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 Definition 1.2 Background 1.3 Description 1.4 Status of Stevia in World 1.5 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 Traditional method 3.2 Improved method 3.3 Product quality 3.4 Taste quality improvement 3.5 Extraction of Steviol glycoside 3.6 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 Need of intense sweeteners 6.2 Classification of sweeteners 6.3 Relative sweetness 6.4 Benefits of Stevia 6.5 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: Legal Regulations regarding the Use of Stevia

8.1 USA 8.2 Canada 8.3 European countries 8.4 Japan 8.5 Australia and New Zealand 8.6 Hong Kong 8.7 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 Topic no. 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.

1

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.

2

Sweeteners such as nutritive (Polyols) and non-nutritive/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.

3

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.

4

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

5

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.

6

1.4 Status of Stevia in World Table 1: List of countries where stevia is grown and researched

1 Commercial production excludes small quantities grown for domestic use 9 .

Country/Location Commercial Production [1]1.

[Agricultural] Research

Non- [Agricultural] Research.

Approved for use

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

- -

-

-

7

Table 2: Climatic Features of Agronomic Research Location[s] for Stevia in various parts of the world.

Ref Location Latitude Degrees

Rainfall (mm)

Altitude (meters)

Topography Comments

[12] St Petersburg Russia, Voronegh

60N* -

8

[20]

[18]

[21]

Taiwan Thailand India-Bangalore

24N* 18N* 13N*

- 1260 -

- 300 930

- Uplands Uplands

- 6mth dry season -

[22]

[18] Indonesia- Bogor Surakata

7 S** 7S**

1800 2300

400 1000

Slopes - 3mth dry season

[18]

[23] Brazil Maringa Paraguay- native locality

23S** 23S**

1260 1500 1800

500 200-400 -

Uplands Inland - Valleys

400 km from coast Amambay Mountain Ranges

* N: North, ** S: South

9

Table 3: Stevia Leaf Analyses in major stevia producing countries

*St: Stevioside, **R-A: Rebaudioside A Table 4: Trial Crop Yield in various countries

Steviosides Ref Trial Location

Yield Total

T/ha- Dry leaf

Total R-A% St% Kg/ha

Stevioss

[16] California 9.0 3.6 7% - - 252 [26] Russia 13.8 5.5 *9% - - 495 [12] Russia - 1.4-3.0 *9% - - 125-270 [14] China-

V9 - 1.40 20.4% - - 265

- Average - - 12.4% - - 186 - -

Jiangsu - - - - - 267

[27] North China

1.3 plus 67kg Seed

- - - - -

[28] Indonesia - - - - - - [21] India - 1.74-

2.16 - -

8.0

297.12-338.16

[15] Canada - 2.85 15-20 - - 425-570 Not given; assumed value used to calculate kg/ha Stevioside.

[T=Ton; ha=Hectare]

Ref Location Cultivar St* %

R-A** %

R-A/St Ratio

Total Sweetener

[23] Paraguay average 8-14 2-4 0.4 10-15% - Paraguay-typical 5-10 2-4 0.4 9-15% [24] Paraguay-wild - - - 10.2-13.5% [25] China-average 6.44 3.86 0.6 10.3%

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1.5 Marketing and Economic Issues

Current markets are restricted to Japan, where there is well-established 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

11

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

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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:

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Table 5: Effect of method of planting on stevia yield30 Treatment Biomass yield

(mg ha-1) Fresh leaf yield

(mg ha-1) Dry leaf

yield (mg ha-1)

Methods of planting a) Flat bed

method 37.38 10.67 2.59

b) Ridges and furrow method

(P=0.05)

38.40

NS*

11.20

NS*

2.70

NS* Fertilizers levels (kg NPK ha-1)

a) 0:0:0 26.69 7.38 1.82 b) 20:10:15 32.67 9.00 2.25 c) 40:20:30 45.46 13.53 3.22 d) 60:30:45 46.72 13.81 3.29

(P=.05) 2.36 1.16 0.29 Interaction

(P=0.05) NS* NS* 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

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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. Pre-treatment of cuttings with 3-Indolbutyric acid (IBA) or a-naphthapene 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.

