods, stevia extracts are mixed with glycyrrhizin, for better sensory

attributes. Purification is not necessary for such an application and most

“stevia extracts” available in the Japanese market are aqueous decoctions

clarified using adsorbent resins (Mizutani and Tanaka, 2002). In Korea, the

major consumers are Soju manufacturers, who use 50% of the total

manufactured stevioside in the country (Kim et al 2002). Soju, a clear liquor,

therefore requires purified steviol glycosides. A laboratory scale process for

producing 63% pure steviol glycosides from stevia leaves was developed at

the Institute for Himalayan Bioresource Technology, Palampur, India.

This technology involves aqueous decoction, filtration and crystallization of

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the glycosides (Megeji et al 2005). Extraction efficiencies of the glycosides as

described in patents, increased with the progress of time (approximately 6.5%

in US Patent No. 3723410, 7.5% in US Patent No. 4599403, 8% in US Patent

No. 4892938). However all the reported processes, except that based on

membrane separation, generate large volumes of sludge, use significant

quantities of chemicals and have complex steps. But the membrane based

process has the disadvantage of increased capital cost including replacement

of fouled membranes periodically and energy costs of operations. The energy

required for evaporation of the extracting solvent plays a significant role in

operating costs. While water is preferred as a cheap, easy to handle and safe

solvent of choice, its latent heat of vaporization is 2257 kJ / kg which is

nearly double that of methanol (1100 kJ / kg). Hence, choice of extraction

solvent determines the energy cost to a large extent. Membrane processes

used for desalination typically provide pure water at 50% of the cost of

distillation units (Malek et al 1996). A survey of processing strategies

employed by companies involved in purification of steviol glycosides reveals

a preference to aqueous extraction and liquid-liquid partitioning for

purification. Recent advances in structure-activity relationship have made the

manufacture of modified glycosides by enzymatic transglycosylation more

lucrative. These products have steviol glycosides with glucose units added on

the C13 for better taste perceptions (Fukunaga et al 1989).

Electrocoagulation for removing the pigments provides an

interesting method of reducing solvent usage to remove chlorophylls

(Jumpatong et al 2006). This process needs further study before it can be used

industrially. Flavonoid components of stevia leaves have been characterized

(Rajbhandari and Roberts 1983; Putieva and Saatov 1997). But tannins, which

comprise nearly 7.8% w/w of stevia leaves, have not been characterized yet.

These tannins contribute substantially to the colour and knowledge of their

chemistry will help in fine tuning the current processing methods available.

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1.2.2.5 Free Radical Scavenging Assays

A number of radical scavenging capacity (RSC) assays have been

established and widely used for the rapid screening and evaluation of novel

antioxidant preparations using peroxyl, hydroxyl, cation ABTS (2,2'-azino-

bis(3-ethylbenzthiazoline-6-sulphonic acid)), peroxide anion, and

2,2-diphenyl-1-picryhydrazyl (DPPH) radicals (Cheng et al 2006). The stable

DPPH and the chemically generated ABTS are still widely utilized in

antioxidant research due to their simple reaction systems, which involve only

the direct reaction between the radical and the antioxidants, and have no other

interference such as enzyme inhibition or the presence of multiple radicals,

although they are not physiologically relevant. In contrast to the chemically

generated ABTS, DPPH may be utilized in aqueous and nonpolar organic

solvents such as benzene and can be used to examine both hydrophilic and

lipophilic antioxidants. The DPPH scavenging capacity assay performed in

organic solvents may evaluate lipophilic antioxidants without any additional

solubilizing agents such as the â-cyclodextrin, required in the oxygen radical

absorbing capacity (ORAC) (Cao et al 1993) and peroxyl radical scavenging

capacity assays, which has been reported to have a strong interference in HO

scavenging capacity estimation. The DPPH scavenging capacity assay is

considered to be a valid and easy colorimetric method for antioxidant

property evaluation. This assay has been successfully utilized for

investigating antioxidant properties of wheat grain and bran, vegetables,

conjugated linoleic acids, herbs, edible seed oils, and flours in several

different solvent systems including ethanol, aqueous acetone, methanol,

aqueous alcohol, and benzene. Hence it was the method of choice in

following the free radical scavenging activity of the stevia extracts.

23

1.2.2.6 Outline of Work

Many physical and chemical processes known for separation of

molecules have been employed and standardized for the purification of steviol

glycosides. All of these processes had the objective of complete purification.

Whole extracts of the plant contain components that have similar

solvation properties; hence multiple solvent fractionation steps are required

for purification. Chromatographic mobilities of these molecules are also

similar requiring stringent choice of adsorbents and mobile phases. Membrane

fouling by the organic components of these extracts increases operational

expenses either in pretreatment of the extracts or in replacement of

consumables.

The whole extracts of the plant contain phytochemicals which

possess qualities that are desirable. The polyphenols contribute to the anti-

oxidant activity of the extracts. In most of the foods that they are added to,

this activity is synergistic. Removal of these polyphenols is wasteful.

Hence a method for modifying or removing the pigments, that

cause the aforementioned problems while retaining the advantages of

phytochemical synergy, was needed. Biological systems provide the means to

selectively modify the colour and odour causing components in an extract

containing mixtures of molecules. Systems based on bioremediation are used

in treatment of industrial and municipal effluents. Similar fermentation based

on fungi was employed to evaluate the utility of this process in decolourising

extracts of Stevia rebaudiana.

24

The efficiency of decolourising the extract was monitored

spectrophotometrically. Enzymes required for decolourising the extract were

profiled during the fermentation. The addition of dextrose and its influence in

decolourisation of the extract was established. The anti-oxidant activity of the

extract was followed to ensure the objective of retaining the beneficial

properties of the whole extract was achieved.