Food Safety Stevia

Stevia:
Prospects as an Emerging
Natural Sweetener
VEENA SHARMA
INTERNATIONAL FOOD DIVISION
Assisted by
Dr D. Chattopadhya
Assistant Director General (IF)
2007
CONTENTS
Chapter 1: Introduction
1.1
1.2
1.3
1.4
1.5
Definition
Background
Description
Status of Stevia in World
Marketing and Economic issues
Chapter 2: Status of Stevia in India

2.1 Effect of method of planting on Stevia
yield
2.2 Cultivation
2.3 Cultivation parameter for Stevia
production in India
2.4 Economy of Stevia as sweetener in
India
Chapter 3: Commercial extracts of Steviol
glycoside
3.1
3.2
3.3
3.4
3.5
3.6
Traditional method
Improved method
Product quality
Taste quality improvement
Extraction of Steviol glycoside
Detailed process
Chapter 4: Chemistry
4.1 Steviol glycosides
4.2 Detailed specifications of
Steviol glycosides as developed by
JECFA
4.3 Studies conducted other than JECFA.
4.4 Sensory evaluation of Stevia species.
4.5 Electrophysiological and behavioral
assays with Mongolian Gerbil
Chapter 5: Toxicological Studies
5.1 Stevioside
5.2 Steviol
5.3 Studies conducted other than JECFA
Chapter 6: Uses of Stevia
6.1
6.2
6.3
6.4
6.5
Need of intense sweeteners

Classification of sweeteners
Relative sweetness
Benefits of Stevia
Recommendation by JECFA
Chapter 7: Stability studies of Stevioside and

Rebaudioside A
7.1 Stability studies of pure sweetener
7.2 Stability of pure sweetener in food
system
Chapter 8:
8.1
8.2
8.3
8.4
8.5
8.6
8.7
Legal Regulations regarding the

Use of Stevia
USA
Canada
European countries
Japan
Australia and New Zealand
Hong Kong
Singapore
Chapter 9: Pharmacological Properties

9.1 Absorption, Distribution and
Excretion
9.2 Biotransformation
9.3 Effects on enzymes and other
Biochemical properties
9.4Antihyperglycemic effect of Stevioside
9.5 Effect of Stevioside on cardiovascular
system
9.6 Antihypertensive effect of Stevioside
9.7 Anti inflammatory and
Immunodulatory
activities
of
Stevioside and its metabolite Steviol
9.8 Hypoglycemic effect of Stevioside

9.9 Cariogenic study of Stevioside and
Rebaudioside.
Chapter 10: Estimation of Stevioside in food
10.1 Simultaneous determination of GA,
RA and St in food
10.2 HPLC method for the determination
of sweet tasting Stevioside in S.
Rebaudiana and in beverages
10.3 Analysis of sweet diterpene
glycoside of Stevia Rebaudiana
10.4 HPLC determination of Steviol in
biological fluids and plant material
with fluorescence detection
10.5 Quantification at the picomol level
by HPLC
Chapter 11: Advantages of Stevia and Steviol
glycosides.
Chapter 12: Proposal for the suitability of
Stevia as sweetener
Chapter 13: References
LIST OF TABLES
Table
no.
Topic
1. List of countries where Stevia is

grown and researched.
2. Climatic
features
of
agronomic
research location of Stevia in world.
3. Stevia leaf analysis in major Stevia
producing countries.
4. Trial crop yield in various countries.
5. Effect of method of planting on Stevia
yield.
6. Herb yield of two accessions of Stevia
rebaudiana.
7. Costing of Stevia cultivation in India.
8. Cost of Stevia leaves as per IHBT.
9. Sweetness equivalence of Stevia in
comparison to sucrose.
10. Organoleptic evaluation for sweetness
of Stevia leaf herbarium samples.
11. Acute toxicity of Stevioside.
12. Genotoxic study of Stevioside.
13. Genotoxic study of Steviol.
14. Nutritional composition of Stevia.
15. Functional properties of Stevia leaf
powder.
16. Uses of Stevioside.
17. Shelf life study of Stevia sweetened
products.
18. GI of Stevia containing products.
19. TLC analysis of Stevioside.

20. HPLC analysis of Stevioside.
21. Interaction of Stevioside with organic
acids.
22. Characteristic of flavobacterium
johnsonae
23. Long
term
storage
studies
of
Stevioside in carbonated beverages.
24. Microbiological studies of Stevioside
in carbonated beverages.
25. Effect of heating on Stevioside
sweetened carbonated beverages.
26. Abstracts from the record of views
formed by the FSANZ Novel Foods
Reference Group.
27. Recovery of artificial sweeteners by
HPLC.
28. Limits of detection for artificial
sweeteners.
29. HPLC characteristics of Stevioside
metabolites.
30. HPLC limit of detection of Stevioside
in food samples.
Introduction
Chapter 1
In the last couple of decades, growing concern about health

and life quality has encouraged people to exercise, eat
healthy food and decrease the consumption of food rich in
sugar, salt and fat.
Omission of added sucrose in foods increases the relative
proportion of polymeric carbohydrates that may have
beneficial effect for a balanced food intake as well as for
human health1.
In addition, there has been an increase in the demand by
consumers for food with functional properties.
Changes in eating habits and lifestyle are mainly due to
incessant search for health. In the past, food science was
concerned with the development of food for human survival,
a goal that was substituted by the concept of production of
quality food2.
More recently, the main concept has become to use food as
a means of promoting health and welfare, while reducing
the risk of disease3 .The food industry has responded to this
demand and as a consequence, there has been a fast
growing increase in diet foods and beverages available to
consumers in many markets of the world4.
With increased consumer interest in reducing sugar intake,
food products made with sweeteners rather than the sugar
have become popular. Sweeteners are alternative
substances to sugars, which give food a sweet taste and are
used to partially or totally replace sucrose 5
The discovery of great number of sweeteners during the last
decade has triggered the development of sugar free
products, particularly for diabetic, obese people and for
dietetic purpose6.
Sweeteners such as nutritive (Polyols) and nonnutritive/intense sweeteners (Artificial and natural) have
become alternatives to replace sucrose and have been widely
used in various food products.
Natural sweeteners are mainly plant constituents. Plants
have contributed to about 75 highly sweet compounds.
These sweet compounds fall mainly within the terpenoid,
flavonoid and protein compound classes, although
altogether nine districts structural groups of potently sweet
molecules have been derived from plants7.
So far, highly sweet compounds have not been documented
as these occurred in lower plants, insects or native
organisms and the taxonomic distribution of plants, known
to biosynthesize highly sweet compounds, is random within
the angiosperm super order as classified according to
Dahlgren7.
Several highly sweet plant constituents are used
commercially as sucrose substitutes in one or more
countries. The plant secondary metabolites of most
widespread interest in this regard are Steviol glycosides i.e.
Stevioside and Rebaudioside A, constituents of the Stevia
rebaudiana bertoni. These two products, made from S.
rebaudiana are widely available in Japan, with Stevioside
approved as a sweetener in Brazil and having limited use in
Korea too. 7
In India, the use of artificial sweeteners in food products
has not been very common so far when compared with the
majority of western countries. However, over the past
decade, there has been a steady increase in many Indian
retail foods that are labeled as diet and /or light.
Contrary to the situation on the late 1980s when only
people with health problems (e.g. diabetes or high blood
cholesterol) used to buy these products, many Indians have
now started to consume low calorie foods and are eating less
sugar and fat as part of their main diet.
Given the reasonably sound track record of plant

constituents and particularly S.rebaudiana (Stevioside
glycoside) as Intense sweetening agents and because of the
great public demand for natural food ingredients,
particularly for diabetic and dietetic applications,
FSDU, PFA has worked on the prospects of Steviol
glycosides as sugar substitute.
This comprehensive document on S.rebaudiana have
included aspects for legal regulations of various countries,
marketing and economic issues, status of stevia in India,
commercial extraction, practical application of Stevioside in
foods and beverages stability and Organoleptic studies ,
estimation of Stevioside when present in food or other
samples. As well as botanical field and literature studies,
chemistry, toxicological, mutagenicity, pharmacological
properties, electrophysiological and behavioral methods for
natural sweetener detection using Mongolian Gerbil and
Cariogenicity study on Stevioside and Rebaudiana A.
1.1 Definition
1.1.1 Stevia [Stevia Rebaudiana]
Stevia is a herb with incredible sweetening property. Its
ability to sweeten is rated between 70 to 400 times that of
white sugar. Typically, it has a mild licorice like taste and is
completely natural in its biochemical profile8.
Synonyms: Sweet herb, sugar leaf and honey leaf.
Parts used: Leaves
1.2
Background
Stevia is a plant, indigenous to mountainous regions of

Brazil and Paraguay. For centuries this herbal sweetener
has been used by native cultures to counteract the bitter
taste of various plant base medicines and beverages8.
S.rebaudiana (Bertoni) was rediscovered by Europeans in

Paraguay in 1888 by Dr. M. S. Bertoni. He later botanically
described and named the plant (1905) in honor of
Paraguayan chemist Dr. Rebaudi.
Historically the natives of Paraguay and Brazil have been
using the leaves of stevia as a sweetening essence for tea.
Half a century later the British tried to cultivate it as a
replacement for sugar, but the idea never materialized.
Three decades later in 1971. Japanese brought the seedling
of stevia from Brazil and six years later Japan marketed a
sweetener extracted from the stevia leaves9
S.rebaudiana has been carried to too many countries since
first described by Bertoni and has subsequently been grown
in latitudes well north of its native tropic of Capricorn
latitude.
Stevia products are used commercially extensively in Japan,
using locally grown and imported (mainly from China) dried
stevia leaves where (at over 2000 tonnes refined products)
they make up over 40% of the non-sucrose sweeteners (the
others being fructose, syrups, honey etc.) and 5 to 6 % of
the total sweetener market.
In most other countries where it is used it is mainly used
directly by consumers, rather than commercially. Domestic
consumers utilize dried leaves, liquid extracts, crystals or
powder to their drinks and cooking as an herbal
supplement9.
The main Stevioside producing countries are China and
Paraguay with adjacent parts of Brazil. China is a main
supplier to Japan, who is the main commercial producer
and user of Stevioside. Paraguay or Brazil is the main center
for the production and distribution of Stevia products direct
to consumer via the health food and herbal product outlets
and by direct order sold around the worlds. There are the
numbers of processors in Paraguay and Brazil who have
company plantation of 2 to 300 hectors or more as well as
numerous small holders suppliers. 10
1.3
Description
Stevia is a small perennial shrub with green leaves that

belongs to the aster (asteraceae) or chrysanthemum family
of plants. They grow primarily in the Amambay mountain
range of Paraguay but over 150 various species of stevia
including Stevia eupatoria, Stevia ovata, Stevia plummerae,
S.rebaudiana, Stevia salicifolia and Stevia serrata have been
identified around the globe8
The distribution range of this taxon extends from
Southwestern U.S.A to Northern Argentina, through Mexico,
Central America, the South American Andes and the
Brazilian highlands.
Eight sweet ent-kaurene glycosides viz Stevioside,
Rebaudiosides A-E, Ducloside A and Stevioside have been
identified from S.rebaudiana Bertoni. High concentrations of
these sweet principles accumulate in the leaves of this
specie, with yields of over 10 % w/w of Stevioside, the most
abundant representative of this series, having been
reported9.
Extracts of S. rebaudiana are currently used commercially
in Japan for sweetening variety of products including
pickled vegetables seafood, soft drinks, and soy sauce and
confectionary products. In addition, S. rebaudiana
sweetening preparations were recently approved for sale in
Brazil.
The use of S.rebaudiana as a sweetener can be found in
many parts of Central and South America, where these
species are indigenous, as well as in Japan.
S.rebaudiana is the only species at present which posses an
inordinate ability to sweeten. Its common form is known as
Stevioside, a fine white powder extracted from the leaves of
the plant10.
1.4 Status of Stevia in World

Table 1: List of countries where stevia is grown and
researched
Country/Location Commercial
Production [1]
1.
South America
Paraguay
Uruguay/Brazil
Central America
Mexico
USA
Canada
China
Vietnam
Taiwan
Japan
South Korea
Thailand
Malaysia
Indonesia
India
Georgia
Russia
Ukraine/Moldova
Spain
Italy
United Kingdom
Germany
Sweden
1
[Agricultura Nonl] Research [Agricultural]

Research.
Approved
for use
Commercial production excludes small quantities grown for domestic use 9
Table 2: Climatic Features of Agronomic Research Location[s] for Stevia in various parts of
the world.
Ref Location
[12]
[13]
[14]
[15]
[13]
[16]
[17]
[14]
[18]
[19]
Latitude
Degrees
60N*
Rainfall
(mm)
-
47N*
45N*
600
-
Altitude
(meters)
<200
<200
<200
-
Canada
Georgia
43N*
43N*
250-300
<200
California
Korea-Suweon
38N*
37.5N*
1250
-
<200
<100
Central China
Japan-Kyushu isl.
-Okanawa
Mexico
32N*
32N*
26N*
25N*
2000
2475
2150
200
<200
<50
<50
<200
Coastal
Coastal
Island
Coastal
St Petersburg
Russia, Voronegh
Moldova/Ukraine
North China
Topography Comments
Plains
Plains
Plains
-
Hielongjjang
Prov.
Irrigated
Black
Sea
Coast
Davis,
irrigated
Jiangsu
Prov.
Tropical
conditions
Irrigated
[20]
[18]
[21]
[22]
[18]
[18]
[23]
Taiwan
Thailand
India-Bangalore
24N*
18N*
13N*
1260
-
300
930
Uplands
Uplands
Indonesia- Bogor
Surakata
7 S**
7S**
1800
2300
400
1000
Slopes
23S**
Brazil Maringa
Paraguaynative 23S**
locality
1260
1500
1800
500
200-400
-
Uplands
Inland
Valleys
6mth
dry
season
3mth
dry
season
400
km
from coast
Amambay
Mountain
Ranges
* N: North, ** S: South
Table 3: Stevia Leaf Analyses in major stevia producing

countries
Ref
[23]
[24]
[25]
R-A** R%
A/St
Ratio
Paraguay average 8-14 2-4
0.4
Paraguay-typical
5-10 2-4
0.4
Paraguay-wild
China-average
6.44 3.86 0.6
Location Cultivar
St*
%
Total
Sweetener
10-15%
9-15%
10.2-13.5%
10.3%
*St: Stevioside, **R-A: Rebaudioside A

Table 4: Trial Crop Yield in various countries
Ref
Trial
Location
Yield
Total
[16]
California
Russia
Russia
ChinaV9
Average
Jiangsu
North
China
[26]
[12]
[14]
[27]
[28]
[21]
[15]
9.0
13.8
-
T/haDry
leaf
3.6
5.5
1.4-3.0
1.40
Steviosides
Kg/ha
Total R-A% St% Stevioss
7%
*9%
*9%
20.4%
252
495
125-270
265
12.4%
-
186
267
Indonesia
India
1.3
plus
67kg
Seed
-
1.742.16
8.0
Canada
2.85
15-20
297.12338.16
425-570
Not given; assumed value used to calculate kg/ha Stevioside.
[T=Ton; ha=Hectare]
1.5 Marketing and Economic Issues

Current markets are restricted to Japan, where there is wellestablished commercial market, and markets for unstructured
diverse health food, natural food and mail orders. The Japanese
market is broadly described as over, 2,000 tones of Stevioside
(1996) or 40% of the non-sucrose sweetener market. The
health food/mail order market is primarily supplied from
processing companies in Brazil and Paraguay often via
distribution centers in the USA.9, 11
Information on prices is not available but ranges, as suggested
relative to the price of sugar (on a sweetening value), being
usually slightly above to 25% above. Prices of chemical
sweeteners, for the sake of comparison are apparently similar to
the equivalent sweetening quantity of sugar. Soft drink
manufacturers can and to sell diet drinks at the same price as
sugar sweetened drinks.
Some processors in Japan use patented procedures. In Canada
2,200 kg leaf/ha of production equals $8,500 (a price of Can
$3.85/kg is indicated for dried Stevia leaves). This is
approximately Au$40,000 per ton of Stevioside (10% leaf
content). One ton of Stevioside is equivalent in sweetening value
to 275 tonnes of sugar, which, at $290 per ton of sugar, values
one ton of Stevioside at $79,750. Therefore it would appear that
the price paid for leaves is approximately 50% of the equivalent
raw sugar value. And so, these prices would leave the other
50% for the cost of extraction and refining. 9, 11
Wholesale prices paid for Stevioside crystal in Australia by
health-food packers and distributors are approximately $
100/kg (e.g. Wonder foods, Brisbane). Retail mail-order prices for
stevia crystals or powders (pure 91% -96% dehydrated water
extract) are quoted from US $ 145.00/kg in bulk packs and
from $280 up to $ 615 /kg is small 15-60 gm packs. 9, 11
10
There is also a range of stevia blend powders and crystals and

also liquid extracts/syrups available. These are generally in
packs of less than 120 gm (but up to 450gm) and include
tablets and tea bags. The concentrations of these packs are
not given and prices vary enormously.
For stevia growers, break-even dried leaf yields in Canada are
suggested to be 2,200kg/ha to cover a cost of $ 8,500/ha. If
multiple harvests per year can be achieved with plants
producing for 2-4 years, costs would be significantly reduced,
even if a seedling nursery with transplanting were necessary.
Direct seeding would reduce costs still further.9, 11
11
Status of Stevia in India
Chapter 2
Stevia is a natural herb native of Paraguay, cultivated as a cash

crop in number of countries. There appears to be no large-scale
mechanized production of stevia due to difficulties in producing
the crop through seeds.
In India cultivation of stevia as a crop is still restricted to the
research level. However Department of Ayurveda, Yoga &
Naturopathy, Unani, Siddha and Homoeopathy (AYUSH)
Government of India has sanctioned proposals for the prospects
of S.rebaudiana cultivation in various states like West Bengal,
Uttranchal, Haryana and Punjab29.
2.1
Effect of method of planting and fertilizer on Stevia
yield
2.1.1 Method of planting
The field experiment for stevia cultivation was conducted in
summer season of 1995 G.K.V.K, University of Agricultural
Sciences, Bangalore on alfisols. The soil was medium in
available nitrogen, phosphorus and potassium with pH 6.5.
The treatments included two methods of planting viz., control,
20:10:15, and 40:20:30 and 60:30:45 kg NPK ha-1. The eight
treatment combinations were replicated thrice in a factorial
randomized block design. Entire fertilizer dose was applied as
basal application. The seedlings were transplanted at a spacing
of 45 cm x 22.5 cm30
The crop was harvested 100 days after transplanting and the
following observations were recorded viz biomass yield, dry leaf
yield, which were then subjected to statistical analysis.
Planting methods did not exhibit significant influence on
biomass yield, fresh leaf yield and dry leaf yields which are as
follows:
12
Table 5: Effect of method of planting on stevia yield30

Treatment
a) Flat bed
method
b) Ridges and
furrow method
(P=0.05)
a) 0:0:0
b) 20:10:15
c) 40:20:30
d) 60:30:45
(P=.05)
(P=0.05)
Biomass yield
(mg ha-1)
Fresh leaf yield

(mg ha-1)
Methods of planting
37.38
10.67
38.40
Dry leaf
yield
(mg ha-1)
2.59
11.20
2.70
NS*
NS*
Fertilizers levels (kg NPK ha-1)
26.69
7.38
32.67
9.00
45.46
13.53
46.72
13.81
2.36
1.16
Interaction
NS*
NS*
NS*
1.82
2.25
3.22
3.29
0.29
NS*
* NS: Not Significant