15

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 : 1300m msl Soil characteristic : 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

Leaf weight (q/ha)

Stem weight (q/ha)

Leaf: stem ratio (w/w)

Herb yield (q/ha)

Accession

Fresh Dry Fresh Dry Fresh

Dry Fresh Dry

Stevioside

content (%)

Stevioside yield of

whole herb (kg/ha)

Accession

1a

69.83

17.46

98.41

19.68

0.71

0.88

168.24

37.14

8.00

297.12

Accession

2b

108.47

21.69

138.69

34.67

0.78

0.63

247.16

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.

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

(a) Establishment cost (Rs /ha) 60,500 - - - (b) Variable costs (Rs/ha) 73,500 82,100 82,200 82,200

(c) Fixed costs (Rs/ha) 27,060 22,389 22,398 22,398 (d) Total production cost

(A+B+C)(Rs/ha) 161,060 104,489 104,598 104,598

(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

170,000 200,000 230,000 250,000

(f) Net income (Rs/ha) 8940 95,511 125,402 145,402 (g) Benefit cost ratio (BCR)

(Gross Income/Total Production Cost)

1,055 1,914 2,198 2,390

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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:

* S.Rebaudiana: Stevia rebaudiana bertoni

Rates for different slab (Rs. /unit)

Plant Species

Common Name

Form of plant

material

Unit

1-99 100-900

1000

Mode of

supply

S.Rebaudiana*

Stevia, Sweet herb

Seed

Seed raised sapling

Cutting raised sapling

g

No.

No.

15.00

3.00

4.00

12.00

2.50

3.50

10.00

2.00

3.00

Poly bags

Bunch pack

Bunch pack

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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:

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1. Dissolving the sweetener in boiling water or in other solvent. 2. Ion exchange separation. 3. Filtration with precipitation / coagulation. 4. 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.

20

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 R-A, 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.

21

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.

22

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

23

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)

24

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

25

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 air-circulating oven38

26

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. Stevioside 2. Rebaudioside A 3. Rebaudioside C 4. 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.

27

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 glycoside as developed by JECFA is as following: 39 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.

28

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 -D- glucopyranosyl ester.

Rebaudioside: 13-[(2-O--D-glucopyranosyl-3--D-

glucopyranosyl--D-glucopyranosyl] oxy. kaur-16-PM-18-oic acid -D- glucopyranosyl ester.

Rebaudioside C: 13-[(2-O--L-rhamnopyranosyl-3-O---D-

glucopyranosyl-- D-glucopyranosyl -- D-glucopyranosyl--D-glucopyranosyl] oxy. kaur-16-PM-18-oic acid -D- glucopyranosyl ester.

DulcosideA: 13-[2-O--L-rhamnopyranosyl--D-

glucopyranosyl]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 secondary Steviol glycosides: Stevioside : 57817-89-7 Rebaudioside A : 58543-16-1 Rebaudioside C : 63550-99-2 Dulcoside A : 64432-06-0

29

4.2.4 Chemical formula The following are the chemical formulas for the principal and secondary Steviol glycosides: 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 secondary Steviol glycosides: O-R2 CH3 CH2 CH3 Coo-R1 Compound R1 R2 Name Stevioside -Glc -Glc--Glc (2 1) Rebaudioside A -Glc -Glc--Glc (2 1) -Glc (3 1) Rebaudioside C -Glc -Glc--Rha (2 1) -Glc (3 1) Dulcoside A -Glc--Rha (2 1)

30

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 : 804.88 Rebaudioside C : 951.03 Rebaudioside A : 967.03 Dulcoside A : 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 in water and in

ethanol Stevioside The material contains not less than

70% of Stevioside and Rebaudioside A Rebaudioside A as identified and

determined in the Method of Assay.

31

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 high-pressure 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.

32

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 A, Rebaudioside A and Rebaudioside C from the formulas:

% Stevioside = [Ws/W] x [Aa/As] x 100 %Dulcoside A = [Ws/W] x Ab x [0.98/As] x 100 %Rebaudioside A = [Ws/W] x Ac x [1.20/As] x 100 %Rebaudioside C = [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.

33

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 and Rebaudioside A.39 4.3 Studies conducted other than JECFA 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

4.4 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

Quantity of stevia (gm)

In 100 ml of water

Sucrose equivalent (gm) in 100 ml of

water

Perception of duration of sweet

stimulus (sec) 1 10 14 1 15 14 1 20 58 1 25 14

34

Unit tested (mm2)

S.No.