However, ridges and furrow method produced marginally higher
biomass yields (38.40 mg ha-1), fresh lead yield (11.20 mg ha-1)
and dry leaf yield (2.70 mg ha-1) compared to flat bed method.
Shock (1982) also reported that the ridges and furrow method
was the best method of planting. 30
2.1.2 Effect of fertilizer levels
Higher biomass yield (46.72 mg ha-1), fresh leaf yield (13.81 mg
ha-1) and dry leaf yield (3.29 mg ha-1) were obtained due to
addition of 60:30: 45 kg NPK ha -1. However, the fertilizer levels
60:30:45 kg and 40:20:30 kg NPK ha-1 were at par with each
other and significantly superior to 20:10:15 kg NPK(Nitrogen,
Phosphorous & Potassium) ha-1 and control. The higher dry leaf
yield with either 60:30:45 or 40:20:30 kg NPK ha-1 was caused
by higher growth attributes, since the dry leaf yield was highly
associated with dry matter production (r=0.987**), number of
branches per plant (r=0.896**) and number of leaves per
plant(r=0.803**). Murayama et al. (1980) and Goenadi (1985)
have showed that application of higher levels of NPK produced
better growth and dry leaf yield than lower levels of NPK. 30
13
2.2 Cultivation:
Stevia is self-incompatible in nature; hence propagation
through seeds is a difficult proposition. Therefore, vegetative
propagation or micro propagation is the means of propagation
of stevia. Chalapathi (1996) standardized the vegetative
propagation technique using stem cuttings. The 15 cm length of
cuttings was found to be optimum and pretreatment of cuttings
with Paclobutrazol at 50-100 ppm resulted in good sprouting
and rooting. Ashwini (1996) standardized the micro propagation
technique.31
Stevia can be grown on a wide range of soil with pH range from
slightly acidic to neutral, but soil should not be saline 32
A field experiment was conducted at University of Agricultural
Sciences, Bangalore to study the effect of length of stem
cuttings and growth regulators on vegetative propagation of
Stevia (Stevia rebaudiana bertoni). The sprouting percentage
and shoot growth of sprouted cuttings were significantly higher
with 15 cm cuttings compared to 7.5 cm cuttings. Pretreatment of cuttings with 3-Indolbutyric acid (IBA) or anaphthapene acetic acid (NAA) or their mixture caused injury to
callus tissue due to higher concentration and resulted in very
poor sprouting even compared to control. The direct planting of
stem in main field was found to have a limited success. 32, 33
Two accessions of S.rebaudiana were successfully introduced in
the experimental farm at the Institute of Himalayan Bioresource
Technology (IHBT), Palampur in 2000. Cultivation trial of these
accessions was conducted during 2001-03. Overall crop
performance was satisfactory for both the accessions and they
were least affected by biotic and abiotic factors like high
rainfall, frost, and infestation by insects and disease.
Quantitative differences were found in Stevioside content of the
two accessions, ranging between 6 and 8%. Accession 1 was
superior in Stevioside content and Accession 2 was superior in
leaf biomass. Higher content of Stevioside was found in the
regenerated crop in January, during the second year of plant
growth.
14
With improved management practices, there is further scope for

improvement in Stevioside content. A laboratory-scale process
was developed for the extraction of Stevioside up to 63% purity.
Although the crop is self-incompatible in its breeding behavior,
the prevalence of two diverse accessions has facilitated seed
production under Palampur conditions. This has triggered the
production of plant material for its introduction amongst
interested growers in large numbers.21
Cultivation Parameters for Stevia Production in India
Altitude
Soil characteristic
: 1300m msl
: Clay loam in texture, low in carbon
(0.2%), high in total nitrogen
(0.15%), medium in
available
P2O5 (0.18%), pH 5.6
Table 6: Herb yield of two accessions of Stevia rebaudiana*21

Accession
Leaf weight
(q/ha)
Fresh
Dry
Stem weight
(q/ha)
Fresh
Dry
Leaf: stem
ratio (w/w)
Fres
Dry
h
Herb yield
(q/ha)
Fresh
Dry
Accession
1a
69.83
17.46
98.41
19.68
0.71
0.88
168.24
Accession
2b
108.47
21.69
138.69
34.67
0.78
0.63
247.16
Stev
iosi
de
cont
ent
(%)
Stevioside
yield of
whole herb
(kg/ha)
37.14
8.00
297.12
56.36
6.00
338.16
*Recorded during crop cycle from September 2001 to

January 2003 (data recorded September 2002 and
January 2003)
a In the form of plantlet, further multiplication of plants
from this accession was achieved through stem
cuttings.
b Accession 2 was raised both through seeds as well as
stem cuttings.
15
2.3
Economy of Stevia as sweetener in India
The total market value of stevia sweetener in Japan is estimated

to be around 2-3 billion yen/yr. The crop has been introduced
in other countries, including Brazil, Korea, Mexico, Indonesia
and Tanzania21 Presently, more than 1300MT raw material i.e.
stevia is being cultivated in China, Taiwan and Malaysia for
marketing in Japan21.
Keeping in view the Stevias increasing popularity along with its
traditional importance all over the world India has also
ventured in the same field. Department of AYUSH, Govt. of
India has accepted a project for financing the stevia cultivation
in Uttaranchal, West Bengal, Haryana and Punjab.
Stevias plant propagation and crop production studies were
conducted in the Chandpur Experimental Field at the Institute
of Himalayan Bioresource Technology (IHBT). Palampur located
in Kangra Valley, Himachal Pradesh, India.
According to IHBT research:
Cost of cultivation for producing an average leaf yield of
17, 20, 23 and 25 q/ha for first, second, third and fourth
year respectively was worked out to be Rs. 4.74 lakhs/ha
during four years.Net returns for four years were
calculated as Rs. 3.75 lakhs accounting for an average
annual income of Rs. 0.93 lakhs/ha at a sale price of Rs.
100/kg dried leave. 21
Table no 7: Costing of stevia cultivation in India21
Particulars
(a) Establishment cost (Rs /ha)
(b) Variable costs (Rs/ha)
(c) Fixed costs (Rs/ha)
(d) Total production cost
(A+B+C)(Rs/ha)
(e) Gross income (Rs/ha)
Sale price of dried leaf @Rs
100/kg dried leaf yield 1700kg
in 1st, 2000 kg in 2nd, 2300 kg
in 3rd and 2500 kg in 4th year
(f) Net income (Rs/ha)
(g) Benefit cost ratio (BCR)
(Gross Income/Total Production
Cost)
1st year 2nd year 3rd year 4th year

60,500
73,500
82,100
82,200
82,200
27,060
22,389
22,398
22,398
161,060 104,489 104,598 104,598
170,000 200,000 230,000 250,000
8940
1,055
16
95,511
1,914
125,402 145,402
2,198
2,390
It was, therefore, concluded that stevia cultivation is a

remunerative venture with a cost benefit ratio at 1.89. Net
profit at Rs. 4.61 lakhs/acre during the third year has
been reported. Keeping the sale price of Rs. 200/kg dried
leaf, through this analysis, reduction in the cost of
production has been achieved by raising seedling at
cheaper rate21.
IHBT has notified the price list of various medicinal and
aromatic seeds of planting material. The price list of S.
rebaudiana is as follows:Table 8: Cost of stevia leaves as per IHBT:
Plant
Species
S.Rebau
diana*
Common
Name
Stevia,
Sweet
herb
Form of
plant
material
Unit
Rates for different slab

(Rs. /unit)
1-99
100900
1000
Mode
of
supply
Seed
15.00
12.00
10.00
Poly
bags
Seed
raised
sapling
No.
3.00
2.50
2.00
Bunch
pack
No.
4.00
3.50
3.00
Bunch
pack
Cutting
raised
sapling
* S.Rebaudiana: Stevia rebaudiana bertoni
17
Commercial Extraction of Steviol Glycosides Chapter 3

Most of the commercial processing of stevia leaves occurs in
Japan and there are dozens of patents describing methods for
the extraction of Steviol glycosides34 categorized into those
based on solvent35 solvent plus a decolorizing agent, adsorption
chromatography ion exchange 36, and selective precipitation of
individual glycosides. The most favored extraction processes
involve four steps: aqueous or solvent extraction, ion exchange,
precipitation or coagulation with filtration, then crystallization
and drying49. New methods based on ultra-filtration have been
disclosed recently37
Traditional Method
The traditional method of use by the Paraguayan Guarani
Indians was to dry the leaves and to use them to sweeten tea
and medicines or to chew the leaves as a sweet treat. Stevia
was regularly used in drinks many times a day, not just
occasionally, with no side effects.21
3.1 Improved Method
The use of dried leaves (pieces or powder) is not unacceptable in
domestic cooking but it does leave sediment in clear drinks etc
and can also leave a green color. There can also be an
unpleasant aroma associated with the dried leaves. Appropriate
processing of the dry herb can remove this aroma, which is due
to specific leaf compounds (not Stevioside) 2, 8.
Aqueous extracts of the leaves obtained by boiling leaves in
water followed by cooling and straining (filtration).
Crystalline powders and extracts are preferred in the
commercial situation as they have a fixed known sweetening
value.
There are a number of patented refining processes registered in
Japan11. They generally use four basic steps:
18
1.
2.
3.
4.
Dissolving the sweetener in boiling water or in other solvent.

Ion exchange separation.
Filtration with precipitation / coagulation.
Crystallizing and drying
Methanol appears to be used in most of the extraction and

purification process, presumably to improve extraction
efficiency and to facilitate the separation of individual
Steviosides. This use of methanol however raised the questions
regarding safety aspect of the stevia extracts.
More recent processing methods used water filtration
procedures and do not use methanol and so produce a more
natural product. Newer factories in Brazil used only water
extraction procedures and claim 96% purity of the product e.g.
Stevia Crystals.21
Boiling water extraction can achieve 93 98 % extraction of
Stevioside. The purification and separation of the various
glycosides can be achieved with resin adsorption and ion
exchange methods. Reverse osmosis, ultrafilteration and
nanofilteration can also be used. Some extraction methods have
been designed to maximize R-A percentage. The need to
separate the various Steviosides could diminish as the ratio of
R-A %: St* % in the leaves is increased by plant breeders from
under 0.8:1 to over 1.2:1.
In pure (crystalline) form the Stevioside mix will be 250-300
times sweeter than sugar and therefore could be valued at $75 90 /kg (cost of equivalent sweetening quantity of sugar at
$300/tonne).
Most primary processing of stevia leaves is carried out in China,
Japan, Korea, Brazil or Paraguay, where factories are located
near original growing areas. Factories in Japan (and Korea) now
import leaves for processing, as growing of stevia has almost
ceased in Japan.
19
The most common processes used for extracting Steviol

glycosides from the leaves consist of:
Soaking leaves in warm/hot water to dissolve the

glycosides (in batches)
Filtering the resultant liquid (often after adding a
precipitation agent)
Concentration by vacuum evaporation
Resin exchange to separate the glycosides into high and
low R-A fractions
Ion exchange purification (sometimes)
Evaporation and spray drying or, less frequently,
crystallization to produce the stevia powder/crystals.
Some secondary processing may be undertaken, especially in

Japan, to further separate high R-A fractions or to convert
Stevioside to Rebaudioside or some other glycoside to improve
taste quality. This process is very similar to the extraction
process of raw sugar from sugar cane11
*St: Stevioside
3.2 Product Quality

Product quality has two elements: purity and absence of
contaminants, which are largely determined by processing
hygiene and method, and taste quality, which is determined
largely by the mix of glycosides present. Purity levels of
commercial Steviol glycosides powders vary from 80% to 95%.
Sweetness and taste quality will vary with the glycoside content
of the leaves processed as well as with the processing method.
High taste quality is generally expressed as the percentage of RA, the higher being the better, with over 50% being enhanced
quality and over 80% premium quality. With modern extraction
technology and the use of crystallization rather than spray
drying, high purity levels are expected9
3.3 Taste Quality Improvement
There are two avenues available to improve taste quality:
(a) Further processing
(b) Plant breeding and selection.
20
A number of factories use a range of further processing

procedures to improve taste by increasing the level of
Rebaudioside-A relative to Stevioside. These processes can
include further separation or modification (e.g. by enzyme
action) of Stevioside (St) to produce Rebaudioside A [R-A] or a
similar tasting glycoside.
Plant breeding and selection, often sponsored by factories, has
been used for many years to increase total glycoside content
(from 10% to 15% or more) and particularly R-A content (from
4% to 10% or more). A few of these improved varieties could be
available and will be tested for suitability in Australia. The
preferred taste improvement approach for Australia is by plant
selection, as this will avoid the need for further processing,
which can involve the use of chemicals and this could
compromise the clean, natural and, possibly, organic status of
stevia grown in Australia. 9
3.4 Extraction of Steviol glycosides
In order to obtain stevia extract of a better quality the process
includes two steps:
1.
Pretreatment of the leaves by Super Critical Fluid
Extraction (SCFE).
2.
Extraction of stevia glycosides by SCFE using CO2 as
solvent and water and / or ethanol as co solvent.
The mean total yield for SCFE pretreatment was 3.0 %. The
yields for SCFE with co solvent of stevia glycoside were below
0.50% except at 120 bar, 160C and 9.5% (Molar) of water.
Under this condition total yield was 3.4%. The quality of the
glycosidic fraction with respect to its capacity as sweetener was
better for the SCFE extracts as compared to extract obtained by
the conventional process.
21
3.5 Detailed process

3.5.1 Pretreatment of leaves: Pretreatment conditions were
set at 200 bar and 300C. The glycosides were obtained at 120
and 200 bar at 16, 30 and 450C.
3.5.2 Extraction of stevia glycosides
3.5.2.1 The Raw Material
Stevia leaves from the crop were bought in Maring (Paran,
Brazil). The solid material was cleaned, selected, packed in
plastic bags, and stored at room temperature (20 to 32oC). The
humidity of the raw material was determined using the toluene
distillation method (Jacobs,M.B; The chemical analysis of foods
& food products; 3rd edition Robert Krieger Publishing Co. New
York; 1973). The glycoside content was determined according to
the phenol sulfur method for total carbohydrates (Alvarez et al.,
1986).
3.5.2.2 Particles and Bed Characterization
The real density of the stevia particles was determined by
picnometry with gas helium (Multivolume Picnometer 1305) at
the Central Analtica, IQ Unicamp. Apparent density was
calculated from the mass used to fill the extraction cell. Bed
porosity was defined using the real density of the particles and
the apparent density of the bed. The mean diameter of the
particles was evaluated using the methodology described by
Pasquel.A (2000) 38
22
3.5.2.3 The Experimental Unit for the Super Critical Fluid

Extraction [SCFE] 38
The experimental unit used was that described by Pasquel et al.
(1999) for the pretreatment of stevia leaves. A cosolvent pump
was added to the system (Figure 1)

23
3.5.2.4 Experimental Procedure: SCFE

The mass of solid used varied from 69.10-3 to 82.10-3 kg. The
triturated solid was packed inside the extraction cell (SS 316,
with a length of 0.375 m and an inside diameter of 0.0283 m).
The extraction cell was adapted to the SCFE unit and the
heating and/or cooling system was turned on. Once the system
reached a temperature of 30oC (approximately 3 hours), valves
2a, 2b, 2c, and 2h were opened. As soon as the system pressure
reached 200 bar, valves 2j, 2m, and micro metering valve 15
were opened. The extracts were collected in 20mL glass flasks.
An adsorption column containing Porapak Q (80 /100 mesh,
Waters Associates Inc., USA) was adapted to prevent losses of
volatile substances in the pretreatment step at the solvent
outlet. The solvent flow rate was continuously monitored.
Samples of the extract were collected every hour. Pretreatment
was carried out at 200 bar, 30oC, and an average solvent flow
rate of 4.82.10-5 kg/s for a period of 12 hours. The extraction
cell containing the pretreated stevia leaves was stored in a
domestic refrigerator.
For extraction of the glycosides, the extraction cell was
readapted in the SCFE unit. The experimental procedure was
similar to the one described above. Samples of the extract were
collected every 30 minutes and the total extraction time was 12
hours. The experimental runs were conducted at 120 and 200
bar at 16, 30, and 45oC. The cosolvents used were 9.5% (molar)
water, ethanol, or an equimolar mixture of water and ethanol.
Because the experiments were very long (12 hours for the
pretreatment, 12 hours for the glycoside extraction plus setup
time), the experimental plan was a fractional factorial design
and only one-third of the total was selected. 38
24
3.5.2.5 Experimental Procedure: Conventional Extraction

Stevia leaves subjected to the SCFE pretreatment and stevia
leaves with no pretreatment were used. The method described
by Alvarez and Couto (1984) and Goto (1997) was used. One
liter of boiling water was added to fifty grams of stevia leaves.
The infusion was kept at room temperature (25 to 30oC) for one
hour. The aqueous extract was vacuum filtered. In a separation
funnel the aqueous extract was mixed with isobutyl alcohol
[99.99%) maintaining the 40:60 (v/v) proportion. The system
was allowed to rest until complete phase separation was
achieved.
The butanolic extract was centrifuged at 3500 rpm for 15
minutes. The extract was heated up to 80oC, and percolated
through a bed of activated carbon (1 g of activated carbon for
every 100 mL of extract).
The extract was concentrated in a rota-evaporator and allowed
to rest for 24 hours to achieve crystallization of the glycosides.
The crystals were washed with methanol (99.9%) and dried in
an air-circulating oven. The crystallization mother liquor was
concentrated and extracted with acetone (99.8%). The crystals
were washed with anhydrous acetone and dried in an aircirculating oven38
25
Chemistry
Chapter 4
4.1 Steviol Glycosides

The Joint Expert Committee on Food Additives (JECFA)
committee in its fifty-first meeting (1999) stated that Before
the substance is reviewed again specifications should be
developed to ensure that the material tested is representative
of the material of commerce. Further information was required
on the nature of the substance that was tested, on the
metabolism of the Stevioside in humans and on the activity of
Steviol in suitable studies of Genotoxicity in- vivo.
JECFA in its 63rd meeting (2004) at Geneva has noted that
Steviol glycosides are natural constituents of the plant Stevia
rebaudiana bertoni which contain at least ten different
glycosides, the major constituents being Stevioside and
Rebaudioside A. The material evaluated at that meeting
contains not less than 95% glycosylated derivatives of Steviol,
primarily Stevioside, Rebaudioside A and C and Dulcoside A,
with minor amounts of Rubausoside, Steviolbioside and
Rebuioside B, D, E and F. In the same meeting the JECFA has
brought out a detailed tentative specifications of the Steviol
glycosides which includes
1.
2.
3.
4.
Stevioside
Rebaudioside A
Rebaudioside C
Dulcoside A
JECFA has allotted a temporary ADI of 0 to 2 mg/kg bw for

Steviol glycoside (expressed as Steviol) on the basis of the
NOEL for Stevioside (970 mg/kg bw/day or 383 mg/kg
bw/day, expressed as Steviol in the two years) after a study on
rats and using a safety factor of 200. This safety factor
incorporates a factor of 100 for inter and intra species
differences and an additional factor of 2 because of the need
for further information.
26
The committee noted that this temporary ADI only applies to

products complying with the specifications. New tentative
specifications were prepared, accompanied by a chemical and
technical assessment.
JECFA also recommended for the collection of following
information for commercially available products39:
Analytical data on distribution and concentration of all

components Steviol glycosides, including those that were
not identified in the tentative specifications.
Method of analysis for the determination of all

components Steviol glycosides, including those that were
not identified in the tentative specifications.
The nature and concentration of the fractions that do not

contain Steviol glycosides.
The quantities of residual solvents from isolation &

purification steps of the manufacturing process.
The hydrolytic stability of the Steviol glycoside in acidic

foods and beverages.
4.2 Detailed specifications of Steviol

developed by JECFA is as following: 39
glycoside
as
4.2.1 Definition:
Steviol Glycosides are obtained by extracting leaves of Steviol
rebaudiana Bertoni with hot water followed by solvent
purification of the water-soluble extract. Ion exchange resins
may also be used during the purification process. Stevioside
and Rebaudioside A are the principles Steviol glycosides of the
specified material. Rebaudioside C and Dulcoside A are
secondary glycosides. Others Steviol glycosides may also be
present.
27
4.2.2
Chemical Name:
The following are the chemical names for the principal and
secondary Steviol glycosides:
Stevioside:
13-[(2-O--D-glucopyranosyl-
-D-glucopyranosyl) oxy]
kaur-16-PM-18-oic
acid
glucopyranosyl ester.
-D-
Rebaudioside:
13-[(2-O--D-glucopyranosyl-3--Dglucopyranosyl--D-glucopyranosyl] oxy.
kaur-16-PM-18-oic
acid
-Dglucopyranosyl ester.
Rebaudioside C:
13-[(2-O--L-rhamnopyranosyl-3-O---Dglucopyranosyl-D-glucopyranosyl -- D-glucopyranosyl-D-glucopyranosyl] oxy. kaur-16-PM-18oic acid -D- glucopyranosyl ester.
DulcosideA:
13-[2-O--L-rhamnopyranosyl--Dglucopyranosyl]oxy.
kaur-16-PM-18-oic
acid -D- glucopyranosyl ester.
4.2.3
C.A.S.(Chemical Abstracts Services) number
The following are the C.A.S. numbers for the principals and
Stevioside
57817-89-7
Rebaudioside A :
58543-16-1
Rebaudioside C :
63550-99-2
Dulcoside A
64432-06-0
28
4.2.4
Chemical formula
The following are the chemical formulas for the principal and
Stevioside
:
C38H60O18
Rebaudioside A
:
C44H70O23
Rebaudioside C
:
C44H70O22
Dulcoside A
:
C38H60O17
4.2.5
Structural formula
The following are the structural formulas for the principal and
O-R2
CH3
CH2
CH3
Coo-R1
Compound
Name
R1
R2
Stevioside
Rebaudioside A
-Glc
-Glc
-Glc--Glc (2
-Glc--Glc (2
-Glc
Rebaudioside C
-Glc
(3
1)
-Glc--Rha (2
1)
-Glc
Dulcoside A
29
1)
1)
(3
1)
-Glc--Rha (2
1)
Steviol (R1=R2=H) is the aglycone of the Steviol glycosides.

Glc and Rha represent, respectively, glucose and Rhamnose
sugar moieties.
4.2.6
Formula weight
The following are the formula weights for the principal and
secondary Steviol Glycosides:
Stevioside
Rebaudioside C
Rebaudioside A
Dulcoside A
:
:
:
:
804.88
951.03
967.03
788.88
4.2.7
Assay
Not less than 95% of total Steviol glycosides.
The sum of the percentages of Stevioside and Rebaudioside A
is not less than 70%.
4.2.8
Description
White crystalline powder, odorless or having a slight

characteristic odour, about 200-300 times sweeter than
sucrose.
4.2.9
Characteristics
4.2.9.1 Identification
Solubility (Vol. 4)
Freely soluble
ethanol
Stevioside
The material contains not less than

70% of Stevioside and
Rebaudioside A as identified and
determined in the Method of Assay.
Rebaudioside A
30
in
water
and
in
4.2.9.2 Purity
Ash (Vol. 4)
Not more than 1%

Test 3 g of the sample (Method I)
Loss on drying (Vol. 4)
Not more than 4 %( 105, 3h)
Residual solvents
Information required
Arsenic (Vol.4)
Not more than 1 mg/kg
Lead (Vol. 4)
Not more than 1 mg/kg

Determine
using
an
atomic
absorption technique appropriate to
the specified level. The selection of
sample size and method of sample
preparation may be based on the
principles of the methods described
in FNP 5, Instrumental methods.
4.2.9.3 Method of Assay:

Determine the percentages of the Steviol glycosides by highpressure liquid chromatography (Volume 4).
Standards: Stevioside, >99.3% purity and Rebaudioside A,
>97%, purity (available from Wako pure Chemical Industries,
Ltd. Japan).
Mobile phase: Mix HPLC grade acetonitrile and water 80: 20).
Adjust the pH to 3.0 with phosphoric acid (85% reagent grade.
Filter through 0.22 m Millipore filter or equivalent.
Standard solution:
Accurately weigh 50 mg of dried
(105C/3h) Stevioside standard into a 100ml volumetric flask
and dilute to volume with mobile phase.
Sample solution: Accurately weigh 60-120 mg of the sample
into a 100 ml volumetric flask and dilute to volume with
mobile phase to volume.
31
Conditions:
Column
Supelcosil LC-NH2 or equivalent

(Length: 15- 30cm;)
Inner diameter:
3.9-4.6mm)
Mobile phase
: A 80:20 mixture of acetonitrile and water
(See above)
Flow rate
:
Adjust so that the retention time of
Stevioside is about 10 min.
Injection volume
: 5-10l
Detector
: UV at 210 nm
Column temperature:
40C
Equilibrate the instrument by pumping mobile phase through

it until a drift free baseline is obtained. Record the
chromatograms of the sample solution and of the standard
solution.
The relative retention time of Dulcoside A and Rebaudioside C
with respect to Stevioside is 0.68-0.76 and 1.15-1.23,
respectively. To obtain the retention time of Rebaudioside A,
use the Rebaudioside A, standard.
Measure the peak areas of Stevioside, Rebaudioside A,
Rebaudioside C and Dulcoside A from the samples solution.
Measure the peak areas of Stevioside from the standard
solution.
Calculate the percentage of Stevioside, Dulcoside
Rebaudioside A and Rebaudioside C from the formulas:
% Stevioside
=
%Dulcoside A
=
%Rebaudioside A =
%Rebaudioside C =
A,
[Ws/W] x [Aa/As] x 100

[Ws/W] x Ab x [0.98/As] x 100
[Ws/W] x Ac x [1.20/As] x 100
[Ws/W] x Ad x [1.18/As] x 100
Where
Ws = Weighed amount (mg) of Stevioside in the standard
solution.
W = weighed amount of sample (mg).
As = Peak area of Stevioside from the standard
solution.
Aa = Peak area of Stevioside from the sample
solution.
Ab = Peak area of dulcoside A from the sample
solution.
32
Ac = Peak area of Rebaudioside A from the sample solution.

Ad = Peak area of Rebaudioside C from the sample solution.
The factors 0.98, 1.20 and 1.18 for, respectively, dulcoside A,
Rebaudioside A, and Rebaudioside C are the ratios of their
formula weights to that of the formula weight of Stevioside.
Calculate
(1) The % Steviol glycosides (sum the four percentages)
(2) The sum of the percentages for Stevioside
Rebaudioside A.39
4.3
Studies conducted other than JECFA
and
40, 41
Department of Rural Home Science, University of Agricultural

sciences Hebbal, Bangalore and University of Agricultural
sciences, GKVK, Bangalore carried out a study on the
sweetness equivalence of leaves of S.rebaudiana, the findings
of which are as follows:
Table 9: Sweetness equivalence of stevia in comparison to
sucrose61
Quantity of stevia
(gm)
In 100 ml of water
1
1
1
1
4.4
Sucrose equivalent
(gm) in 100 ml of
water
10
15
20
25
Perception of
duration of sweet
stimulus (sec)
14
14
58
14
Sensory evaluation of Stevia species
Organoleptic data are presented recording the sensation of

sweetness exhibited by fragments of herbarium leaf samples of
species in the genus Stevia, including S. rebaudiana .
Table 10: Results from organoleptic evaluation for
sweetness of 184 Stevia leaf herbarium samples, inclusive
of 110 species and 121 taxa.42
33
S.No.
Species
S.
ambylolepis
Robins
S.
ambylolepis
Robins
S.
amplexicaulis
Hassler
S. aristata D. Don
S. aristata D.Don
S.
aschenborniana
Sch. Bip
S.
ashenborniana
Sch. Bip
S. balansae Hieron
S. bangii Rusby
10
S.
benthamiana
Hieron
S berlandieri A. Gray
2
3
11
12
13
14
S.
berlandieri
A.
Gray
var. berlandieri
S. berholdii Robins
18
S. boliviensis Sch.
Bip. Ex Rusby
S. boliviensis Sch.Bip. Ex Rusby
S.
breviarishtata
Hook. & Arnott.
S.
calderillensis
Hieron
S. camporum Baker
19
S. caracasna DC
20
S. cathartica Poepp.
& Endl
S.
chamaedrys
Griseb
S. cinerascens Sch.
Bip
15
16
17
21
22
Voucher
Specimen, Unit
Country, Collection Year
(mm2)
Pringle 13655
Mexico, 1905
Jorgensen 4902
Paraguay, no date
Hassler
1011
Paraguay,
1908
Cabrera 11777
Argentina, 1954
Hassler 9033a
Paraguay, 1905
Pringle 9120
Mexico, 1900
Cronquist & Sousa 10408
Mexico, 1965
Jorgensen 4702
Paraguay, 1931
Rusby 6885
Bolivia, no date
Apolinar-Maria 274
Colombia, 1938
Pringle 11573
Mexico, 1903
Barkley, Webster & Rowell
7139
Mexico, 1947
Acosta-Solis 7849
Ecuador, 1944
Holway & Holway 542
Bolivia, 1920
Buchtien s.n.
Bolivia, 1912
Cabrera 3068
Argentina, 1933
Fiebrig 3425
Bolivia, 1904
Irwin 2749
Brazil, 1959
Molina 30283
Guatemala, 1974
Espinosa E-787
Ecuador, 1946
Lorentz & Hieronymus 171
Argentina, 1873
Matzenbacher 510
Brazil, 1976
34
tested
1-2
4-6
Na
810
B
BB
BB
--
--
--
BB
BB
--
BB
BB
--
BB
BB
BB
--
BB
BB
--
BB
--
BB
BB
B
N
23
24
25
S. clausseni Sch.Bip
S. clausseni Sch.Bip
S.
clinopidioides
Greenm
26
S. collina Gardn
27
S. compacta Benth
28
S. conmixta Robins
29
S. connata Lag
30
S. connata Lag
31
S. connata Lag
32
S. crenulata Baker
33
S. cuneata Hassler
34
S. cuzcoensis Hieron
35
S. cuzcoensis Hieron
36
37
S.
dianthoidea
Hieron
S. elatior HBK
38
S. elatior HBK
39
S. elatior HBK
40
S. elationr HBK
41
S. elongate HBK
42
S. elongate HBK
43
S.
entreriensis
Hieron
S.
eupatoria
(spreng.) Willd
S. filipes Rusby
44
45
46
47
S.
galeopsidifolia
Hieron
S.
galeopsidifolia
Hieron
Barreto 8353
Brazil, 1936
Lenarte 2570
Brazil, 1950
Sesse, Mocino & Maldondo
2613
Mexico, ca. 1804
Warming 618
Brazil, 1865
Buchtien 186
Bolivia, 1912
Hatschbach 18764
Brazil, 1968
Pringle 4580
Mexico, 1893
Smith 255
Mexico, 1894
Pringle 4580
Mexico, 1893
Hatschbach 18770
Brazil, 1968
Hassler 10286
Paraguay, 1908
Pennell 13553
Peru, 1925
Herrera 2353
Peru, 1929
Penland & Summers 564
Ecuador, 1939
Acosta-Solia 10184
Ecuador, 1945
Apolinar-Maria 448
Colombia
O. Kuntze s.n.
Bolivia, no date
Breedlove 7036
Mexico, 1964
Haenke 329
Mexico, 1791
Linden 475
Venezuela, 1842
Cabrera 3922
Urugua, 1936
Taylor 13
Mexico, 1936
Brooke 6209
Bolivia, 1950
Vargas 9531
Peru, 1950
Vargas 4310
Peru, 1944
35
BB
BB
B
B
BB
B
BBB BB
B
BB
B
--
BB
BB
--
BB
BB
--
BB
BB
--
BB
--
BB
--
--
--
--
BB
--
BB
BB
--
BB
--
BB
--
48
50
S. glandulosa Hook.
& Arnott
var. gentryi Grashoff
S. glandulosa Hook.
& arnott
var. glandulosa
S. glutinosa HBK
51
S. heptachaeta DC
52
S. herrerae Robins
53
56
S. hirsute DC. var

hirsute
S. hirsute DC. var
hirsute
S.
hypomalaca
Robins
S. hyptifolia gardn
57
S. iltisiana Grash off
58
S. iltisiana Grashoff
59
62
S.
incongnita
Grashoff
S.
incongnita
Grashoff
S. incognita Grash
off
S. ialiscensis Robins
63
S. jorullensis HBK
64
S. jorullensis HBK
65
S. latifolia Benth
66
S. latifolia Benth
67
S. lehmannii Hieron
68
S. lehmannii Hieron
69
S. lemmonii Hieron.
var.
hispidula
Grashoff
S. lemmonii Hieron.
var.
hispidula
Grashoff
S. leptophylla SchBip. ex Robins
49
54
55
60
61
70
71
Gentry 1204
Mexico, 1934
BB
Palmer 1821
Mexico, 1892
BBB --
--
Lehmann 4727
Colombia
No Collector
Brazil, 1863
Vargas 4139
Peru, 1944
Hinton 2392
Mexico, 1932
Molina & Molina 26641
Guatemala, 1971
Pringle 9976
Mexico, 1902
Mexia 5540
Brazil, 1931
Fisher s.n.
Mexico, 1924
Pringle 11576
Mexico, 1903
Ton 491
Mexico, 1966
Williams 41681
Guatemala, 1972
Guatemala, 1974
No Collector
Mexico, 1892
Williams et al. 41438
Guatemala, 1972
Pringle 13085
Mexico, 1904
Anderson & Laskowski 4432
Mexico, 1966
Smith 253a
Mexico, 1894
Guatemala, 1971
Purpus 3133
Mexico, 1908
Gentry 1414
Mexico, 1935
BB
BB
BB
BB
--
BB
BB
B
BB
BB
BB
BBB
BB
BB
BB
BB
BB
BBS BB
S
SS
BBB
S
Palmer 96
Mexico, 1906
Hasler 6617
Paraguay, 1990
36
BB
B
--
BBB
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
S. lucida Lag
Killip 38078
Columia, 1944
S. lucida Lag
Vilbur 15405
Panama, 1971
S. lucida Lag
Steyeermark 55645
Venezuela, 1944
S. lucida Lag. var. Morley 653
lucida
Mexico, 1946
S. lucida Lag. var. Powell & Edmondson 665
bipontini Robins
Mexico, 1961
S. lucida Lag. var. Williams & Williams 21703
oaxacana
Mexico, 1962
(DC.) Grashoff
S. lundiana DC
Hatschbach 12529
Brazil, 1965
S. macbridei Robins Soukup 3002
Peru, 1964
S. macbridei Robins Isert 2064
Peru, 1863
s. mandonii Sch.-Bip Pennell 13372
Peru, 1925
S. mandonii Sch.- Soukup 236
Bip
Peru, 1935
S.
melissaefolia Macbride 5965
(Lamk.)
Peru, 1923
Sch.-Bip
S.
melissaefolia Cerrate 865
(Lamk.)
Peru, 1951
Sch.-Bip
S.
menthaefolia Dusen 16948
Sch.-Bip
Brazil, 1915
S. menthaeflia Sch.- Martius 771
Bip
Brazil, ca. 1800
S.
menthaefolia Buchtien s.n.
Sch.-Bip
Bolivia, 1914
S.
mercedensis Lossesn 202
Hieron. ex Reiss
Argentina, 1925
S.
micradenia Pringle 4543
Robins
Mexico, 1893
S. micrantha Lag
Sesse et al. 2589

Mexico, ca. 1800
S. micrantha Lag
Orcutt 4293
Mexico, 1910
S. micrantha Lag
Cronquist 10246
Mexico, 1965
S. microchaeta Sch.- Botteri 407
Bip
Mexico, no date
S. monardifolia HBK Pringle 11580
Mexico, 1903
S. monardifolia HBK Balls & Gourlay 5288
Mexico, 1938
37
BB
BB
BB
BB
BT
BT
BT
TB
BB
BBB
--
BBB BB
SS
BS
S
N
N
--
BB
BB
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
S.
multiaristata Cabrera 3341
Spreng
Uruguay, 1935
S. nelsonii robins
Leavenworth & Hoogstraal
1121
Mexico, 1941
S. nepetifolia HBK
Breedlove 14153
Mexico,1965
S. nepetifolia HBK
Pringle 11581
Mexico 1903
S. nepetifolia HBK
Smith 1996
Colombia, 1900
S. oligocephala DC
Glaziou s.n.
Brazil, 1907
S. origanoides HBK
Mexia 9043
Mexico, 1937
S. origanoides HBK
Pringle 1780
Mexico, 1888
S. origanoides HBK
Shedon s.n.
Mexico, 1892
S.
ovalis(Robins.) Pringle 4491
Robins
Mexico, 1893
S.
ovalis(Robins.) Barnes & Land 187
Robins
Mexico, 1908
S. ovata Willd
Williams et al. 41694
Guatemala, 1972
S. ovata Willd
Stewart 1227
Mexico, 1941
S. ovata Willd. var. Molina & Molina 26935
ovata
Guatemala, 1971
S. ovata Willd. var. Pringle 6624
reglensis
Mexico, 1896
(Benth.) Grashoff
S. oxylaena DC
Hassler 12154
Paraguay, 1913
S. oxhlaena DC
Lorentz 952
Uruguay, 1877
S. pallida (Sch.-Bip.) Killip & Smith 17269
Hieron
Colombia, 1927
S. palmeri A. Gray Standley 2475
var. palmeri
Mexico, 1936
S. palmeri A. Gray Ortega 7132
var.
Mexico, 1993
Constricta Grashoff
S. pauciradiata Bab
Glaziou 11025
Brazil(?)
S. perfoliata Crongq
Cronquist 11229
Mexico, 1974
S. phlebophylla a. Pringle 2291
Gray
Mexico, 1889
S. pilosa Lag
Barkley et al. 2828

Mexico, 1947
38
BB
BB
SS
N
SS
B
S
SSB
B
SB
SB
SB
SB
SB
SB
SBB
BB
BBB BB
B
N
B
--
BB
SS
SSB
BBB BB
SS
BS
S
BS
BB
ST
-BB
120
S. pilosa Lag
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
Powell & Edmondson 735