Species

Voucher Specimen, Country, Collection Year

1-2

4-6

8-10

1

S. ambylolepis Robins

Pringle 13655 Mexico, 1905

Na B B

2 S. ambylolepis Robins

Jorgensen 4902 Paraguay, no date

BB BB --

3 S. amplexicaulis Hassler

Hassler 1011 Paraguay, 1908

B B --

4 S. aristata D. Don Cabrera 11777 Argentina, 1954

T T --

5 S. aristata D.Don Hassler 9033a Paraguay, 1905

BB BB --

6 S. aschenborniana Sch. Bip

Pringle 9120 Mexico, 1900

B BB BB

7 S. ashenborniana Sch. Bip

Cronquist & Sousa 10408 Mexico, 1965

B B --

8 S. balansae Hieron Jorgensen 4702 Paraguay, 1931

N B B

9 S. bangii Rusby Rusby 6885 Bolivia, no date

N N T

10 S. benthamiana Hieron

Apolinar-Maria 274 Colombia, 1938

B B B

11 S berlandieri A. Gray Pringle 11573 Mexico, 1903

B BB BB

12 S. berlandieri A. Gray var. berlandieri

Barkley, Webster & Rowell 7139 Mexico, 1947

B BB --

13 S. berholdii Robins Acosta-Solis 7849 Ecuador, 1944

B B BB

14 S. boliviensis Sch. Bip. Ex Rusby

Holway & Holway 542 Bolivia, 1920

N N B

15 S. boliviensis Sch.-Bip. Ex Rusby

Buchtien s.n. Bolivia, 1912

B BB --

16 S. breviarishtata Hook. & Arnott.

Cabrera 3068 Argentina, 1933

N N N

17 S. calderillensis Hieron

Fiebrig 3425 Bolivia, 1904

B BB --

18 S. camporum Baker Irwin 2749 Brazil, 1959

B B B

19 S. caracasna DC Molina 30283 Guatemala, 1974

N N S

20 S. cathartica Poepp. & Endl

Espinosa E-787 Ecuador, 1946

B BB BBB

21 S. chamaedrys Griseb

Lorentz & Hieronymus 171 Argentina, 1873

N N N

22 S. cinerascens Sch. Bip

Matzenbacher 510 Brazil, 1976

N N B

35

23 S. clausseni Sch.-

Bip Barreto 8353 Brazil, 1936

BB BBB

BBB

24 S. clausseni Sch.- Bip

Lenarte 2570 Brazil, 1950

B BBB

--

25 S. clinopidioides Greenm

Sesse, Mocino & Maldondo 2613 Mexico, ca. 1804

BBB BBB

--

26 S. collina Gardn Warming 618 Brazil, 1865

BB BB --

27 S. compacta Benth Buchtien 186 Bolivia, 1912

N N B

28 S. conmixta Robins Hatschbach 18764 Brazil, 1968

N N B

29 S. connata Lag Pringle 4580 Mexico, 1893

BB BB --

30 S. connata Lag Smith 255 Mexico, 1894

B B BB

31 S. connata Lag Pringle 4580 Mexico, 1893

B BB --

32 S. crenulata Baker Hatschbach 18770 Brazil, 1968

B BB --

33 S. cuneata Hassler Hassler 10286 Paraguay, 1908

B BB --

34 S. cuzcoensis Hieron Pennell 13553 Peru, 1925

B B B

35 S. cuzcoensis Hieron Herrera 2353 Peru, 1929

B B --

36 S. dianthoidea Hieron

Penland & Summers 564 Ecuador, 1939

N N --

37 S. elatior HBK Acosta-Solia 10184 Ecuador, 1945

N N --

38 S. elatior HBK Apolinar-Maria 448 Colombia

N B B

39 S. elatior HBK O. Kuntze s.n. Bolivia, no date

N N N

40 S. elationr HBK Breedlove 7036 Mexico, 1964

B B B

41 S. elongate HBK Haenke 329 Mexico, 1791

N N N

42 S. elongate HBK Linden 475 Venezuela, 1842

B BB --

43 S. entreriensis Hieron

Cabrera 3922 Urugua, 1936

N B B

44 S. eupatoria (spreng.) Willd

Taylor 13 Mexico, 1936

BB BB --

45 S. filipes Rusby Brooke 6209 Bolivia, 1950

B BB --

46 S. galeopsidifolia Hieron

Vargas 9531 Peru, 1950

N N N

47 S. galeopsidifolia Hieron

Vargas 4310 Peru, 1944

B B BB

36

48 S. glandulosa Hook.

& Arnott var. gentryi Grashoff

Gentry 1204 Mexico, 1934

BB BBB

--

49 S. glandulosa Hook. & arnott var. glandulosa

Palmer 1821 Mexico, 1892

BBB -- --

50 S. glutinosa HBK Lehmann 4727 Colombia

B B B

51 S. heptachaeta DC No Collector Brazil, 1863

N N N

52 S. herrerae Robins Vargas 4139 Peru, 1944

B BB BB

53 S. hirsute DC. var hirsute

Hinton 2392 Mexico, 1932

B B B

54 S. hirsute DC. var hirsute

Molina & Molina 26641 Guatemala, 1971

N B B

55 S. hypomalaca Robins

Pringle 9976 Mexico, 1902

B B BB

56 S. hyptifolia gardn Mexia 5540 Brazil, 1931

BB BBB

--

57 S. iltisiana Grash off Fisher s.n. Mexico, 1924

BB BB BBB

58 S. iltisiana Grashoff Pringle 11576 Mexico, 1903

BB BB BBB

59 S. incongnita Grashoff

Ton 491 Mexico, 1966

B B B

60 S. incongnita Grashoff

Williams 41681 Guatemala, 1972

B BB BB

61 S. incognita Grash off

Molina & Molina 30047 Guatemala, 1974

B B B

62 S. ialiscensis Robins No Collector Mexico, 1892

B B BB

63 S. jorullensis HBK Williams et al. 41438 Guatemala, 1972

B B BB

64 S. jorullensis HBK Pringle 13085 Mexico, 1904

B B BB

65 S. latifolia Benth Anderson & Laskowski 4432 Mexico, 1966

B B B

66 S. latifolia Benth Smith 253a Mexico, 1894

N B B

67 S. lehmannii Hieron Molina & Molina 26296 Guatemala, 1971

N B B

68 S. lehmannii Hieron Purpus 3133 Mexico, 1908

N B B

69 S. lemmonii Hieron. var. hispidula Grashoff

Gentry 1414 Mexico, 1935

BBSS

BBSS

BBBS

70 S. lemmonii Hieron. var. hispidula Grashoff

Palmer 96 Mexico, 1906

B B B

71 S. leptophylla Sch-Bip. ex Robins

Hasler 6617 Paraguay, 1990

N B B

37

72 S. lucida Lag Killip 38078

Columia, 1944 N B BB

73 S. lucida Lag Vilbur 15405 Panama, 1971

N B B

74 S. lucida Lag Steyeermark 55645 Venezuela, 1944

N B B

75 S. lucida Lag. var. lucida

Morley 653 Mexico, 1946

N B BB

76 S. lucida Lag. var. bipontini Robins

Powell & Edmondson 665 Mexico, 1961

N B B

77 S. lucida Lag. var. oaxacana (DC.) Grashoff

Williams & Williams 21703 Mexico, 1962

B B B

78 S. lundiana DC Hatschbach 12529 Brazil, 1965

N N B

79 S. macbridei Robins Soukup 3002 Peru, 1964

B B BB

80 S. macbridei Robins Isert 2064 Peru, 1863

B B BB

81 s. mandonii Sch.-Bip Pennell 13372 Peru, 1925

BT BT BT

82 S. mandonii Sch.-Bip

Soukup 236 Peru, 1935

B B B

83 S. melissaefolia (Lamk.) Sch.-Bip

Macbride 5965 Peru, 1923

T T T

84 S. melissaefolia (Lamk.) Sch.-Bip

Cerrate 865 Peru, 1951

T T B

85 S. menthaefolia Sch.-Bip

Dusen 16948 Brazil, 1915

N B TB

86 S. menthaeflia Sch.-Bip

Martius 771 Brazil, ca. 1800

B BB BBB

87 S. menthaefolia Sch.-Bip

Buchtien s.n. Bolivia, 1914

N B B

88 S. mercedensis Hieron. ex Reiss

Lossesn 202 Argentina, 1925

B B --

89 S. micradenia Robins

Pringle 4543 Mexico, 1893

BBBSS

BBBSS

--

90 S. micrantha Lag Sesse et al. 2589 Mexico, ca. 1800

N N N

91 S. micrantha Lag Orcutt 4293 Mexico, 1910

N B B

92 S. micrantha Lag Cronquist 10246 Mexico, 1965

N B B

93 S. microchaeta Sch.- Bip

Botteri 407 Mexico, no date

N N N