Mexico, 1961
A. Gentry 1948
Mexico, 1935
BS
BS
S. plummerae
Gray
var. plummerae
S. plummerae A. LeSueur 961
Gray
Mexico, 1936
var. plummerae
S. pohliana Baker
Dusen 16944
Brazil, 1915
S.
polycephala Breedlove 7995
Bertol
Mexico, 1964
var. polycephala
S. porphyrea Mc Waterfall 15446
Vaugh
Mexico, 1959
S. porphyrea Mc Palmer 456
Vaugh
Mexico, 1896
BB
BB
BB
BB
SB
SB
BB
S
BBB
S
S. puberula Hook
BB
SBB
BB
BS
SB
SSS
S
N
--
--
BB
BB
BBB
BB
BB
BB
BB
--
--
BB
BB
BB
BB
BB
Pennell 14367
Peru, 1925
S. puberula Hook
Pennell 14336
Peru, 1925
S. purpusii Robins
Purpus 1486
Mexico, 1905
S. quitensis HBK
Bonpland s.n.
Ecuador, no date
S.
rebaudiana Gosling s.n.
(Bertoni) Bertoni.
Paraguay, 1919
S.
reticulate No collector
Grashoff
Mexico, 1893
S. revolute Robins
Purpus 3842
Mexico, 1909
S. rhombifolia HBK
Williams 10629
Venezuela, 1938
S. rhombifolia HBK
Cuatrecasas 20845
Colombia, 1946
S. rhombifolia HBK
Holmgren 526
Ecuador, 1920
S. rhombifolia HBK
Stork & Horton 10884
Peru, 1939
S. rhombifolia HBK
Dorantes et al. 767
Mexico, 1972
S. rhombifolia HBK
Jimenez 1382
Costa Rica, 1963
S. saliciafolia Cav. Cronquist & Fry 10823
var.
Mexico, 1970
salicifolia
S. salicifolia Cav. Purpus 2541
var. callodes
Mexico, 1908
(Greenm.) Robins
S. salicifolia Cav. Palmer 931
var.
Mexico, 1896
Rirgulifera Robins
39
143
S. sanguinea Hieron
144
S.
satureifolia(Lamk.)
Sch.-Bip
S.
satureifolia
(Lamk.)
Sch.-Bip
S.
satureifolia
(Lamk.)
Sch.-Bip
S. seemannii Sch.Bip
S. seleriana Robins
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
Fabris-Crisci 7385
Argentina, 1968
Pastore 1146
Argentina, 1937
BB
BB
BB
Herter s.n.
Uruguay, 1925
Cabrera 6839
Argentina, 1940
BB
BB
BB
BB
BB
BB
BB
BB
Smith 254
Mexico, 1894
Cronquist & Fay 10876
Mexico, 1970
S.
selloi Hassler 3910
(Spreng.)Sch.-Bip
Paraguay, no date
S. serrata Cav
Gehriger 246
Venezuela, 1930
S. serrata Cav
Black 46-653
Colombia, 1946
S. serrata Cav. var. Williams et al. 41771
serrata
Guatemala, 1972
S. serrata Cav. var. Powell & Edmondson 623
arguta
Mexico, 1961
Robins
S. soratensis Hieron Pennell 13536
Peru, 1925
S. soratensis Hieron Weberbauer 6920
Peru, 1909-1914
S. stenophylla A. Johnston 8907
Gray
Mexico, 1941
S. subpubescens Lag Cronquist & Sousa 10417
Mexico, 1965
S.
subpubescens Anderson & Laskowski 4430
Lag. var.
Mexico, 1966
Intermedia Grashoff
S.
subpubescens Mexia 1615
Lag. var opaca
Mexico, 1927
(sch.-Bip) robins
S.
subpubescens Haenke 1703
Lag. var.
Mexico, 1791
subpubescens
S.
subpubescens Cronquist & Fay 10906
Lag. var.
Meixco, 1970
subpubescens
S.
tapacariensis Eyerdam 24784
Hieron
Bolivia, 1939
S. tephra Robins
Pringle 7965
Mexico, 1899
40
164
S. tephra Robins
165
S.
Blake
166
S. tomentosa HBK
167
S. tomentosa HBK
168
S. trifida Lag
169
S. triflora DC
170
S. triflora DC
171
172
S. urticaefolia Billb.
In Thumb
S. vaga Griseb
173
S. vaga Griseb
174
S. venosa A. Gray
175
S. vernicosa Greenm
176
S. veronicae DC
177
179
S. villaregalis
Vaugh
S. villaregalis
Vaugh
S. viscida HBK
180
S. viscida HBK
181
S. wageneri Hieron
182
S.
weberbaueri
Hieron
S. yaconensis Hieron
178
183
tephrophylla
MC
Mc
Purpus 4830
Mexico, 1910
Breedlove
&
raven
13643
Mexico, 1965
Pupus 2550
Mexico, 1907
Mueller 2397
Mexico, 1935
Rzedowski 27054
Mexico, 1970
Pringle 11835
Mexico, 1935
Williams
&
Molina
42299
Nicaragua, 1973
Williams & Assis 7238
Mexico, 1945
Cabrera
&
Fabris
19930
Argentina, 1969
Lossen 238
Argentina, 1925
Gentry 1948
Mexico, 1935
Pringle 10349
Meixco, 1907
Herter 1971
Uruguay, 1947
Pringle 2486
Mexico, 1889
Barnes & Land 198A
Mexico, 1908
LeSueur 347
Mexico, 1935
Guatemala, 1971
Vogl 505
Venezuela
Stafford 438
Peru, 1937
Cabrera 3032
Argentina, 1933
BB
BB
BB
BB
BB
BB
BB
--
BB
BBB --
BB
BB
--
BB
BB
--
TT
--
BB
--
--
BBS
BBS
BBS
SB
SB
TT
TT
B, slightly bitter; BB, moderately bitter; BBB, strongly bitter;

N, neutral (not bitter and not sweet); T, salty; S, slightly sweet;
SS, moderately sweet; SSS, strongly sweet. Where mixed taste
sensations are recorded (SB, etc.), tastes are coded in the order
in which they were experienced.
41
A taste considered sweet was evident in 24 samples,

representing 18 Stevia species and varieties.
The herbarium samples of S. lemmonii var. hispidula. S.
micradenia, S. oligocephala, S. perfoliata and S. phlebophylla
leaves exhibited the strongest sensations of sweetness,
including the S. rebaudiana sample, of the 18 species and
varieties listed above. Since none of these samples produced
the intensity and duration of taste response exhibited by the
S. rebaudiana specimen, it may be inferred that if ent-kaurene
glycosides are responsible for their sweet effects, the
concentrations of these compounds are in all probability much
lower than in S. rebaudiana 42
It may be seen from table10 that the sweet taste of these
samples was in many cases accompanied by bitterness. This
may be due to the masking of the taste or ent-kaurene
glycosides by sesquiterpene lactones. Sesquiterpene lactones
are bitter43 and have been isolated previously from species in
the genus Stevia 44-48
Phillips (1989)49 summarized the early sensory research.
Stevioside was between 110 and 270 times sweeter than
sucrose, Rebaudioside A between 150 and 320 times sweeter,
and Rebaudioside C between 40 and 60 times sweeter,
Dulcoside A was 30 times sweeter than sucrose. Rebaudioside
A was the least astringent, the least bitter, had the least
persistent aftertaste and was judged to have the most
favorable sensory attributes of the four major steivol
glycosides49. It was also confirmed that Rebaudioside-A is less
bitter than Stevioside. Bitter notes in Stevioside and
Rebaudioside A is an inherent property of the compounds and
not necessarily the result of impurities in whole-plant extracts.
Relative to other high potency sweeteners such as aspartame,
bitterness tends to increase with concentration for both
Stevioside and Rebaudioside A50
Both Stevioside and Rebaudioside A are synergistic in
mixtures with other high-potency sweeteners such as
aspartame and are good candidates for inclusion in blends. 50
42
It is possible to improve on the somewhat unpleasant hedonic

characteristics of Stevioside by the production of sugartransformed steivol, in which a bacterial enzyme is used to
alter this natural product structurally, by transglycosylation of
its saccharide moieties, to afford a pleasant-tasting mixture of
sweet substances7
4.5 Electrophysiological
Mongolian Gerbil
and
behavioral
assays
with
Gerbil methods when used in combination with other

procedures could be used to detect the presence or absence of
sweet tasting terpenoid glycosides of extracts of different
polarities from S. rebaudiana leaves. It was found that the
Gerbils Chorda tympani nerve did not respond to
Rebaudioside B and C and Steviolbioside from S. rebaudiana
in
electrophysiological
experiments.
However,
electrophysiological concentration-response curves were
obtained for Stevioside and Rebaudioside A from S.
rebaudiana. Both of these compounds were more effective
stimuli in the gerbil than sucrose. In the subsequent
conditioned-avoidance test, gerbil trained to avoid these
stimulatory compounds generalized an avoidance to sucrose
but not to hydrochloric acid, and it was concluded that these
substances taste like sucrose to gerbils. 7, 50
43
Toxicological Studies
Chapter 5
The toxicological evaluation of Stevioside and Steviol was

investigated by JECFA in its fifty-first meeting at Geneva
(1999). The detailed report is annexure I. The abstracts from
the same are given below:
5.1
Stevioside
5.1.1
Acute toxicity51
No lethal effect was seen within 14 days after administration,

and no clinical signs of toxicity or morphological or
histopathological changes were found. The JECFA study
concluded that Stevioside is totally devoid of acute extra renal
effects (such as hypoxaemia, which could contribute to
nephrotoxicity) and direct renal effects during the 6-h period
following intravenous administration.
Table 11: Acute toxicity of Stevioside (purity 96%) given
orally to rodents.
Species
Sex
Mouse
Male &
Female
Male
Mouse
Rat
Hamster
5.1.2
LD50(g/kg
bw)
>15
>2
Male &
Female
Male &
Female
>15
>15
Reference
Toskulkao et
al. (1997)
Medon al.
(1982)
Toskulkao et
al. (1997)
Toskulkao et
al. (1997)
Short term studies of toxicity on rats
A 13-week study of toxicity was carried out on Fischer 344

rats with doses of 0, .31, .62, 1.25, 2.5 or 5 % in the diet
(equivalent to 160, 310, 630, 1300 and 2500 mg/kg bw per
day) for two year carcinogenicity study. The rats were
randomally allocated to 6 groups, each consisting of 10 male
and10 female.
44
None of the animals died during the administration period and

there was no difference in body weight gain between the
control and treated groups during administration or in food
consumption in later part of the studies. The study concluded
that a concentration of 5% in diet was a suitable maximum
tolerable dose of Stevioside for a 2-year study in rats.
5.1.3
Long
term
studies
carcinogenicity on rats52
of
toxicity
and
A Group of 45 male and 45 female inbred Wistar rats were fed

with diets containing Stevioside (purity 85%) at 0, .2, .6 or 1.2
% (equivalent to 100,300 and 600mg/kg bw/day) for two
years. The study suggested that the acceptable daily intake of
Steviosides for human was 7.9 mg/kg bw/ day, on the basis of
Stevioside consumption of the rats during the first 3 months
(the average for males & females being 790 mg/kg bw/day)
and a safety factor of 100.
Stevioside (purity 95.6%) was added to the powder diet at
concentrations of 0, 2.5or 5% (equivalent to 0, 970 & 2000mg
/kg bw/day for males and 0, 1100 and 2400 for females) and
pelleted every three months. There was no significant
histopathological evidence of neoplastic or non-neoplastic
lesions attributable to treatment in any organ or issue, except
for a decreased incidence of mammary adenomas in females
and a reduced severity of chronic nephropathy in males.
The study concluded that the Stevioside is not carcinogenic in
Fischer 344 rats under these experimental conditions.
45
5.1.4
Genotoxicity
Table 12: Genotoxic study of Stevioside53

With and without metabolic Activation
A positive response towards TA 98 was seen without
metabolic activation at 50 upto 20 mg/ml.
c.
Without metabolic activation.
d.
The same results were cited in an earlier abstract (Matsui
et al 1987).
e.
With metabolic activation.
End-point
Test object
Concentration
Reverse mutation
S.
typhimurium 50mg/platea
(purity
TA98, TA100
99%)
Reverse mutation
S. typhimurium TA 5mg/plate c
97, TA98, TA100, 1 mg/pate e
TA102, TA 104, TA purity 83%
1535, TA1537
Forward mutation S. typhimurium TM 10 mg / plate a
677
Forward mutation S. typhimurium TM Not specified a
677
a.
b.
Forward mutation
S. typhimurium TM
677
Umu
Gene S. typhimurium TA
mutation
1535/psk1002
Gene mutation
B. subtilis H17 rec +,
M45 recChromosomal
Chinese
hamster
aberration
lung fibroblasts
Chromosomal
Human lymphocytes
aberration
Chinese
hamster
Chromosomal
lung
Chinese
aberration
hamster
lung
fibroblasts
46
10 mg / plate
5 mg / plate
10mg/disc
8mg/ml c
12 mg/ml
10 mg/ml
12mg/mlc
12 mg/mlc
(Purity, 85%)
5.1.5
Reproductive toxicity54
5.1.5.1 Hamsters
Groups of 10 male and 10 female one-month-old golden
hamsters (Mesocricetus auratus,) were force-fed with
Stevioside (purity, 90%) at 0, 500, 1000, 2500 mg/kg bw per
day daily. Histological examination of reproductive tissues
from anilas of all three-generations revealed no abnormality
that could be linked to treatment. The study concluded that
Stevioside at doses up to 2500 mg/kg bw per day affected
neither growth nor reproduction in hamsters.
5.1.5.2 Rats
A group of 11 male Wistar rats were given Stevioside (purity,
96%) in the diet at 0, 0.15, 0.75, or 3% for 60 days. The
authors concluded (Mori et al., 1981). The Committee noted
that Stevioside had no adverse effect on fertility or on the
development of fetuses but there is a slight statistically non
significant increase in the number of dead or resorbed fetuses
at the highest dose.
The effects of aqueous S. rebaudiana extracts corresponding
to 0.67 g dried leaves per ml, given at a dose of 2 ml/rat twice
a day for 60 days, were studied in prepubertal (25-30 days old)
rats. The end-points were glycaemia; serum concentrations of
thyroxine and tri-iodothyronine; available binding sites in
3Hthyroid
hormone-binding
proteins;
binding
of
methyltrienolone (a specific ligand of androgen receptors) to
prostate cytosol; zinc content of the prostate, testis,
submandibular salivary gland, and pancreas; water content of
testis and prostate; body-weight gain; and the final weights of
the testis, prostate, seminal vesicle, submandibular salivary
gland, and adrenal. None of these parameters were
significantly different from those in the control group, with the
exception of the seminal vesicle weight, which fell by about
60%.
The study concluded that if the Stevia extract can decrease
fertility in rats, the effect is almost certainly not exerted on
males.
47
5.1.6
Teratogenicity55
Stevioside (purity, 95.6%) dissolved in distilled water was given

to four groups of 25 or 26 pregnant Wistar rats by gavage once
a day on days 6-15 of gestation at doses of 0, 250, 500 or
1000 mg/kg bw. No treatment related effect on general or
reproductive toxicity was observed up to the highest dose. The
study concluded that orally administered Stevioside is not
teratogenic in rats
5.1.7
Mutagenicity
56, 57
A weak mutagenic effect on Steviol (only 90% purity) in one

sensitive Salmonella typhimurium TM 677 strain does not
mean that Stevioside used as a sweetener should be
carcinogenic per se, even if the Stevioside might be
transformed to Steviol by bacteria in the colon. The safety of
oral Stevioside in relation to carcinogenic activity is evidenced
by the work of many studies (as mentioned in JECFA report)
with rats. Very significant inhibitory effects of Stevioside were
reported on tumor promotion by 12-O-tetradecanoylphorbol13-acetate in carcinogenesis in mouse skin. In 1999 the
JECFA clearly stated that there was no indication of
carcinogenic potential of Stevioside (WHO, 1999).
5.2
5.2.1
Steviol
Acute toxicity51
In male and female mice and rats Steviol (purity 90%) was
given orally, the LD50 was more than 15g/kg bw. The LD50
values in hamster was 5.2 g/kg bw in males and 6.1 g/kg bw
in females. Histopathological examination of the kidneys
revealed severe degeneration of the proximal tubular cells, and
these structural alterations were correlated with increased
serum blood urea nitrogen and creatinine. The study
concluded that the cause of the death was acute renal failure.
48
5.2.2
Genotoxicity
Table 13: Studies of genotoxicity of Steviol51

End-point
Reverse
mutation
Reverse
mutation
Forward
mutation
Forward
mutation
Umu Gene
mutation
S.typhimurium
TA1535/psk1002
Gene
mutation
B.subtilis H17 rec+M45 recChinese hamster lung

fibroblasts
Chinese hamster lung
fibroblasts
Human lymphocytes
0.5g/mlc
1-1.5 mg/mld
0.2 mg/ml
MS/Ae mice
1000mg/kgbwg
Chromosomal
aberration
Chromosomal
aberration
Micronucleus
formation
a.
b.
c.
d.
e.
f.
g.
Test object
Concentration
S.typhimurium TA98 &
20mg/platea
TA100
S.typhimurium TA97,TA98,
5mg/platea
purity, 99%
TA100,TA102,TA104,TA1535
and TA1537
S.typhimurium TM677
10mg/platec
0.510mg/plate
S.typhimurium TM677
10mg/platec
10mg/platee
6251250g/pla
12592500g/pl
10mg/disca
400g/mld
With and without metabolic activation.

The same results are cited in an earlier abstract (Matsui
et al., 1987).
Without metabolic activation.
With metabolic activation.
With metabolic activation derived from Phenobarbital- or
Aroclor
1254-pretre.3-methlycholanthrene-pretreated
rats were ineffective.
Diphtheria toxin-resistant colonies.
Toxic: 4/6 mice at highest dose given intraperitoneally
died.
49
On the basis of various studies, JECFA concluded that:
Steviol is a metabolite of an integral component of

Stevioside that is mutagenic. The structural features
necessary for the expression of mutagenic activity include
a hydroxyl group at position 13 and unsaturated bond
joining the carbon atoms at position 16 and 17.
No significant difference in DNA fragment length was
found between the wild type and spontaneous or Steviol
induced mutants. 53
Metabolically activated Steviol interrupts DNA synthesis
around nucleotide 280, thereby stimulating duplications,
deletion and untargeted mutagenesis in the defined
region of the gpt gene downstream from the site of
interruption.
5.2.3
Developmental toxicity: Steviol58
Groups of 20 pregnant golden hamsters were fed with Steviol

(purity, 90%) at doses of 0, 250, 500, 750, or 1000 mg/kg bw
per day (only 12 animals at the highest dose) by gavage in corn
oil on days 6-10 of gestation. A significant decrease in bodyweight gain and increased mortality (1/20, 7/20, and 5/12)
was observed at the three highest doses, and the number of
live fetuses per litter and mean fetal weight decreased in
parallel. Histopathological examination of the maternal
kidneys showed a dose-dependent increase in the severity of
effects on the convoluted tubules (dilatation, hyaline droplets).
No dose-dependent teratogenic effects were seen. The NOEL
was 250mg/kg bw per day for both maternal and
developmental toxicity.
5.3
Studies conducted other than JECFA
51, 58-60
Mutagenic effects of Steviol, the aglycone of Stevioside, and

/or its metabolites were reported in Salmonella typhimurium
TM 677. After metabolic activation it was shown that so far
unknown
Steviol
metabolites
caused
mutations
in
S.typhimurium TM 677.
50
However, Stevioside and Steviol were inactive in various TA

strains of S.typhimurium, E.coliWP to uvrA/pKM101 in the
rec-assay using Bacillus subtilis even when activated
microsomal fraction was present. The direct mutagenic activity
of 15-oxo-Steviol was refuted by Procinska et al 1991 but
confirmed by Terai et al 2002. The activity of Steviol in S.
typhimurium TM 677 was very low and was only about
1/3000 that of 3, 4- benzopyrene and that of Steviol methyl
ester 8, 13 lactone was 1/24500 that of furyl furamide (Trai et
al 2002). Although a weak activity of Steviol and some of its
derivates were found in the S.typhimurium TM 677 strain, the
study concluded that the daily use of Stevioside as a
sweetener is safe.
Moreover, the presence in the blood of the chemically
synthesized Steviol derivatives after feeding Stevioside is not
proven at all. Very high dozes of Steviol (purity 90%) incubated
to hamsters (4gm/kg bw), rats and mice (8 gm/kg bw) did not
induce micronucleus in bone marrow erythrocytes of both
male and female animals. However, these dozes showed some
cytotoxic effect to the female but not to the male of all treated
animal species. It is not excluded that the toxicity is due to the
10% impurities present.
It has to be said that of all animals tested hamster had the
most sensitive response. Moreover, in hamster several
metabolites of Stevioside were found that are not formed in
rats or human.
51
Uses of Stevia
Chapter 6
6.1 Need of intense sweeteners

In early days, honey and fruits have been used for their
sweetness. It is only in the 14th century that sugar was refined
and considered as a special food item. The main source of
sugar for long has been cane sugar with beet sugar
contributing a small percentage.
The production of cane sugar has been of the order of 262
million tonnes and that of beet sugar 19,500 tonnes in India.
These sugars along with sweetening qualities also have been
found to contribute calories, which can lead to obesity or may
act as a risk factor for some chronic diseases such as diabetes
mellitus, hypertension and cardiovascular diseases.61
In a sweet-toothed society, in which sugar and chemical
sweeteners are considered unsuitable for health, the interest is
now focused on natural sweeteners31
Hence, the craving for sweetness led man to discover several
forms of alternative intense sweeteners, which have made
possible to offer consumers the sweet taste without the
calories. Intense sweeteners add to food a taste that is similar
to that of sucrose and is generally several hundred to several
thousand times sweeter than sucrose. Most of them do not
contain any calories, and those that do contain calories, are
used in very small amounts because of their, concentrated
sweetening property61
Non-nutritive sweeteners serve a number of purposes.
They are used to: 62
6.1.1.1 Expand food and beverage choices for those who must
or want to control calories, carbohydrate, or specific sugar
intake. A significant number of people in affluent countries are
overweight, and obesity is frequently cited as a serious health
problem.
6.1.1.2 Assist weight control or reduction.
6.1.1.3 Aid the management of Diabetes.
52
6.1.1.4 Assist the control of Dental caries.