94 S. monardifolia HBK Pringle 11580 Mexico, 1903

B BB BB

95 S. monardifolia HBK Balls & Gourlay 5288 Mexico, 1938

N B B

38

96 S. multiaristata

Spreng Cabrera 3341 Uruguay, 1935

N N T

97 S. nelsonii robins Leavenworth & Hoogstraal 1121 Mexico, 1941

N S S

98 S. nepetifolia HBK Breedlove 14153 Mexico,1965

N B BB

99 S. nepetifolia HBK Pringle 11581 Mexico 1903

N B BB

100 S. nepetifolia HBK Smith 1996 Colombia, 1900

N B B

101 S. oligocephala DC Glaziou s.n. Brazil, 1907

SS SSB

SSBB

102 S. origanoides HBK Mexia 9043 Mexico, 1937

N S SB

103 S. origanoides HBK Pringle 1780 Mexico, 1888

N SB SB

104 S. origanoides HBK Shedon s.n. Mexico, 1892

SB SB SB

105 S. ovalis(Robins.) Robins

Pringle 4491 Mexico, 1893

N B SBB

106 S. ovalis(Robins.) Robins

Barnes & Land 187 Mexico, 1908

N N S

107 S. ovata Willd Williams et al. 41694 Guatemala, 1972

N N N

108 S. ovata Willd Stewart 1227 Mexico, 1941

N N B

109 S. ovata Willd. var. ovata

Molina & Molina 26935 Guatemala, 1971

N B B

110 S. ovata Willd. var. reglensis (Benth.) Grashoff

Pringle 6624 Mexico, 1896

B B BB

111 S. oxylaena DC Hassler 12154 Paraguay, 1913

B B B

112 S. oxhlaena DC Lorentz 952 Uruguay, 1877

N N N

113 S. pallida (Sch.-Bip.) Hieron

Killip & Smith 17269 Colombia, 1927

BBB BBB

--

114 S. palmeri A. Gray var. palmeri

Standley 2475 Mexico, 1936

N B B

115 S. palmeri A. Gray var. Constricta Grashoff

Ortega 7132 Mexico, 1993

N B BB

116 S. pauciradiata Bab Glaziou 11025 Brazil(?)

N N N

117 S. perfoliata Crongq Cronquist 11229 Mexico, 1974

S SS SSB

118 S. phlebophylla a. Gray

Pringle 2291 Mexico, 1889

BBBSS

BBBSS

--

119 S. pilosa Lag Barkley et al. 2828 Mexico, 1947

BS BBST

BB

39

120 S. pilosa Lag Powell & Edmondson 735

Mexico, 1961 N BS BS

121 S. plummerae A. Gray var. plummerae

Gentry 1948 Mexico, 1935

N B B

122 S. plummerae A. Gray var. plummerae

LeSueur 961 Mexico, 1936

N N B

123 S. pohliana Baker Dusen 16944 Brazil, 1915

B BB BB

124 S. polycephala Bertol var. polycephala

Breedlove 7995 Mexico, 1964

B BB BB

125 S. porphyrea Mc Vaugh

Waterfall 15446 Mexico, 1959

N SB SB

126 S. porphyrea Mc Vaugh

Palmer 456 Mexico, 1896

B BBS

BBBS

127 S. puberula Hook Pennell 14367 Peru, 1925

B BB SBB

128 S. puberula Hook Pennell 14336 Peru, 1925

N B BB

129 S. purpusii Robins Purpus 1486 Mexico, 1905

N N BS

130 S. quitensis HBK Bonpland s.n. Ecuador, no date

N B SB

131 S. rebaudiana (Bertoni) Bertoni.

Gosling s.n. Paraguay, 1919

SSSS

-- --

132 S. reticulate Grashoff

No collector Mexico, 1893

N N S

133 S. revolute Robins Purpus 3842 Mexico, 1909

BB BB BBB

134 S. rhombifolia HBK Williams 10629 Venezuela, 1938

B BB BB

135 S. rhombifolia HBK Cuatrecasas 20845 Colombia, 1946

BB BB --

136 S. rhombifolia HBK Holmgren 526 Ecuador, 1920

N B --

137 S. rhombifolia HBK Stork & Horton 10884 Peru, 1939

B B B

138 S. rhombifolia HBK Dorantes et al. 767 Mexico, 1972

N B BB

139 S. rhombifolia HBK Jimenez 1382 Costa Rica, 1963

N N N

140 S. saliciafolia Cav. var. salicifolia

Cronquist & Fry 10823 Mexico, 1970

B BB BB

141 S. salicifolia Cav. var. callodes (Greenm.) Robins

Purpus 2541 Mexico, 1908

N N B

142 S. salicifolia Cav. var. Rirgulifera Robins

Palmer 931 Mexico, 1896

B BB BB

40

143 S. sanguinea Hieron Fabris-Crisci 7385 Argentina, 1968

B BB BB

144 S. satureifolia(Lamk.) Sch.-Bip

Pastore 1146 Argentina, 1937

B B BB

145 S. satureifolia (Lamk.) Sch.-Bip

Herter s.n. Uruguay, 1925

N N N

146 S. satureifolia (Lamk.) Sch.-Bip

Cabrera 6839 Argentina, 1940

N T T

147 S. seemannii Sch.-Bip

Smith 254 Mexico, 1894

N B BB

148 S. seleriana Robins Cronquist & Fay 10876 Mexico, 1970

N B B

149 S. selloi (Spreng.)Sch.-Bip

Hassler 3910 Paraguay, no date

N B B

150 S. serrata Cav Gehriger 246 Venezuela, 1930

B B B

151 S. serrata Cav Black 46-653 Colombia, 1946

N N B

152 S. serrata Cav. var. serrata

Williams et al. 41771 Guatemala, 1972

N N N

153 S. serrata Cav. var. arguta Robins

Powell & Edmondson 623 Mexico, 1961

B BB BB

154 S. soratensis Hieron Pennell 13536 Peru, 1925

N N B

155 S. soratensis Hieron Weberbauer 6920 Peru, 1909-1914

N N N

156 S. stenophylla A. Gray

Johnston 8907 Mexico, 1941

B BB BB

157 S. subpubescens Lag Cronquist & Sousa 10417 Mexico, 1965

N B B

158 S. subpubescens Lag. var. Intermedia Grashoff

Anderson & Laskowski 4430 Mexico, 1966

N N N

159 S. subpubescens Lag. var opaca (sch.-Bip) robins

Mexia 1615 Mexico, 1927

N B B

160 S. subpubescens Lag. var. subpubescens

Haenke 1703 Mexico, 1791

N N N

161 S. subpubescens Lag. var. subpubescens

Cronquist & Fay 10906 Meixco, 1970

B B BB

162 S. tapacariensis Hieron

Eyerdam 24784 Bolivia, 1939

N N N

163 S. tephra Robins Pringle 7965 Mexico, 1899

B BB BB

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164 S. tephra Robins Purpus 4830

Mexico, 1910 N B B

165 S. tephrophylla Blake

Breedlove & raven 13643 Mexico, 1965

BB BB BB

166 S. tomentosa HBK Pupus 2550 Mexico, 1907

BB BB BB

167 S. tomentosa HBK Mueller 2397 Mexico, 1935

B BB --

168 S. trifida Lag Rzedowski 27054 Mexico, 1970

N B B

169 S. triflora DC Pringle 11835 Mexico, 1935

BB BBB --

170 S. triflora DC Williams & Molina 42299 Nicaragua, 1973

B B BB

171 S. urticaefolia Billb. In Thumb

Williams & Assis 7238 Mexico, 1945

B BB --

172 S. vaga Griseb Cabrera & Fabris 19930 Argentina, 1969

N N S

173 S. vaga Griseb Lossen 238 Argentina, 1925

B B BB

174 S. venosa A. Gray Gentry 1948 Mexico, 1935

N B B

175 S. vernicosa Greenm Pringle 10349 Meixco, 1907

B BB --

176 S. veronicae DC Herter 1971 Uruguay, 1947

T TT --

177 S. villaregalis MC Vaugh

Pringle 2486 Mexico, 1889

B BB --

178 S. villaregalis Mc Vaugh

Barnes & Land 198A Mexico, 1908

N B --

179 S. viscida HBK LeSueur 347 Mexico, 1935

BBS BBS BBS

180 S. viscida HBK Molina & Molina 26559 Guatemala, 1971

N SB SB

181 S. wageneri Hieron Vogl 505 Venezuela

N N S

182 S. weberbaueri Hieron

Stafford 438 Peru, 1937

T TT TT

183 S. yaconensis Hieron Cabrera 3032 Argentina, 1933

N N N

aB, 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.