6.1.1.5 Enhance the usability of pharmaceuticals and
cosmetics. As these are often superior top sugars in making
the unpleasant taste of drugs.
6.1.1.6
Provide sweetness when sugar is not available.(e.g.
in various countries during world war I and II)
6.1.1.7 Assist the cost effective use of limited resources.
6.2 Classification of sweeteners.
62
Sweeteners
Nutritive Sweeteners
sweeteners.
Non-Nutritive/Intense
Those which are calorie free.
*Those which are essentially

significantly reduced in
calories.
*Intense sweeteners are

generally not or produce
negligible
*Carbohydrates are nutritive,

because in human body they
metabolized are metabolized
to produce energy energy.
*e.g. Sugars and Polyols.
Non- Nutritive (OR) Intense (OR) High Potency Sweeteners
NATURAL
ARTIFICIAL
*Thaumantin
*Monellin
*Miraculin
*Brazzein
*Stevioside
*Glycyrrhizininc acid
*Mogroside
*Dihydrochalcones
*Saccharin
*Aspartame
*Cyclamate
*Acesulfame k
*Cyclamate
*Sucralose
*Dulcin
53
6.3 Relative Sweetness:

The evaluation of the sweetness of a given substance in
relation to sucrose on weight basis is termed as relative
sweetness. An arbitrary scale expresses sweetness of these
sweeteners, where sweetness of sucrose is taken as unity. 62
6.4
Benefits of Stevia61

Science, Hebbal, Bangalore India conducted a study on
nutritional and functional importance of stevia in various food
products.
6.4.1Nutritional benefits of Stevia61
Table 14: Nutritional composition of stevia
Nutrient composition
Per 100 gm
Proximate
Moisture(g)
7
Energy(K cal)
270
Protein(g)
10
Fat(g)
3
Total Carbohydrate(g)
52
Ash(g)
11
Crude fibre(g)
18
Minerals
Calcium(mg)
464.4
Phosphorous(mg)
11.4
Iron(mg)
55.3
Sodium(mg)
190.0
Potassium(mg)
1800.0
Anti-Nutritional Factors
Oxalic acid(mg)
2295.0
Tannins(mg)
0.010
Nutrient composition of stevia on dry wt. basis indicate that
energy value being 2.7 kcal/g entails it the status of low
calorific sweetener due to its intense sweetness in comparison
to other available low calories sweeteners1 e.g. Aspartame (4
Kcal/g).
54
In this context, the use of stevia as a low calorie sweetener

could be of immense help in restricting the calorie intake in
the diet of affluent and also wherein restricted diets are
prescribed.
Stevia contains protein, ash of crude fiber, which are essential
for the maintenance of good health and are comparable to
commonly used cereals in India.
However stevia was found to have higher percentage of antinutritional factors-Oxalic acid, which may hinder the
bioavailability of minerals and other nutrients.
6.4.2 Functional benefits of Stevia61
Functional properties of any food aid in determining its
suitability in various methods of cooking and in different
aspects of handing.
Table 15: Functional properties of stevia leaf powder.
Properties
Bulk density
Water absorption capacity
Fat absorption capacity
Emulsification value
Swelling
Solubility
pH
Values
0.443 gm/ml
4.7ml/gm
4.5 ml/gm
5 ml/gm
5.01 gm/gm
0.365/gm
5.95
6.4.2.1 Water binding Capacity: - Water binding capacity is

an important property of protein in viscous foods such as
soups, gravies, doughs and baked products. Hence increased
water binding capacity of stevia appears to be advantageous
may be due to its higher protein content.
55
6.4.2.2 Fat absorption capacity; - This is important since

fat acts as a flavour retainer and increases the mouth feel of
food. Stevia appears to have adequate fat absorption capacity.
6.4.2.3 Emulsifying property: - The ability of protein to aid
the formation and stabilization of emulsion is critical for many
applications such as cake, batters, coffee whiteners, milk,
frozen desserts etc. depending on the composition and
stresses during processing under which it is subjected.
6.4.2.4 Baked Foods: - [Crammer and Ikan. R; Sweet glycosides
from the Stevia plant; Chem.Brit, 22:915-917 (1986)] expressed
that since Stevioside is stable at 950C. It is not much suitable
for baked foods.
6.5
Recommendation by JECFA:
39
According to new tentative specifications prepared at 63rd

meeting of JECFA (2004), published in FNP 52 and ADD
12(2004); Steviol glycosides are functionally designated as
sweetener not as a food additive.
Food Uses63
Stevia and its extracts are used in a wide range of food
products.
6.6.1 Stevia
There are three distinct traditions for using Stevia:
6.6.1.1 Flavor Enhancement: Stevia whole leaf or whole leaf
extract is combined with other herbs to enhance the flavor and
nutritive value of other herbs. Stevia is traditionally
considered nutrient rich containing calcium, phosphorous
alongwith moderate source of protein.
6.6.1.2 Herbal Tea: Stevia is appropriate for use in
conjunction with a variety of other herbal teas. One can mimic
the South American practice of combining stevia with verba
mate, lapacho, and other native herbs, or one can experiment
with stevia in altering the taste parameters of any number of
traditional teas.
56
6.6.2
Steviol glycosides11
Steviol glycosides (the purified extract) are good alternative to

sugar in nearly all of its uses and can replace all or some of
the Sugar. Unlike some man-made chemical sweeteners
Steviol glycosides can be used in conjunction with other
sweeteners (e.g., sugar, fructose, intense sweeteners) and they
act as a flavor enhancer as well as a sweetener. They have
been used in other countries for many years (e.g. Japan for
more than 35 years).
Food products containing stevia extracts, which have been
successfully produced and consumed includes:
Aerated soft drinks, mineral waters and cordials etc
Juices and juice drinks
Ice creams, yoghurts, milk and other dairy products
Sauces, chutneys, pickles
Biscuits, cakes and pastries
Preserved fruits, jams and spreads
Processed and frozen vegetables and meats
Confectioneries, chocolate
Cereals and muesli bars.
Other
stevia-containing
products
have
included
toothpastes, chewing gums and medicinal tablets.
It is often recommended for inclusion in weight loss diets and

for diabetic foods and as part of diets to combat a range of
chronic disorders and infections.
The largest consumers of stevia include Japan, China, Korea,
Paraguay and Brazil, with both Japan and China having peak
consumptions reaching 2,000 tonnes of Steviol glycosides per
year in 1995 2003.
57
Some beneficial side effects of Steviol glycoside consumption

have been demonstrated in a number of scientific studies and
papers. These include reducing high blood pressure, reducing
high blood glucose levels, a reduction in dental caries bacteria
and other bactericidal effects. These effects, combined with the
non-calorie sweetness, the all natural nature and flavor
enhancing properties, add to the attractiveness of using
Steviol glycosides in foods and beverages11
58
Table 16: Uses of Stevioside

Form of stevia
used
Stevioside
chrysantaR)*
(Stevia
Stevioside
(Stevia chrysantaR)*
Stevioside
Stevioside
Stevioside
Food / Non- food

items
Beverages
Ice cream
Chewing gums &
candies
Canned fruits and
fruit jellies
Marron glaces
Stevioside
Beverages
Stevioside
Frozen foods
Stevioside
Canned fruits
Stevioside
Stevioside
Sea food, minced

fish meat &sea
tangle products
Fruit preserves &
cakes
Condiments
Stevioside
Wine and alcohol
Stevioside
Cake, biscuits and

moon-cakes
Sausages, hams, and
salted-dried meat
Chewing gum & daily
chemical products
Pharmaceutical
industry
Stevioside
Stevioside
Stevioside
Stevioside
Level
Can replace 5080% of sucrose
Can replace 50%
of sucrose
Can replace 50%
of sucrose
* Brand name of Stevioside used by Dainippon Ink & Chemicals Inc.

70
59
64-
6.6.3
Stevia: Uses in Indian Indigenous food
61

Sciences, Hebbal, Bangalore India developed various products
such as fruit custard, jam, chikki, basen ladu, wheat ladu,
biscuit, grape juice, bun, tea and milk shakes using stevia (0.25
to 1.0 g per 100 gm) in substitution to sugar. These products
were evaluated for appearance, flavours, texture, and taste
and over all acceptability by trained and semi-trained judges
using 5- point hedonic scale.
Storage study was conducted to study the shelf life for a
period of 3 months. The study was carried out in airtight
polythene pouch and tested for its sensory attributes weekly.
The observations are as follows:
Table 17:
products61
Shelf life study of stevia sweetened Indian
Products
Wheat ladu, besan
ladu& chikki
Mixed fruit jam
Biscuit
Bun
Observations during shelf life

studies
(3 months)
Acceptable upto 12 weeks of storage
Completely spoiled at 5th week (fungal
growths)
Spoiled at the 1st week
Not more that two days
Other products such as grape juice, milk shake, tea and fruit
custard were not considered for shelf life study because of the
perishable nature of ingredients used.
The concept of Glycemic Index (GI) of various food has
emerged as a boon to diet therapy for diabetes mellitus
indicated beneficial aspect of food consumed both individually,
supplemented or in mixed diet.
The Glycemic response of stevia containing product was also
estimated in order to know the effectiveness of the product in
the diet of diabetic people.
60
The GI of stevia bun with 75 gm of carbohydrate was tested in

comparison to the bun without stevia and for glucose. The
observations are as follows:
Table 18: GI of stevia containing products61
Test Products
GI in Normal
GI in Diabetic
Subjects
people
Glucose
100
100
Control bun
60.4
72
Stevia bun
55.5
62.12
This study showed that stevia has hypoglycemic response.
61
Stabilities studies of Stevioside and Rebaudioside A Chapter 7
To date few data are available on the practical applications of

Stevioside in foods and beverages and there is a lack of
detailed relevance regarding its stability during different
processing and storage conditions and its interactions with
other food ingredients or food additives with special regard to
its application in appropriate food categories.
7.1 Stability studies of the pure sweetener:
Stevioside has been reported for good stability when stored in
solution with water, soy sauce and vegetable protein
hydrolysates at 30 oC for 30 days. Some loss, however, was
found when stored in vinegar and after heating at 80 oC.
7.1.1
Stability
temperatures:
studies
of
Stevioside
at
elevated
Incubation of the solid sweetener (0.5g/l) at elevated

temperatures for 1 hr showed good stability up to 120oC,
whilst at temperatures exceeding 140oC forced decomposition
was seen which resulted in total decomposition by heating
upto 200oC 71
7.1.2 Stability studies of Stevioside at different pH at
elevated temperatures:
In aqueous solution Stevioside (0.5g/l) is remarkably stable
over a wide range of pH values and temperature.
Under thermal treatment in a pH range of 2-10 over 2 hr
practically no degradation of Stevioside (0.5g/l) could be
observed at 60oC and only slight losses up to 5% (pH 2 and 10)
occurred on heating to a temperature of 80oC .
Under strong acidic conditions (pH 1) forced decomposition of
Stevioside (0.5g/l) was observed which resulted in total
decomposition after incubation at a temperature of 80oC for 2
hr.
Only traces of Steviolbioside and glucose, degradation
products of Stevioside, were detected which could be
attributed to the rupture of C- 19 ester bond in the sweetener. 71
62
7.1.3 Stability studies of pure Stevioside (6.5 mg/ml)

when heated in neutral solutions:
HPLC and TLC showed that prolonged heating of Stevioside
solution at 100oC resulted in a decrease in the Stevioside
concentration with the appearance of the two degradation
products Steviolbioside and glucose.
Steviolbioside appeared in the TLC chromatogram after
heating for 4 hrs and Glucose was detected following 8 hr of
heating.
Rebaudioside: Similar results were obtained for Rebaudioside
A, yielding Rebaudioside B and Glucose as the degradation
products. Thus, the C-19 ester linking appeared to be the
most heat-sensitive bond in Stevioside and in Rebaudioside
A.72
7.1.4 Heating in acidic solutions:
Table 19: TLC Analysis of Stevioside72
Steviol
Temperature Time
Effect
o
component
( C)
(hr)
Stevioside (in citric acid beverage)
60
137
No appreciable degradation
100
4
Steviolbioside, glucose and two
unknown components (A, B)
100
10
Component C appears
100
13
Traces of two components D, E
Stevioside (in phosphoric acid beverage)
60
137
No appreciable degradation
100
4
Components A, B and C,
Steviolbioside and glucose
appeared.
100
13
Significant amount of
components D and E were seen.
Rebaudioside (in citric acid beverage)
100
4
Rebaudioside B, glucose, one
unknown (N), and a trace of
another unknown (M) were
detected.
100
13
Concentration of degradation
products increased with time,
no additional product.
Rebaudioside (in phosphoric acid beverage): Similar results were
obtained except that degradation products were more pronounced,
higher concentration of component M
63
Table 20: HPLC Analysis of Stevioside72

Steviol
component
Temperature
Time (hr)
Effect
o
( C)
Stevioside (in citric acid beverage)
100
4
One unknown
component (2)
100
10
Three
unknown
components
(1,2 and 5)
100
13
Four unknown
components
(4,10 and13)
Stevioside (in phosphoric acid beverage)
100
4
Two unknown
components (2
and 5)
100
10
Two unknown
components (1
and 4)
100
13
Two unknown
components (1
and 4)
Rebaudioside (in citric acid beverage)
100
4
One unknown
components
(iii)
100
10
Two unknown
components (ii,
iii)
100
13
Two unknown
components (ii,
iii)
Rebaudioside (in phosphoric acid beverage)
100
4-10
Unknown (i)
100
13
No component
found
Both Stevioside and Rebaudioside A degraded much faster

in acidic solutions than in neutral solutions. Moreover,
both sweeteners were slightly less stable in phosphoric
acid than in citric acid, possibly partly due to slightly
lower pH
64
pH stability and degradation rate of stevioside in a pH range of 1-10 under thermal

treatment at 60 and 80oC for 1 and 2 hr.
100
90
80
70
60
stevioside% 50
40
30
20
10
0
60dc/1hr
60dc/2hr
80dc/1hr
80dc/2hr
5
pH
9 10
Degradation rate of stevioside in aqueous solutions of organic acids(10g/l) for

storage at room temperature
100
90
80
70
60
stevioside % 50
40
30
20
10
0
Acetic acid
Citric acid
Tartaric
acid
Phosphoric
acid
30
60
90
Storage time(days)
120
7.1.5 Stability studies in organic acids:

Table 21: Interaction of Stevioside with organic acids71
Stevioside
concentration
1 g/l
10 g/l
Temp of
storage
Room
temperat
ure
Room
temperat
ure
Time of
storage
(months)
Medium
Effect
Acetic acid
(pH 3.1)
No evidence
Citric acid
of
(pH 2.6)
degradation
Tartaric
acid (pH
2.6)
Phosphori 30% losses in
c acid (pH
Stevioside
2.2)
concentration
Acetic acid 2% losses in
(pH 2.6)
Stevioside
concentration
Citric acid 22% losses in
(pH 2.1)
Stevioside
concentration
Tartaric
33% losses in
acid (pH
Stevioside
2.1)
concentration
Phosphori 75% losses in
c acid (pH
Stevioside
1.6)
concentration
7.1.6 Interaction with vitamins71:

Incubation of Stevioside for up to 4 hr with individual watersoluble B-group vitamins and vitamin C in aqueous solution at
80oC showed no significant change in sweetener or in vitamin
concentration for the B-vitamins under thermal treatment and
in untreated samples.
In the case of vitamin C, however, incubation at 80oC resulted
in time dependent degradation of ascorbic acid, where - as a
protective effect of Stevioside on the degradation of ascorbic
acid was observed, resulting in a delayed degradation of
vitamin C in the presence of Stevioside (27% after 4 hr
incubation) compared to the analogous treated substance
(13% after 4 hr incubation).
65
7.1.7 Interaction with other low calorie sweeteners71:

With regard to the practical application of low calorie
sweeteners in synergistic mixtures, binary aqueous solutions
of Stevioside with other individual low calorie sweeteners,
Saccharin, Cycalmate, Aspartame, Acesulfame K and
Neohesperidin dihydrochalcone, were investigated.
Excellent stability were found in the course of thermal
treatment at 80oC for up to 4 hrs as well as over 4 months of
incubation at room temperature, indicating that there are no
chemical interference to the simultaneous use of Stevioside
with low calorie sweeteners. 71
7.1.8
Interaction with enzymes73
7.1.8.1Flavobacterium johnsonae was isolated from a
microorganism that produced a -glucosidase with hydrolytic
activity of -glucosyl ester linkages in Steviol glycosides. The
molecular mass of the purified enzyme was about 72 kDa by
SDS-PAGE.
Table 22: Characteristics of Flavobacterium Johnsonae73
S.No.
1.
2.
3.
Characteristics
pI (Isoelcetric point)
:
Most Active at
pH
:
Stable between pH
:
Optimum temperature :
Stable below
:
8.8
7.0
3.0 - 9.0
45C
35C
7.1.8.2 Action of enzyme: The enzyme hydrolyzes glucosyl

ester linkages at site 19 of Rebaudioside A, Stevioside, and
rubusoside, although it could not degrade -glucosidic
linkages at site 13 of Rebaudioside B or Steviolbioside.
7.1.8.3 Enzyme inhibition: The enzyme activity on
rubusoside was inactivated completely by Hg2+, and reduced
Cu2+,
p-chloromercuric
benzoate,
and
by
Fe3+,
phenylmethylsulfonyl fluoride (residual activity; 67.9--84.8%).
73
66
7.2
Stability of pure sweetener in food system:
7.2.1 Stability and interaction in hot beverages:

Thermal treatment of Stevioside sweetened coffee and tea at
80oC over a time period of 4 hr resulted in no significant
influence on the Stevioside content.
Only minimal losses of Stevioside, up to 5% were seen after 4
hr incubation of tea or coffee, indicating that no significant
effects should be expected under practical conditions of the
preparation and consumption of the hot beverages. 71
7.2.2 Stability studies in carbonated beverages:
7.2.2.1 Table 23: Long term storage studies of Stevioside
in carbonated beverages72
0.1% Stevioside as sole sweetener in carbonated
beverages
o
Temp ( C)
Time of storage
Effect
(months)
4 or at room
5
No significant changes
temperature
37
4
36% loss in Stevioside
concentration
(phosphoric acid
beverage)
17% loss in Stevioside
concentration (citric acid
beverage)
0.1% Rebaudioside as sole sweetener in carbonated
beverages
4
4
No significant change in
Rebaudioside A
Room
3
concentration
(in both
temperature
citric and or phosphoric
37
1
acid beverages)
7.2.2.2 Table 24: Microbiological Studies of Stevioside in

carbonated beverages72
Microbiological parameter
Initial total count
Yeast & mold count
67
Value
Under 100 counts/ml
Zero
7.2.2.3 Organoleptic studies:

Organoleptically no significant differences other than
sweetness were found between the aged sweetened products
and the aged unsweetened products with 0.1% sweetener
added to the latter after storage.
Tasters generally could not easily detect a loss in sweetness
resulting from a 10% or less decrease in either the Stevioside
or Rebaudioside A concentration.
7.2.2.4 Table 25: Effect of heating
sweetened carbonated beverages72
Steviol
glycoside
Temperature
o
C
Stevioside
60
Rebaudioside
A
60
Time of
storage
(hr)
137
137
on
Stevioside
Effect on
concentration
No decrease
(citric acid
beverages)
4% decrease
(phosphoric
acid beverages)
3% decrease
(citric acid
beverages)
6% decrease
(phosphoric
acid beverages)
Both the sweeteners appeared to be quite stable when the

beverages were heated at 60oC for 137 hr.72
7.2.2.5 Effect of sun light exposure
sweetened carbonated beverages:
on
Stevioside
No significant changes in Stevioside concentration were

observed in either the phosphoric or citric acid beverages
when exposed to 3000 langleys of sunlight.
On the other hand, Rebaudioside A underwent considerable
degradation under similar conditions with 22 and 18% losses
in the phosphoric and the citric acid beverages, respectively 72
68
Regulations Regarding Stevia and Steviol glycosides
8.1
Chapter 8
USA(See Annexure II) 74:
In the United States, any consumable food or drink that

contains stevia is considered adulterated.
According to Federal Food, Drug and Cosmetic Act of
1994(Revised April 2000), Section 402(a) (2) (c), Section 409
and 21 CFR 170 and 21 CFR 189-1, Stevia is a substance
Prohibited from use in human food. As per 21 CFR 190,
Stevia may be sold in the United States as a stand-alone
dietary supplement or an herb, but not as a sweetener.
Dietary supplements as defined by Dietary Supplement
Health and Education Act (DSHEA) do not include food
products or products intended to replace a meal.
Food products are consumed as part of an individuals daily
dietary intake for the purpose of satiety, taste[,] basic
nutritional needs, and pleasure.
Dietary supplements are currently defined as products that
are intended to supplement the diet that contains one or more
of certain dietary ingredients, such as:
A vitamin or mineral
An herb or other botanical
An amino acid
A dietary substance for use by man to supplement the

diet by increasing the total dietary intake, or
A concentrate, metabolite, constituent, extract, or
Combination of the preceding ingredients, and, that meet

other criteria specified in Section 201(ff) (2)-(3)
Dietary supplements by definition must be taken orally. They
come in many dosages forms including pills, liquids, powders,
or granules.
As of March 23, 1999, all dietary supplements must bear
nutrition information entitled Supplement Facts. This
labeling is similar to nutrition content labeling for
conventional foods but is tailored to the special characteristics
of dietary supplements. Since the mid 1980s the US Food and
Drug Administration (FDA) has labeled Stevia as an unsafe
food additive.
69
Stevia use in dietary supplements:As per Food and Drug Administration (FDA) Oct 18, 2000 If
Stevia is used in a dietary supplement for a technical effect,
such as use as a sweetener or flavoring agent, and is labeled
as such, it is considered an UNSAFE FOOD ADDITIVE
The following products (partial list) may be seized/detained if
they contain Stevia: Tea, drinks, seafood, fruits, vegetables
and candies.
8.2
Canada:
The Canadian Food Inspection Agency (CFIA) has not approved