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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

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It is possible to improve on the somewhat unpleasant hedonic characteristics of Stevioside by the production of sugar-transformed 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 and behavioral assays with Mongolian Gerbil 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

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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 LD50(g/kg bw)

Reference

Mouse Male & Female

>15 Toskulkao et al. (1997)

Mouse Male >2 Medon al. (1982)

Rat Male & Female

>15 Toskulkao et al. (1997)

Hamster Male & Female

>15 Toskulkao et al. (1997)

5.1.2 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.

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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 of toxicity and carcinogenicity on rats52 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.

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5.1.4 Genotoxicity Table 12: Genotoxic study of Stevioside53 a. With and without metabolic Activation b. 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

TA98, TA100 50mg/platea (purity 99%)

Reverse mutation S. typhimurium TA 97, TA98, TA100, TA102, TA 104, TA 1535, TA1537

5mg/plate c 1 mg/pate e purity 83%

Forward mutation S. typhimurium TM 677

10 mg / plate a

Forward mutation S. typhimurium TM 677

Not specified a

Forward mutation

S. typhimurium TM 677

10 mg / plate a

Umu Gene mutation

S. typhimurium TA 1535/psk1002

5 mg / plate a

Gene mutation B. subtilis H17 rec +, M45 rec-

10mg/disc a

Chromosomal aberration

Chinese hamster lung fibroblasts

8mg/ml c 12 mg/ml e

Chromosomal aberration

Human lymphocytes 10 mg/ml

Chromosomal aberration

Chinese hamster lung Chinese hamster lung fibroblasts

12mg/mlc 12 mg/mlc (Purity, 85%)

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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 thyroid hormone-binding proteins; binding of 3H-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.

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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-tetradecanoylphorbol-13-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 Steviol 5.2.1 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.

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5.2.2 Genotoxicity Table 13: Studies of genotoxicity of Steviol51

End-point Test object Concentration Reverse

mutation S.typhimurium TA98 &

TA100 20mg/platea

Reverse mutation

S.typhimurium TA97,TA98, TA100,TA102,TA104,TA1535

and TA1537

5mg/platea purity, 99%

Forward mutation

S.typhimurium TM677 10mg/platec 0.5-

10mg/plate Forward mutation

S.typhimurium TM677 10mg/platec 10mg/platee

Umu Gene mutation

S.typhimurium TA1535/psk1002

625-1250g/pla

1259-2500g/pl

Gene mutation

B.subtilis H17 rec+M45 rec- Chinese hamster lung

fibroblasts

10mg/disca 400g/mld

Chromosomal aberration

Chinese hamster lung fibroblasts

0.5g/mlc 1-1.5 mg/mld

Chromosomal aberration

Human lymphocytes 0.2 mg/ml

Micronucleus formation

MS/Ae mice 1000mg/kgbwg

a. With and without metabolic activation. b. The same results are cited in an earlier abstract (Matsui

et al., 1987). c. Without metabolic activation. d. With metabolic activation. e. With metabolic activation derived from Phenobarbital- or

Aroclor 1254-pretre.3-methlycholanthrene-pretreated rats were ineffective.

f. Diphtheria toxin-resistant colonies. g. Toxic: 4/6 mice at highest dose given intraperitoneally

died.

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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 body- weight 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.

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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.

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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.

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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 Non-Nutritive/Intense sweeteners. *Those which are essentially significantly reduced in calories.

Those which are calorie free.

*Carbohydrates are nutritive, because in human body they metabolized are metabolized to produce energy energy. *e.g. Sugars and Polyols.

*Intense sweeteners are generally not or produce negligible

Non- Nutritive (OR) Intense (OR) High Potency Sweeteners NATURAL ARTIFICIAL *Thaumantin *Saccharin *Monellin *Aspartame *Miraculin *Cyclamate *Brazzein *Acesulfame k *Stevioside *Cyclamate *Glycyrrhizininc acid *Sucralose *Mogroside *Dulcin *Dihydrochalcones

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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 Department of Rural Home Science, University of Agricultural 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).

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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 anti-nutritional 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 Values Bulk density 0.443 gm/ml

Water absorption capacity 4.7ml/gm Fat absorption capacity 4.5 ml/gm

Emulsification value 5 ml/gm Swelling 5.01 gm/gm Solubility 0.365/gm

pH 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.

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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