Stevia as a sweetener in Canada.
The CFIA has issued a notice of detention to companies in
Canada who attempt to move, sell, alter or dispose of Stevia
products (1999). In Toronto, the CFIA issued a notice of
detention to a company regarding a product containing Stevia
(December 1999).
The company was banned from selling, moving, or disposing of
the Stevia product.
8.3 European Countries
The Commission of the European Communities, European
Parliament, and all Member States has disapproved the use of
Stevia as food or a food ingredient in consumable foods.
The Commission of the European Communities
Having regard to the Treaty establishing the European
Community, Regulation (EC) No. 258/97 of the European
Parliament regarding S.rebaudiana Bertoni:
Plants and dried leaves on the market as a novel food or novel
food ingredient, the Commission forwarded the following
notice to all Member States on 18 August 1998.
70
The measures provided for in this decision are in accordance

with the opinion of the Standing Committee for Foodstuffs;
Article 7 of the Regulation, June 17, 1999; Regulation (EC) No.
258/97;Article 3(1) of the Regulation. On February 22, 2000,
committee has adopted the following decision Stevia
rebaudiana bertoni: plants and dried leaves may not be
placed on the Community market as food or food
ingredient.
Official document was delivered in September 2000 and as per
the document 300DO196, 2000/196/EC; Commission
decision dated 22nd February, 2000 refused to place Stevia
rebaudiana bertoni plant and dried leaves as a novel food or
novel food ingredient under Regulation (EC) No. 258/97 of the
European Parliament and the Council [notified under
document number C) (2000) (77) Official Journal L 061,
08/03/2000 p. 0014].75
8.4
Japan(See Annexure III) 76
In 1947, Ministry of health, labour and welfare enacted the

food sanitation law as the first comprehensive law for food
safety /hygiene, and introduced a positive list system for food
additives.
Under the system, only additives designated as safe by the
Ministry of health, labour and welfare may be used in foods.
However, designated system had been applied only to
chemically synthesized additives until 1995 when the Food
Sanitation Law was amended. Currently, all types of additives
are equally subject to the designation system, synthetic or
non-synthetic with minor exceptions.
Exemption from the designation system:
There are three substance categories that are exempted from
the designated system:
1.
Existing food additives (substances that were already
marketed or used on the date of the amendment of the
Food Sanitation Law and appear in the list of existing food
additives.
2.
Natural flavoring agents.
3.
Substances that are both generally provided for eating or
drinking as foods and used as food additives have been
excluded from the designation system.
71
The list of existing food additives from natural origin was

compiled and published by the Ministry of Health Labour and
Welfare (Japan) on 16th April 1996. According to the
published list stevia extract (sequence no. 209), a
substance composed mainly of Steviol glycoside obtained
by extraction of stevia leaves was included as a part of
existing food additives. The list was effective from
February 25, 2005.
8.5
Australia and New Zealand(See Annexure IV) 77
Food standards Australia and New Zealand (FSANZ: Food

Standards Code). Chapter 1 (General Food Standards) of the
Australia New Zealand Food Standard Code deals with
standards which apply to all foods, however, New Zealand
regulates its own Maximum Residue Limit (MRLs) for food and
standard 1.4.2 regulates MRLs in Australia only.
As per part 1.3: Substances Added to Food, standard: Food
Additives, 1.3.1: schedules 2,3,4,5) of chapter 1, a record of
views formed and actions taken in relation to standard 1.5.1
Novel Food of the Code.
The listed foods and food ingredients with views as to their
status as non-traditional / novel foods reached either by the
Novel Food Reference Group (NFRG) alone or in consultation
with Senior Food Officers (SFOs) of the Australian, State,
Territory and New Zealand Governments, as well as the
Australian Quarantine and Inspection Service.
Table 26: Abstract form the record of views formed by the
FSANZ Novel Foods Reference Group:
Food or
Food
Ingredient
Stevia
(crushed
leaf)
Outcome View
Nontraditional food
Novel food
Stevioside
and Stevia
extract
considered
as a food
additive
72
Comment
Previous applications for
Stevioside (A397 & A457)
as a food additive have
previously been
submitted but there have
been deficiencies in some
of the safety data. Both
applications were
withdrawn.
8.6
Hong Kong(See Annexure V) 78
In formulating food safety standards and performing safety

assessment, Hong Kong draws reference from the evaluation
made by JECFA and the standards and guidelines set by the
CAC, an international foods standard setting authority.
Although the food standards set by Codex are not binding to
its member states, they serve as the World Trade
Organizations international trade reference standards.
In Hong Kong, the import and sale of Stevioside is subject to
the control of the sweeteners in Food Regulations, Chap 132.
According to the Regulations, sweetener means any chemical
compound, which is sweet to the taste, but does not include
any sugars or other carbohydrates or polyhydric alcohols.
Stevioside is not one of the permitted sweeteners under the
Regulations.
By virtue of the Food and Drugs (Composition and Labeling)
Regulations, Chap.132, prepackaged food should be labelled if
it contains sweeteners.
Sale of food containing non-permitted sweeteners or
prepackaged food not properly labeled in accordance with the
requirements of the regulations is liable to a maximum fine of
HK$50,000 and imprisonment for 6 months.
8.7
Singapore
According to Ministry of the Environment, the use of stevia in

food products will be approved in Singapore until its safety
has been fully established and recognized by international
bodies, such as JECFA.
(Relevant legislations: Sale of Food Act Chap 283, Pt III: sale of
food)79
73
Pharmacological studies
Chapter 9
JECFA in its 51st meeting (1999) revised the study on

Absorption, distribution and excretion of Steviol glycosides.
The abstracts from the same are as following:
9.1
Absorption, Distribution, and Excretion.
9.1.1 Rats
(specific activity, 13 or 46 Ci /mg), was
administered with gavage to groups of three to seven Wistar
rats at a dose of 125 mg/kg bw (10-120 Ci/kg bw), Stevioside
was absorbed slowly and a maximum blood concentration of
4.8g/ml being reached by 8 h.
3H-Stevioside
As per the studies mentioned in JECFA report, committee

concluded that Stevioside is absorbed from the gut very slowly,
that enterohepatic circulation occurs in rats, and faecal
excretion is the major route of elimination.
The renal excretion of Stevioside and its effect on the renal
excretion of several other substances was studied in groups of
10 male Wistar rats, which received intravenous infusions of
Stevioside at doses of 4, 8, 12, or 16mg/kg bw per hour for 30
min, after a control period of 30 min.
No significant change in insulin clearance was observed, but
there was a significant increase in para-aminohippuric acid
clearance, fractional sodium excretion (FeNa+), urinary flow as
percent of glomerular filtration rate, and glucose clearance
when compared with controls at doses greater than 4 mg/kg
bw per hour; Stevioside clearance was greater than insulin
clearance and smaller that para-aminohippuric acid clearance
at all doses tested.
The study concluded that Stevioside is secreted by the renal
tubular epithelium and induces diuresis and natriuresis and a
fall in renal tubular reabsorption of glucose.
74
9.2
Biotransformation
Thin-layer chromatography of the intestinal contents, faeces,

and bile of groups of three to seven Wistar rats fed with 3H
Stevioside (specific activity, 13 or 46 Ci/mg), by gavage at a
dose of 125 mg/kg bw (10-120 Ci /kg bw) revealed that
Stevioside is decomposed by rat faecal flora to Steviol and
sugar.
The JECFA study concluded that orally administered
Stevioside is not readily absorbed from the upper part of the
small intestine, but metabolites, formed primarily by the
bacterial flora in the caecum, are absorbed from the lower part
of the intestine; they also concluded that most of the
Stevioside is excreted as Steviol in the faeces (Nakayama et al.,
1986). The committee concluded further that the faecal Steviol
could also have formed from deconjugation of biliary
conjugates by the gut flora.
When 2.5-mg/ml Stevioside were incubated under anaerobic
conditions with whole-cell suspensions of bacteria from rat
caecum, Stevioside was completely degraded to Steviol within
two days. The JECFA report authors concluded that similar
degradation of Stevioside occurs in humans.
131I-
Stevioside (specific activity, 3.7 MB/q/mg, equivalent to

100Ci/mg; 1.1 MBq, 30Ci, equivalent to 1mg/kg bw) was
injected intravenously to male Wistar rats.
The results of reverse-phase high-performance liquid
chromatography (RP-HPLC) of the bile showed that Stevioside
was degraded in vivo and that Steviol was the major metabolite
(47% of radiolabel); 37% of the radiolabel was on Stevioside,
and the remaining 15% was on an unidentified metabolite.
RP-HPLC analysis of urine 90 min after injection showed the
presence of Stevioside and the same unidentified metabolite
found in bile, but no Steviol.
The study concluded that Stevioside is metabolized in rat liver
to Steviol, which is excreted through the bile, and that similar
degradation occurs in humans.
However, the JECFA Committee considered that this study
was of limited value since introduction of a large 131I atom into
Stevioside might significantly affects its absorption,
distribution, metabolism, and excretion in bile or urine.
75
9.3
Effects
parameters
on
enzymes
and
other
biochemical
Stevioside given to female RCR/Ha mice did not induce

glutathione S- transferase activity in liver or intestinal mucosa
(Pezzuto et al., 1986). Stevioside (1 mmo1/L, equivalent to 0.8
mg/ml) inhibited oxidative phosphorylation and the activity of
ATPase (50% inhibition), succinate oxidase (8% inhibition),
and succinate dehydrogenase (10% inhibition).
No inhibition of NADH-oxidase or L- glutamate dehydrogenase
activity was seen. The ADP:O ratio was slightly decreased.
Substrate respiration was increased at low concentrations (up
to 0.5 mmo1/L, equivalent to 0.8 mg/ml).
The study concluded that Stevioside acts as a weak uncoupler
of oxidative phosphorylation (kelmer- Bracht et al., 1985).
9.3.1 Effect on citric acid cycle and ketogenic pathway
The effect of Stevioside, an inhibitor of long chain fatty acid
transport, on ketogenesis and on 14C-carbon dioxide
production from [1-14C]-palmitate (100-300 mo1/L) was
investigated in isolated and haemoglobin free perfused rat
liver.
Stevioside (2.5 mmo1/L, equivalent to 2 mg/ml) inhibited both
parameters but had a smaller effect on 14 C-carbon dioxide
production.
At 300 mo1/L palmitate and 150 mo1/L albumins,
ketogenesis was inhibited by 66%, whereas no significant
inhibition of 14 C-carbon dioxide was seen.
The study concluded that these results reflect different degrees
of saturation of the citric acid cycle and the ketogenic pathway
and those changes in the redox state of the mitochondrial NAD
(+) NADH complex occur after infusion of Stevioside
The JECFA Committee noted that the concentrations used in
the studies conducted in vitro were very high relative to those
achieved in blood after oral administration, when the major
intestinal metabolite that enters the circulation is Steviol.
These studies may therefore be of limited significance.
76
9.3.2 Effect on Glycogen synthesis:

When single doses of 200 mo1/L Stevioside, equivalent to
650 mg/kg bw, were given orally to 24-h-fasted male Wistar
rats, either alone or simultaneously with fructose, Stevioside
increased the initial glycogen deposition in the liver.
When it was given to the rats in the drinking-water at 1 or 2
mmo1/L, equivalent to 81 and 160 mg/kg bw, at the
beginning of a fasting period of 24 or 48 h, increased hepatic
glycogen concentrations were found at 48 h (1mmo1/L and at
24 h (2mmo1/L).
The JECFA report authors concluded that Stevioside
stimulates hepatic glycogen synthesis under gluconeogenic
conditions.
9.3.3 Effect of Stevioside on metabolism of D-Glucose and
D- Fructose:
The effect of Stevioside on the transport and metabolism of Dglucose and D-fructose was investigated in isolated perfused
rat liver.
The maximal exchange rate of D-glucose was 700 mol/L per
min/ml, and the Km was 38 mmol/L. Stevioside inhibited Dglucose and d-fructose transport across the cell membrane.
Stevioside had no effect on D-glucose metabolism, except to
cause transient changes in D-glucose release, which reflected
changes in the intracellular concentration.
D-Fructose consumption, however, was specifically affected, as
were all parameters that depend on D-fructose transformation:
D-glucose production, L-lactate and pyruvate production, and
extra oxygen uptake.
In liver the released D-glucose from endogenous glycogen,
strong inhibition of transport increased the intracellular: extra
cellular ratio of D-glucose concentration. The control values
for this ratio, representing an average over the total
intracellular water space, were all below unity.
77
9.3.4 Effect of Stevioside on Gluconeogenesis:

Stevioside had no effect on gluconeogenesis or oxygen uptake
in isolated Wistar rat renal cortical tubules at concentrations
up to 3 m mol/L, equivalent to 2.4 mg/ml.
The study concluded that the lack of activity was due to the
inability of Stevioside to penetrate the cell membrane
(Yamamoto et al., 1985).
9.3.5 Effect of Stevioside on amino acids:
Infusion of Stevioside at 15 mol/L, equivalent to 12 g/ml,
for 20 min did not significantly alter the arginine-induced
secretion of insulin or glucagons in the pancreas of male
Wistar rats (Usami et al., 1980).
Stevioside inhibited the action of atractyloside, a known
inhibitor of the adenine nucleotide carrier of mitochondria and
in consequence and inhibitor of energy metabolism, in isolated
perfused rat liver. It decreased the effects of atractyloside on
glycolysis, glycogenolysis, gluconeogenesis, and oxygen
uptake. The concentration for half-maximal action of
Stevioside was 0.5 mmol/L, equivalent to 0.4 mg/ml.
The authors concluded that it acts on the outside of the cell,
as labeled Stevioside did not penetrate the cell membranes
(Ishii & Bracth, 1986).
Studies other than JECFA are as following:
9.4
Antihyperglycemic Effect of Stevioside:
Department of Endocrinology and Metabolism C, Denmark

studied Antihyperglycemic effect of Stevioside in type 2
diabetic subjects.
The acute effects of Stevioside in type 2 diabetic patients were
studied. 12 type 2 diabetic patients were included in an acute,
paired crossover study. A standard test meal was
supplemented with either 1 g of Stevioside or 1 g of maize
starch (control). Blood samples were drawn at 30 minutes
before and for 240 minutes after ingestion of the test meal.
78
The authors concluded that as compared to control, Stevioside

reduce the incremental area under the glucose response curve
by 18% (P= 0.013). The insulinogenic index (AUC (i insulin)/
AUC (i, glucose) was increased by ~ 40% by Stevioside
compared to control (P<0.001)82-83
Therefore Stevioside reduces postprandial blood glucose levels
in type 2 diabetic patients indicating beneficial effects on the
glucose metabolism. Stevioside may be advantageous in the
treatment of type 2 diabetes.82-84
9.5
Effect of Stevioside on Cardiovascular System: -
IsoSteviol is a derivative of Stevioside, a constituent of S.

rebaudiana , which is commonly used as a non- caloric sugar
substitute in Japan and Brazil.
The study was conducted on rat: cultured rat aortic smooth
muscle cells which were pre incubated with isoSteviol, then
stimulated with angiotensin II, followed by [(3) H] thymidine
incorporation and endothelin-1 Secretion was examined.
IsoSteviol (1-100micromol/l) inhibits angiotensin-II-induced
DNA synthesis and endothelin-1secertion.
Measurement of 27-dichlorofluorescin diacetate, a redox
sensitive fluorescent dye, showed an isoSteviol-mediated
inhibition of intracellular reactive oxygen species generated by
the effects of angiotensin II.
The inductive properties of angiotensin II on extra cellular
signal-regulated kinase (ERK) phosphorylation were found
reversed with isoSteviol and antioxidants such as Nacetylcysteine.
In conclusion this study speculates that isoSteviol inhibits
angiotensin-II-induced cell proliferation and endothelin-1
secretion via attenuation of reactive oxygen species generation.
Thus, this study provides important insights that may
contribute to the effects of isoSteviol on the cardiovascular
system85
79
9.6
Antihypertensive effect of Stevioside:
A double-blind placebo-controlled study of the effectiveness

and tolerability of oral Stevioside in human hypertension was
studied and no significant adverse effect was observed and
quality of life assessment showed no deterioration.
The tolerability of Stevioside appears satisfactory in this study
as only a few patients reported minor side effects, such as
dizziness or nausea after taking these capsules and adverse
effects reported from the placebo group were similar.
Although the hypotensive effect of Stevioside was not better
than other antihypertensive drugs, it appears comparable and
almost all the active drugs. Almost all the active treatment
group patients showed significant lowering of blood pressure86
The antihypertensive effect of crude Stevioside obtained from
the leaves of S.rebaudiana (Bertoni) Bertoni (Compositae) on
previously untreated mild hypertensive patients was
examined.
Patients with essential hypertension were submitted to a
placebo phase for 4 weeks.
All adverse events were prospectively recorded but no major
adverse clinical effects were observed ruing the trial. Systolic
and diastolic blood pressure decreased (p<0.05) during the
treatment with crude Stevioside, but a similar effect was
observed in the placebo group. Therefore, cured Stevioside up
to 15.0 mg/kg/day did not show an antihypertensive effect.
Moreover, the results suggest that oral crude Stevioside is safe
and supports the well-established tolerability during long-term
use an as sweetener in Brazil87.
Inhibitory effect of Stevioside on calcium influx to produce
antihypertension was investigated.
80
Previous studies have shown that Stevioside lowers blood

pressure in spontaneously hypertensive rats (SHRs) when
administered
intravenously.
This
study
shows
that
intraperitoneal injections of Stevioside 25mg/kg also have
antihypertensive effect in SHRs.
In isolated arotic rings from normal rats, Stevioside could
dose-dependently
relax
the
vasopressin-induced
vasoconstriction in both, presence and absence of
endothelium. However, Stevioside had no effect on
phenylephrine- and Potassium Chloride (KCL)- induced phasic
vasoconstriction.
In addition, Stevioside lost its influence on vasopressininduced vasoconstriction in Ca2+ free medium. The result
indicates that Stevioside caused vasorelaxation via an
inhibition of Ca2+ influx into the blood vessel.
This phenomenon was further confirmed in cultured aortic
smooth muscle cells A7r5 using 10-5 M methylene blue for 15
min, Stevioside could still relax 10-8 M vasopressin-induced
vasoconstriction in isolated rat aortic rings, showing that this
vasorelaxation effect was not related to nitric oxide.
The study shows that the vasorelaxation effect of Stevioside
was mediated mainly through Ca2+ influx inhibition. 88
9.7
Anti-Inflammatory
and
Immunomodulatory
Activities of Stevioside and Its Metabolite Steviol:
S. rebaudiana bertoni possesses anti- inflammatory and
antitumor promoting properties; however, not much
information was available to explain its activity.
Stevioside at 1mM significantly suppress lipopolysaccharide
(LPS) - induced release of INF- alpha, Interleukin (IL)-1beta
slightly suppressed nitric oxide release in THP-1 cells without
exerting any direct toxic effect, whereas Steviol at 100M did
not.
Activation of Ikappa B kinases (IKK) beta and transcription
factor NF-kappa B were suppressed by Stevioside as
demonstrated by Western blotting.
81
Furthermore, only Stevioside induced Tumor Necrosis Factor

(TNF)-alpha, IL-1beta and nitric oxide release in
unstimulated THP-1cells. Release of TNF-alpha could be
partially neutralized by anti- TLR 4 antibody.
This study suggested that Stevioside attenuates synthesis of
inflammatory mediators in LPS- stimulated THP-1cells by
interfering with the IKKbeta and NF- kappa B signaling
pathway and Steviosideinduced TNF-alpha secretion is
partially mediated through TLR489 .
9.8 Hypoglycemic effect of Stevioside:
The study was conducted to view the effect of Stevioside on the
glucose and insulin metabolism in 2 models of diabetes in rats
i.e.; Streptozotocin (STZ)-induced diabetic rats and NonInsulin Dependent Diabetes Mellitus (NIDDM) diabetic rats
induced by feeding with fructose.
Stevioside (0.5 mg/kg) lowers the blood glucose levels in STZinduced diabetic rats, peaking at 90min.
Stevioside when administered twice daily, demonstrated dose
dependent effects in lowering the glucose levels in both
diabetic rat models.
Stevioside reduced the rise in glucose during glucose tolerance
testing in normal rats.
Stevioside also reduced insulin resistance in the diabetic
animals as shown by the glucose lowering effects of
tolbutamide.
In conclusion, Stevioside was able to regulate blood glucose
levels by enhancing not only insulin secretion, but also insulin
utilization in insulin-deficient rats; the latter was due to
decreased PEPCK gene expression in rat liver by Steviosides
action of slowing down gluconeogenesis90
82
9.9 In Vivo Cariogenicity

Rebaudioside A
Study
on
Stevioside
and
With the Department of Pediatric Dentistry, College of

Dentistry, and university of Illinois at Chicago, tested the
commercially used natural sweeteners Stevioside and
Rebaudioside A for cariogenicity in albino Sprague-Dawley
rate. Rat pups were colonized with Streptococcus sobrinus, and
four groups used in the experiment were fed for 5 weeks with a
diet incorporating either 30% sucrose, 0.5% Stevioside, 0.5%
Rebaudioside A, or no sweetener additions. By using Keyes
technique to evaluate caries production, it was concluded that
neither Stevioside nor Rebaudioside A was cariogenic under
these conditions.7
83
Estimation of Stevioside in food
Chapter 10
Several procedures for the assay of Stevioside and other

diterpene glycosides of Stevia have been reported including
gas-liquid chromatography,5 thin-layer chromatography,6 and
thin chromatography7 a combination of rod-type thin-layer
chromatography with flame ionization detection. A number of
high performance liquid chromatography (HPLC) methods have
been applied to plant material 8,9 and to food and beverage
products 10,11 containing these sweet glycosides. A capillary
electrophoresis (CE) method 12 has also been reported.91
10.1 Simultaneous Determination of Glycyrrhizinic acid
(GA), Stevioside (St) and Rebaudioside A (RA) in food by
Solid Phase Extraction and HPLC:
Various sweeteners (GA, St, and RA) were extracted with a
methanol - 0.1% ammonium hydroxide (40-60) mixture using
a homogenizer. After centrifugation of the homogenate, a part
of the supernatant was diluted with the water, added to tetra
butyl ammonium bromide (TVA), and then the mixture was
loaded into a C-18 cartridge. The cartridge was washed with
25% acetone- water (containing 5 mM TVA) and water, and the
sweetener were eluted by acetonitrile- 0.1%(v/v) Phosphoric
acid (80-20). The elute was analyzed by HPLC equipped with
an NH 2 column and UV detective (254 nm for GA/210nm for
St and RA).92
Table 27: Recoveries of artificial sweeteners when spiked
at 10 or 50 mg/g are as follows 93
Compound
Recovery (%)
Glycyrrhizinic acid (GA)
82 - 104
Stevioside (St)
84 - 104
Rebaudioside A (RA)
54 - 101
Detection limits of GA, ST, and RA

KG
0.0001Gm /
This method was applied to 16 commercially available foods

with indications of both licorice extracts (GA is dominating
component) and Stevia extracts (ST and RA are
dominating components).
84
Table 28: Limits of detection for artificial Sweeteners

Compound
Limit of
detection(mg/g)
Samples
evaluated
out of total
16
Glycyrrhizinic acid
5.1- 406.7
16
Stevioside (St)
5.4 1.67
14
Rebaudioside A (RA)
4.2 170.2
14
(GA)
10.2 HPLC method for the determination of sweet tasting

Stevioside (STS) in the leaves of the plant S. rebaudiana
and in beverages (e.g. tea, orange juice) 94
10.2.1 Pre separation procedure: This procedure was
consisting of extracting STS from the plant material using
boiling water and a solid phase extraction (SPE).Recovery
rates of the SPE for the analyzed matrices ranged from 92.8%97.8% (for concentrations of TS of 105,210 and 300 g/ml;
Relative standard Deviation (RSD) 3.3%).
10.2.2 Chromatographic separations: The Chromatographic
separations were done using a C18 column, mobile phase
consisting of methanol and water, and with UV detection at
210 NM.
Table 29: HPLC Characteristics of Some of Stevioside
metabolites91
Compound
Retention
Limit of
Time(min)
Detection (g)
3.60
0.4
Stevioside
5.87
0.4
15 - hydroxylSteviol
9.23
0.6
Steviolbioside
10.18
0.4
Isosteviol
11.63
0.6
Steviol
21.29
0.4
Steviol -16
17 - epoxide
85
Table 30:
Sample94.
HPLC limits of detection of Stevioside in Food
Steviol Compound
Food Samples
STS*( g/ml)
Stevioside
Leaf extract
57
Stevioside
Tea
57
Stevioside
Orange Juice
87
*STS: Sweet tasting Stevioside
10.3
Analysis
of
sweet
diterpene
S.rebaudiana: Improved HPLC method95:
glycoside
of
An improved analytical method was developed which may be

applied to quality control of Stevioside and Rebaudioside A
contents in dried leaves of S.rebaudiana before processing.
The procedure developed involves two steps:
10.3.1
Solvent extraction:
The samples (1 gm) of dried leaves of S. rebaudiana , is

grounded and solvent extracted with ethyl alcohol 70% (w/w)
in Erlenmeyer flask by shaking for 30 minutes in a 70O C
water bath.
10.3.2
Isocratic HPLC analysis:
After the extract was cooled it was filtered and analyzed by
HPLC using a NH (2) column (250 x 4.6 mm) and a mixture of
acetonitrile/water (80:20 v/v) as mobile phase, pH 5 adjusted
with acetic acid. The detection was in the UV range at 210 nm.
Quantitation was performed by means of external standard
calibration curve for each analyte which had been obtained
from standard solutions of pure Stevioside and Rebaudioside
A. The method saves time in sample preparation, and reduces
sample handling and chromatographic analysis time, while
having little loss of precision (coefficient of variation (CV %)
between 1.8% and 3.0%) and recovery (between 98.5% and
100.5%).
The method was applied to 30 samples S. rebaudiana from
Misiones (North eastern Argentina), and Stevioside content
found ranged between 3.78 and 9.75 % (weight) various
Rebuadioside A contents ranged between 1.62 and 7.27 % (
weight).
86
10.4 HPLC determination of Steviol in biological fluids and

plant material with fluorescence detection96.
A simple method is described for determination of Steviol (SV)
by reversed-phase high-performance liquid chromatography
(RP-HPLC) using dihydroisoSteviol (DHISV) as internal
standard (IS). SV and DHISV were derivatized with 4(bromomethy1)-7- methoxycoumarin in an aprotic solvent (N,
N- dimethyl from amide or acetone). Separation of the
resulting ester derivatives was achieved on an ODS column
(250 x 4.6 mm i.d., 5 m particle size) at a flow-rate of 1
mL.min-1 using acetonitrile- water (80:20 v/v) as the mobile
phase. Using fluorescence detection with excitation at 321 mm
and emission at 391 nm, a linear relationship was observed
for concentrations between 0.5 and 50 g mL -1 of SV and the
detecting limit was 100 pg. The intra-and inter day variations
(n=9) were 0.64 and 0.88%, respectively. The application of the
method to beer, urine and faeces samples involved a simple
procedure of extraction by diethy1 ether and derivatisation in
DMF. Plant samples required preparation of an acid fraction
containing the SV analyte, derivatisation and sample clean-up
using small silica columns made of pipette tips and thin layer
chromatography. A sensitive determination of 5.9 g of SV
present in 1 g of dry plant material was done with high
precision and accuracy.
10.5 Quantification at the picomol level by HPLC
97
A simple and highly sensitive reversed-phase highperformance liquid chromatographic method (RP-HPLC) has
been developed for the determination of steivol (SV) using
dihydro -isoSteviol (DHOSV) as an internal standard (IS). SV
and DHISV were derivatized by reaction of the acids with 4(bromomethy1)- 7- methoxycoumarin in an aprotic solvent
(DMF or acetone). The resulting ester derivatives were
separated on an ODS column (250 x 4.6 mm i.d., 5m
practical size) using fluorescence detection with excitation at
321 mm and emission at 391 nm. The mobile phase consisted
of acetonitrile-water (80:20 v/v) with a flow rate of 1 mL.min-1.
A liner relationship was observed for concentrations between
0.5 and 50 g/mL of SV and the detection limit was found out
to be 100 picogram.
87
For application of this method to samples of beer fortified with

Stevioside sample procedure for extraction of the beer with
diethy1 ether and derivatisation in DMF was applied. Whereas
beer samples spiked with SV gave a linear response over the
range 0.1 15 g/mL beer, no SV could be detected in beer
samples enriched in Stevioside that had been stored for over 3
years.
The application of the method to plant samples involved
preparation of an acid fraction containing the SV analyte,
derivatisation and sample clean-up using small silica columns
and thin layer chromatography. A sensitive determination of
594 mg of Steviol present in 100 mg of dry plant material was
performed with high precision and accuracy. 97
88
Advantages of Stevia & Steviol Glycosides
Chapter 11
What makes the stevia plant so special is that it can be used

to replace sugar (sucrose). Indeed, the leaves contain diterpene
glycosides with a sweet taste but which are not metabolized
and contain no calories. The biggest part of the sweet
glycosides consists of the Stevioside molecule.
The principal advantages of stevia and Steviol glycosides are
the following 98-102:-
I It is a natural non-synthetic product.

II Stevioside (the Steviol glycosides) contains no calories as
like of other sweeteners.
III Stevia leaves can be used in their natural state (fresh as
well as dried form).
IV The plant has been reported to be non-toxic.
V Thanks to its high sweetening intensity, only small
quantities need to be used in its applications.
VI The leaves as well as the pure Stevioside extract can be
used as such or while cooking.
VII Studies suggested it to be safe for diabetics,
Phenylketonurian (PKU) patients as compared to other
sweeteners.
VIII Stevia does not increase the blood sugar therefore can be
used by diabetics without adverse glycemic responses.
IX Due to its almost negligible calorie contribution, it is useful
for overweight, obese and for health conscious individuals.
X Stevia in the form of pure extract has been reported to give
200 to 300 times sweetness than sugar.
XI Stevia is non-fermentable.
XII Stevia acts as plaque retardant anti-caries and prevents
cavities.
XIII Stevia is non-toxic but, on the contrary, it is healthful, as
shown by long experience
XIV The human body does not recognize the sweet glycosides
and they pass right through the normal excretion channels
and thus the body obtains no calories from stevia therefore
stevia may be safe for diabetic and hypoglycemic patients in
its pure, unadulterated form.
XV Stevia can enhance the effect of other sweeteners such as
honey and maple syrup, so adding it to recipes might be
helpful in reducing the amount of total sweetener needed.
89
XVI Stevia is comparatively more intense sweetener than other

available artificial sweetener in market.
XVII Cheaper as compare to sugar as well as the rest of the
available sweeteners.
90
Conclusions and Recommendation
Chapter 12
Sweeteners were developed because of their possible benefits

in special diets, health and economy; they can be used in the
formulation of foods and beverages whole and/or in part
without affecting the quality of the product.
Keeping in view the global economic aspects for Stevia
cultivation and by comparing it with Indian scenario, it
appears that it may be suitable as a source of comparatively
cheaper sugar substitute.
Organoleptic evaluation of Stevia herbarium species42 revealed
that S.Rebaudiana is distinctly sweet specie owing to its eight
ent-kaurene glycosides.
The stability and sensory properties of low calorie foods can be
influenced by the type of the sweetener used, and this effect
can limit their addition to a product.
1.
According to IHBT research Stevia cultivation is a

remunerative venture with a cost benefit ratio at 1.89. A
net profit of Rs 4.61 lacs/acre can be achieved when the
sale price of dried leaves is Rs 200/kg. Therefore, reduction
in the cost of production has been achieved by raising
seedling at cheaper rate.21
2. Calorie contribution to the diet by commonly used sucrose

being considered high as it gets completely utilized by the
body and has a potential to escalate towards over weight
status.
Calorific value of Stevia was found out to be 2.7 Kcal/g
which entail it the status of low calorific sweetener due to
its intense sweetness in comparison to other available low
calorie sweeteners61.
In this context, the use of Stevia as a low calorie sweetener
could be of immense help in restricting the calorie intake in
the diet of affluent and also where in calorie restricted diet
are prescribed.
91
3. Relative to other sweeteners such as Aspartame, bitterness

tends to increase with concentration for both the Steviol
glycosides (Stevioside and Rebaudioside A)7. This should be
taken as a regulatory measure while permitting the
concentration of these sweeteners for commercial
applications.
4. Calcium, Phosphorous, Iron and Potassium of Stevia were
found out to be 464.6, 11.4, 55.3,190 and 1800 mg/100 g
respectively. This further establishes Stevia as a mineral
loaded ingredient required to protect the body, regulate and
to maintain various metabolic processes61.However, the
contribution of minerals to the body may be marginal due to
small quantities used in the final product.
5.
Rule 47 of Prevention of Food Adulteration (PFA) Act

has already permitted the use of following intense
sweeteners i.e. Saccharin Sodium, Aspertame (methyl
ester), Acesulfame Potassium and Sucralose in
Carbonated Water, Soft drink concentrate, Supari, Pan
masala, sweets(carbohydrate based and milk based
products), Halwa, Mysore Pak, Boondi ladoo, Chocolates,
Sugar
based/Sugar
free
confectionery,
Chewing
gums/Bubble gum.
By considering these guidelines and revelations of various
studies, it appears that Stevioside as a sweetener has been
reported to be used following food products61:
S.No
3.
Food
Product
Wheat
ladu
Besan
ladu
Chikki
Concentration
used
0.25-1.0
g/100g
0.25-1.0
g/100g
0.25-1.0
g/100g
4.
Hot Coffee
0.5 g/litre
5.
Hot tea
0.5 g/litre
1.
2.
92
Critical
Parameters
3 month shelf life
achieved when
packed in airtight
polythene pouch
without any
appreciable effect
on sensory
properties.
80oC/4hrs
80oC/4hrs
6.
Mixed
jam
fruit
7.
Biscuit
0.25-1.0
g/100g
8.
Carbonated
beverages*a
0.1% Stevioside
Carbonated
beverages
9.
0.25-1.0
g/100g
0.1% Stevioside
Carbonated
beverages
0.1% Stevioside
Baked
product
0.25-1.0
g/100g
Shelf life of only

one month can
be achieved
Shelf life of only
one week can be
achieved.
At 4 oC/ Room
Temp leads to
shelf life of 5
month in citric
acid beverages
At 37 oC lead to
shelf life of 4
month in citric
acid beverages.
Not suitable for
phosphoric acid
beverages.*b
*c
*a: Stevioside is quite stable when heated at 60 oC for 1

hr in a ph range of 2-10, so it can be used for mild
acidic food products71.
However it was observed that under thermal treatment
Stevioside is best stable in the pH range of 6-7 i.e. at/or
around neutral pH71.
No significant changes were observed in Stevioside
(0.1%) stability during long-term storage in carbonated
beverages (phosphoric and citric acid acidified) at room
temperature, whilst some degradation occurred at 37 oC
72
.
0.1% of Rebaudioside A was found to be more stable in
carbonated beverages at room temperature, 4 oC and 37
oC for 3, 4 and 1 months respectively72.*a
93
*b: Stability studies of Stevioside at room temperature

in dilute solutions of the organic acids (1 & 10g/l)
acetic acid, citric acid, tartaric acid and phosphoric
acid over time period of up to 4 months showed a
tendency towards enhanced decomposition of the
sweetener at lower pH values, depending on the acidic
medium71.
Stevioside stability in various organic acid solutions is
in decreasing order as follows: Acetic acid> Citric acid>
Tartaric acid> Phosphoric acid71.
Therefore when Stevioside is to be use in phosphoric
acid acidified beverages, then extra measures need to be
taken for reducing the rate of degradation71.*b
*c: In pure form solid Stevioside is stable up to 120oC

for 1 hr; however the same undergoes forced
decomposition when heated at 140 oC and total
decomposition at 200 oC. As a consequence, the
application of Stevioside as sweetening agent might
not be suitable or recommended in baking or other
processes requiring very high temperatures72.*c
6.
The functional properties of Stevia includes Bulk density,

Water absorption capacity, Emulsification value, Swelling
power, Solubility and pH which were found to be
0.443g/ml, 4.7ml/g, 4.5ml/g , 5 ml/g, 5.012 g/g, 0.365 g/g
and 5.95 respectively61.
(i) Due to appreciably good water absorption capacity and
swelling ability, it can be used in soups and gravies etc61.
(ii) Stevia appears to be a good source of protein, ash, and
crude fiber which are the essential factors for the
maintenance of good health and are comparable to
commonly used cereals in India.
(iii) Good protein content aid in the formation and stabilization
of emulsion, and is critical for many applications such as
cake batters, coffee whiteners, milks and frozen desserts
etc.
94
Both Stevioside and Rebaudioside A are synergistic in

mixtures with other high potency sweeteners such as
Aspartame therefore; they are good sweeteners for inclusion in
blends71.
7. TLC and HPLC analysis of Stevioside and Rebaudioside A has
indicated that these Steviol glycosides are stable when heated
at 60 oC for 137 hr but decomposition takes place when
temperature is raised to 100 oC in merely 4-10 hrs72.
Therefore, it may be inferred that food products not requiring
very high heat treatment may use Stevioside as sweetener.
8 Both Stevioside and Rebaudioside A degrade under thermal
treatment much faster in acidic solutions than in neutral
solutions.
9. Few Studies have shown that Stevioside has a protective effect
on the degradation of Vitamin C in the presence of Stevioside
to other sweeteners71.
10 There were no significant changes in Stevioside concentration
in either the phosphoric or citric acid beverages when exposed
to 3000 Langleys of sunlight72.
On the contrary Rebaudioside A underwent considerable
degradation when exposed to similar conditions with 22% and
18% losses in the phosphoric and citric acid beverages
respectively72.
11Good stability for Stevioside has been reported for storage in
solution with water, soy sauce and for vegetable protein
hydrolysates at 30 oC for 30 days. Some loss, however, was
found for storage in vinegar and after heating at 80 oC.
12. The modern pharmacological and stability studies of Stevia
and Steviosides were found to be few. Moreover the available
data was mainly global data with little contribution from
Indian data. Therefore stress should be on collection of more
relevant Indian data for pharmacological and stability of Stevia
and its products.
13. As per the data available of various countries, Stevioside is
approved as a sweetener in Japan, China, Brazil and
Singapore 7, 11.
95
USFDA has approved stevia as a dietary supplement or an

herb but not as a sweetener75.
Canada, European Commission and FSANZ have not yet
approved use of Stevia in any form for the commercial
applications 74-78.
13. Stevioside when present in food samples can be estimated by
HPLC, Solid phase extraction with HPLC, Improved HPLC and
can be quantified at the picomol level by HPLC 91-95.
14. It may be suitable for use in preserved fruits, spreads, jams
and jellies; however, the results will be good when these
sweeteners are used in combination with bulking agents
other than sucrose as the body of these food products needs
to be taken care.
96
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Anonymous (2000); Low calorie sweeteners and

health; IFIC Review, Washington; 1-12.
Pinheiro M.V.S, Oliveria M.V et al; The effect of
different sweeteners in low-calorie yougurts-a review;
International Journal of Diary technology; 2005; Vol.
58(4); 193-199.
Matsubara ; Alimentos funcionais: Uma tendencia que
abre perspectives aos laticinios; Industria de
Laticinios; 2001; 10-18
Tammie et al; 1994; Siveri and Oliveirs; 2002
Zdemir & Sadikoglu; Characterization of Rheological
properties of systems containing sugar substitutes &
carrageenam; International Journal of food Science
and Technology; 1998; Vol. 33; 439-444
Ministerioda Saude; Journal of food Science and
Technology 1997;Vol 32
Kinghorn et al; Non-Cariogenic intense natural
sweeteners; Inc. Med. Res. Rev; 1998; Vol 18(5); 347360.
Antonie Al-Achi et al (Campbell University School of
Pharmacy);
Stevia:
A
plant
for
sweetness;
U.S.Pharmacist- a Johnson publication; 2000; Vol 25.
Mindore.D.J; Rank.A.M; RIRDC (Rural Industries
Research Development Corporation) publication
Australia and New-Zealand; August 2002 report.
Elkins.R;
Stevia-Natures
sweet;
woodland
publication; 1999.
Midmore J.D., Rank A.H.; An intense natural
sweetener-laying the ground work for a new rural
industry; May 2006 RIRDC Publication No 06/020
RIRDC Project No UCQ-17A.
Dzyuba.O.O; Stevia rebaudiana (Bertoni) Hemsley-A
New Source of Natural Sugar Substitute for Russia;
Rastitel Nye Resursy; 1998; Vol 34(2); 86-95.
Gvasaliya.V.P, Kovalenko.M.C et al; Studies on the
Possibility of Growing Honey Grass in Abkhazia
conditions; Subtropiches kie Kul Tury; 1990; Vol 5;
149-156.
Shu.S; Stevia Rebaudiana Variety Trails; Zuocou
Pinzhong Ziyuan; 1989; Vol 1; 17-18.
Colombus.M; The Cultivation of Stevia Natures
Sweetener; Ontario Canada; 1997; 4.
97
16. Shock.C.C; Rebaudis Stevia: Natural Noncalorie

Sweeteners; California Agriculture; 1982; Vol 36(9); 45.
17. Lee.J.I et al; Studies on the New Sweetening Source
Plant; Stevia; Research Reports of the office of Rural
Development Crop; 1980; Vol 22; 22.
18. Matejka.V; Climatic Requirements and Possibilities of
Growing the Herb Stevia rebaudiana bertoni in Czech
Republic; Agriculturra Tropica et Subtropica; 1992;
Vol 25; 21-32.
19. Fors.A.L; A new character in the sweetener scenario;
Sugar Journal; 1995; Vol 58(2); 30.
20. Shyu.Y.T et al; Effects of harvesting dates on the
characteristics, yield and sweetness; Journal of
Agricultural Research of China; 1994; Vol 43(1); 2939.
21. Megeji.N.W et al; Introducing Stevia Rebaudiana-A
natural Zero calorie Sweetener; Current Science;
2005; Vol 88(5).
22. Goenadi.D.H; Effect of slope position on the growth of
Stevia in Indonesia; communications in soil science
& plant analysis; 1987; Vol 18(11); 1317-1328.
23. Oddone.B; How to grow Stevia; Gaurani botanicals
Inc: Pawcatuck, Connecticut; 1999; 1-30.
24. Tateo.F et al; Stevioside content and morphological
variability
in
a
population
of
Stevia
Rebaudiana(bertoni) Bertoni form Paraguay; Italian
Journal of Food Science; 1998; Vol 10(3); 261-267.
25. Huang.Y.S et al; Studies on the variation of
Stevioside
content
and
selection
of
type
Rebaudioside A in Stevia Rebaudiana; Journal of
Plant Resources and Environment; 1995; Vol 4(3); 2832
26. Korneienko.A.V et al; Stevia Cultivation; Sakharnaya
svekla (Russain); 1995; Vol 10; 22-24.
27. Jia.G.N; An experiment on the cultivation of Stevia
Rebaudiana; Shaxi Agricultural science (Chinese);
1984(1); 20-21.
28. Suhendi.D; Mass selection of Stevia rebaudiana
bertoni; Menara Perkebunan; 1989; Vol 56(4); 93-95.
29. National Medicinal Plants Board-Department of
AYUSH (Ministry of Health and Family Welfare, Govt.
of India); Enquiry on present policies and prospects
of
cultivation
of
Stevia
Rebaudiana;
F.NO.
th
A.11019/4/2005-NMPB Dated 19 May 2005
30. Chalapathi.M.V, Shivraj.B et al; Nutrient uptake and
yield of Stevia (Stevia rebaudiana bertoni) as
98
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
influenced by methods of planting and fertilizers

levels; Crop Research; 1997; Vol 14(2); 205-208.
Chalapathi.M.V, Thimmegowda et al; Natural Noncalorie Sweetener (Stevia rebaudiana bertoni)-A future
crop of India; Crop Research; 1997; Vol 14(2); 347350.
Chalapathi.M.V et al; Vegetative Propagation of
Stevia; Crop Research; 1999; Vol 18(2).
Ramesh.K, Singh V, Megeji.N.W; Cultivation of Stevia
(Stevia rebaudiana bertoni: A comprehensive Review;
Advances in Agronomy; 2006; Vol 89; 137-177.
Kinghorn .A.D, Soejarto.D.D; Current status of
Stevioside as a sweetening agent for human uses;
Progress in Economic and Medical Plant Research;
1985; Vol 1; 1-52.
Haa et al; 1976, Itagaki and Ito 1979.
Uneshi.H. Kobayashi.T et al; Purification of a stevia
sweetening agent; Jap. Patent; Vol 54-030199.
Brandle.J.E,
Starratt.A.N,
Gijzen.M;
Stevia
Rebaudiana: Agricultural biological and chemical
properties; Canadian Journal of Plant Science; 1998;
Vol 78; 527-536.
Pasquel.A,
Meireles.MA.A,
Marques.M.O.M
and
Patenate.A.J; Extraction of Steviol glycosides with
Co2+water, Co2+ethanol and Co2+water+ethanol;
Brazilian Journal of Chemical Engineering; 2000; Vol
17(3).
Combined
Compendium
of
Food
Additive
Specifications; Tentative specification prepared at
sixty third JECFA meeting; Published in FNP 52 Add
12; 2004.
Darise.M; Kohda.H, Mizutani et al ; Chemical
Constituents of Flowers of Stevia rebaudiana bertoni;
Agriculture Biological Chemical; 1983; Vol 47; 133135.
Kinghorn.A.D, Fullas.F, Hussain.R.A; Studies in
natural
products
chemistry,
structure
and
function(c); Elsevier Science Publishers, 1995; Vol 15;
3.
Soejarto. A. D, Kinghorn. D et al ; Potential
sweetening agents of plant origin-III- Organoleptic
evaluation of stevia leaf Herbarium samples for
sweetness; Journal of Natural Products; 1982; Vol
45(5); 590-599.
Rodriguez. E, Latinoamer. Q; 1977; Phytochemistry.
Rios.T.A, Vivar.R.D, Romo.J et al; 1967; Vol 23,
4265.
99
45. Salmon.A, Diaz.E, Ortega.A; J. Org. Chem.; (1973);

Vol 38; 1759
46. Salmon. A, Ortega. A., Diaz.E; Rev. Latinoamer. Quim
; (1975); Vol 38; 1759
47. Salmon.A, Diaz.E, Ortega.A; Rev. Lationoamer. Quim;
(1975); Vol 6; 45
48. Salmon.A,
DuttaL.N,
Dorner.W,
King.R.M,
Robinson.H., Phytochemistry; (1979); Vol 18; 673
49. Phillips. K.C; Stevia: Steps in developing a new
sweetener; T. H. Grenby; 1989; 1-43
50. Choi. Y. H, Kinghorn. A .D; Evaluation of plant
extracts sweetness using the Mongolian gerbil;
Journal of Natural Products; 1990; Vol 53(1); 190195.
51. Geuns. J.M.C., Buyse. J et al; About the safety of
Stevioside used as a Sweetener; Journal of Food,
Agriculture and Environment; 2004; Vol 2(3&4); 290291.
52. Toyoda. K, Matsui. H, Shoda. T et al; Assessment of
the carcinogenicity of Stevioside in F344 rats; Food
and Chemical Toxicology; 1997; Vol 35; 597-603.
53. Matsui.M, Matsui.K, Kawasaki.Y et al; Evaluation of
the genotoxicity of Stevioside and Steviol using six in
vitro and one in vivo mutagenicity assays;
Mutagenesis; 1996; Vol 11(6);573-579.
54. Gardana.C, Simonetti.P, Canzi. E et al; Metabolism of
Stevioside and Rebaudioside A from stevia rebaudiana
extracts by human micro flora; Journal agriculture
Food Chemistry; 2003; Vol 51; 6618-6622.
55. Geuns. J.M.C.; Molecules of interest- Stevioside;
Phytochemistry; 2003; Vol 64; 913-921.
56. Maitree. S, Vinitketkaumnuen.U et al; Mutagenicity
and Human chromosomal effect of Stevioside- a
sweetener
from
Stevia
rebaudiana
bertoni;
Environmental Health Perspective Supplements;
1993; Vol 101(3); 53-56.
57. Koyama. E, Ohori. Y et al; In vitro metabolism of the
glycosidic
sweeteners,
stevia
mixture
and
enzymatically modified stevia in human intestinal
micro flora; 2003; Vol 41; 359-374.
58. Abudula.R, Jeppesen.P.B et al; Rebaudioside A
potently stimulates insulin secretion from isolated
mouse islets-Studies on the dose glucose and calcium
dependency; Metabolism; 2004; Vol 53(10); 13781381.
59. Tolstikova.T.G, Bryzgalov. A.O et al; Stevioside is a
novel stimulator of pharmacon clathration with
100
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
glycosides; Doklady Biological science; 2005; Vol 403;

244-246.
Geuns.J.M.C, Bruggeman.V Buyse.J.G; Effect of
Stevioside and Steviol on the developing broiler
embryos; Journal of Agriculture and Food Chemistry;
2003; Vol 51; 5162-5167.
Savita.S.M, Sheela.K, Sunanda. S et al; Stevia
Rebaudiana-A functional Component for Food
Industry; Journal of Human Ecology; 2004; Vol 15(4);
261-264.
Food Additives by L.Branen [incomplete ? ]
Stevia-history & uses/ documented; Georgiana
Duncan,2000;http://www.nspforum.com/faq/bbs.cgi
?read=75
http://www.dic.co.jp/eng/products/stevia/merit/ind
ex.html accessed on 8.8.2006
www.gzstevia.com/english/yingyong.htm ; The uses
of Stevioside products.
Abe K. and Sonobe M.; New Food Industry(Japan);1977;
Vol 19(1); 19
Ochi. Y.; New Food Industry(Japan); 1979; Vol 21(9);
28
Kazuyama. S.; Shokuhin to Kagaku; 1979; Vol 21(4);
90
Fujita. H and Edahiro.T.; Shokuhin Kogyo; 1979; Vol
22(20); 66
Kroyer. G.T.; The low calorie sweetener Stevioside:
stability and interaction with food ingredient;
Lebensm. Wiss. Und. Technologies; 1999; Vol 32; 509512.
Chang. S.S.; Cook.M.J.; Stability studies of Stevioside
and Rebaudioside A in carbonated beverages; Journal
of Agriculture, Food & Chemistry; 1983; Vol 31; 409412.
OkamotoK et al; Purification and some properties of a
beta-glucosidase from flavobacterium johnsonae;
Bioscience. Biotechnology and Biochemistry; 2000;
Vol 64(2); 333-340.
Annexure no I
Regulation (EC) No. 258/97 of the European
Parliament and the Council (notified under document
number C (2000) (77) Official Journal L 061,
08/03/2000 p. 0014.
Annexure no II
Annexure no III
Annexure no IV
101
79. Fact sheet: Stevioside, Research and Library Division,

FS04/01-02.
80. Kochikyan.V.T, Markosyan.A.A, Abelyan.L.A et al;
Combined
enzymatic modification of Stevioside
and Rebaudioside A; applied Biochemistry and
Microbiology; 2006; Vol 42(1);
81. Paul.C, Brian.T et al; A double blind placebo
controlled study of the effectiveness and tolerability
of oral Stevioside in human hypertension; Blackwell
Science
Ltd.,
Brazilian
Journal
of
Clinical
Pharmacology; 2006; Vol 50; 215-220.
82. Gregersen.S, Jeppesen .P.B, Holst.J.J, Hermansen.K
et al; Antihyperglycemis effect of Stevioside in Type
2 Diabetic Subjects; Metabolism, Vol 53(1); 2004; 7376.
83. Gougeon.R, Spidel.M; Canadian Diabetes Association
national Nutrition Committee Technical Review: onnutritive
intense
sweeteners
in
Diabetes
management; Canadian Journal of diabetes; 2004;
28(4); 385-399.
84. Chen.J, Jeppesen.P.D; Stevioside does not cause
increased Basel insulin secretion or beta cell
desensitization
as
does
the
sulphonylurea,glibenclancida:studies in vitro; Journal
of Life Sciences; 2006; Vol 78; 1748-1753.
85. Wong K.L, Lin.J.W, Liu.J.C et al; Antiproliferative
effect of isoSteviol on angiotensin-II-treated rat
arotic smooth muscle cells; Pharmacology; 2006;
76(4); 163-169.
86. Clinical pharmacology; 2000; Vol 20; 215-220.
87. Ferri.L.A.F, Alves.W, Prado et al; Investigation of the
antihypertensive effect of oral crude Stevioside in
patients
with
mild
essential
hypertension;
Phytotherapy Research; 2006; Vol 20; 732-736.
88. Lee.C.N; Wong.K.L; Liu.J.C et al; Inhibitory effect of
Stevioside
on
calcium
influx
to
produce
Antihypertension; Planta Med; 2001; vol 67(9); 796799.
89. Boonkaewwan.C,
Toskulkao.C,
Vongsakul.M;
Antihypertensive and Immunodulatory activities of
Stevioside and its metabolite Steviol on THP-1 cells;
Journal Agriculture Food Chemistry; 2006; Vol 54(3);
785-789.
90. Chen.T.H, Chen.S.C, Chan Paul et al; Original paper
planta med; pharmacology; 2005; Vol 71; 108-113.
91. Hutapea.A.M, Toskulkao.C, Wilairat.P, Buddhasukh.D;
High Performance Liquid Chromatography Separation
102
and Quantitation and its Metabolite; Journal Liquid

Chromatography & Rel. Technology; 1999; Vol 22(8);
1161-1170.
92. Shokuhin Eiseigaku Zasshi.; 2004; Vol 45(2); 81-86.
93. Shokuhin.E.Z;
Simultaneous
determination
of
glycyrrhizinic acid, Stevioside and Rebaudioside A in
food by solid phase extraction and HPLC;
Ann.Rep.Tokyo.Metr.Inst.P.H; 2003; Vol 54; 93-98
94. Vanek.T, Nepovim.A, Valieck.P; Determination of
Stevioside in plant material and fruit teas; Journal of
Food Composition and Analysis; 2001; Vol 14; 383388.
95. Kolb.N, Herrera.J.I, Ferrevra.D.J, Uliana.R.F; Analysis
of sweet diterpene glycosides of stevia rebaudiana:
Improved HPLC method; Journal Agriculture food and
Chemistry; 2001; Vol 49(10); 4538-4541.
96. Minne.V.J.Y, Frans.C; Sensitive HPLC determination
of Steviol in biological fluids and plant material with
fluorescence
detection;
Geuns.J.M.C
and
Buyse.J(Eds.); Proceedings of the first symposium
safety of Stevioside Leuven, April 16; 2004;
EuprintEd Parkbosstraat 3,3001 Heverlee; Belgium.
ISBN: 9074253024; 127.
97. Minne.V; Compernolle.F, Toppet.S, Geuns.J.M.C;
Steviol quantification at the picomol level by HPLC;
Journal of Agriculture and Food Chemistry; 2004; Vol
50; 2445-2449.
98. The Australian New Crops Newsletter issue no 11;
January 1999.
99. http://www.stevia.net/history.htm
100. The Bittersweet Story of The Stevia Herb; Nexus Magazine;
2003; Vol 10(2)
101. www.nexusmagazine.com
102. http://bio.kuleuven.be/biofys/ESC/English/framestart_e
.htm
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Stevia:

Chapter 2: Status of Stevia in India

Need of intense sweeteners

Chapter 7: Stability studies of Stevioside and

Legal Regulations regarding the

Chapter 9: Pharmacological Properties

9.8 Hypoglycemic effect of Stevioside

1. List of countries where Stevia is

19. TLC analysis of Stevioside.

In the last couple of decades, growing concern about health

Given the reasonably sound track record of plant

Stevia is a plant, indigenous to mountainous regions of

S.rebaudiana (Bertoni) was rediscovered by Europeans in

Stevia is a small perennial shrub with green leaves that

1.4 Status of Stevia in World

[Agricultura Nonl] Research [Agricultural]

Commercial production excludes small quantities grown for domestic use 9

Table 3: Stevia Leaf Analyses in major stevia producing

*St: Stevioside, **R-A: Rebaudioside A

Not given; assumed value used to calculate kg/ha Stevioside.

1.5 Marketing and Economic Issues

There is also a range of stevia blend powders and crystals and

Status of Stevia in India

Stevia is a natural herb native of Paraguay, cultivated as a cash

Table 5: Effect of method of planting on stevia yield30

Fresh leaf yield

* NS: Not Significant

With improved management practices, there is further scope for

Table 6: Herb yield of two accessions of Stevia rebaudiana*21

*Recorded during crop cycle from September 2001 to

Economy of Stevia as sweetener in India

The total market value of stevia sweetener in Japan is estimated

1st year 2nd year 3rd year 4th year

It was, therefore, concluded that stevia cultivation is a

Rates for different slab

* S.Rebaudiana: Stevia rebaudiana bertoni

Commercial Extraction of Steviol Glycosides Chapter 3

Dissolving the sweetener in boiling water or in other solvent.

Methanol appears to be used in most of the extraction and

The most common processes used for extracting Steviol

Soaking leaves in warm/hot water to dissolve the

Some secondary processing may be undertaken, especially in

3.2 Product Quality

A number of factories use a range of further processing

3.5 Detailed process

3.5.2.3 The Experimental Unit for the Super Critical Fluid

was added to the system (Figure 1)

3.5.2.4 Experimental Procedure: SCFE

3.5.2.5 Experimental Procedure: Conventional Extraction

4.1 Steviol Glycosides

JECFA has allotted a temporary ADI of 0 to 2 mg/kg bw for

The committee noted that this temporary ADI only applies to

Analytical data on distribution and concentration of all

Method of analysis for the determination of all

The nature and concentration of the fractions that do not

The quantities of residual solvents from isolation &

The hydrolytic stability of the Steviol glycoside in acidic

4.2 Detailed specifications of Steviol

13-[(2-O--L-rhamnopyranosyl-3-O---Dglucopyranosyl-D-glucopyranosyl -- D-glucopyranosyl-D-glucopyranosyl] oxy. kaur-16-PM-18oic acid -D- glucopyranosyl ester.

C.A.S.(Chemical Abstracts Services) number

Steviol (R1=R2=H) is the aglycone of the Steviol glycosides.

White crystalline powder, odorless or having a slight

The material contains not less than

Not more than 1%