Stevia: Nature’s Wonder Herb In recent years, there has been a growing shift towards healthier dietary choices and reduced consumption of refined sugars, owing to the raising concerns about obesity, diabetes and other lifestyle related diseases. Paraguayan sweet herb, Stevia rebaudiana Bertoni, emerged as a promising solution, providing an all-natural, low-calorie alternative for those seeking to satisfy their sweet cravings without compromising their well-being. This plant is native to South America that has been cultivated for hundreds of years (Khiraoui et al., 2018). The leaves and extracts made from them have a sweet taste. They are source of complex mixture of diterpene glycosides including stevioside, steviolbioside, rebaudiosides (A, B, C, D, E) and dulcoside A but the major sweet constituents are stevioside and rebaudioside A. Beyond its sweetening capabilities, stevia’s potential health benefits have sparked a great deal of interest in the scientific community. Reports suggest that stevia may offer anti-oxidant, anti-inflammatory, anti-diabetic, anti-obesity, anti-cariogenic, anti-microbial and anti-hypertensive properties (Ahmad and Ahmad, 2018). Stevia: Nature’s Wonder Herb In recent years, there has been a growing shift towards healthier dietary choices and reduced consumption of refined sugars, owing to the raising concerns about obesity, diabetes and other lifestyle related diseases. Paraguayan sweet herb, Stevia rebaudiana Bertoni, emerged as a promising solution, providing an all-natural, low-calorie alternative for those seeking to satisfy their sweet cravings without compromising their well-being. This plant is native to South America that has been cultivated for hundreds of years (Khiraoui et al., 2018). The leaves and extracts made from them have a sweet taste. They are source of complex mixture of diterpene glycosides including stevioside, steviolbioside, rebaudiosides (A, B, C, D, E) and dulcoside A but the major sweet constituents are stevioside and rebaudioside A. Beyond its sweetening capabilities, stevia’s potential health benefits have sparked a great deal of interest in the scientific community. Reports suggest that stevia may offer anti-oxidant, anti-inflammatory, anti-diabetic, anti-obesity, anti-cariogenic, anti-microbial and anti-hypertensive properties (Ahmad and Ahmad, 2018). Safety regulations for stevia consumption have been established by various regulatory authorities worldwide. The Joint FAO/WHO Expert Committee on Food Additives initially defined the recommended daily consumption of 4 mg/kg body weight in 2004. The US FDA in 2008-09 issued safe status for steviol glycosides, which were followed by the European food safety authority. In 2015, the FSSAI approved its use as a non-calorie sweetener in a variety of food products in India. Stevia is presently recognized as safe in more than 150 nations (Anonymous, 2023). Due to its numerous health benefits and versatility in culinary applications, stevia h

1. 8 November 2023 Department of PSM&AC
UNIVERSITY OF HORTICULTURAL SCIENCES, BAGALKOT
COLLEGE OF HORTICULTURE, BENGALURU-65
Speaker: Shivanand D Ainapur,
UHS21PGD422, II Ph. D.
Department of PSMAC
Major Advisor: Dr. Maruthi Prasad B. N.
Assistant Professor,
Dept. of PSMAC
1

2. Ancient sweetener: Honey In the 18th century, the process of extracting
sucrose from sugar beet & sugar cane
Global sucrose consumption came to 180 million metric tons
https://www.statista.com/statistics/report-content/statistic/249681
151
366
537
643
784
2000 2011 2021 2030 2045
155.8
165.7
167.9
170.8 172.1 171.5
176.01
180.05
2010-11 2012-13 2014-15 2016-17 2018-19 2020-21 2022-23 2023-24**
Consumption of sugar in million metric tonnes
People with diabetes (in Millions)
Global diabetes data report 2000 - 2045
8 November 2023 Department of PSM&AC 2

3. Became the object of controversies and conflicts The need for natural substitutes is crucial
High-potency & low-calorie sweeteners
8 November 2023 Department of PSM&AC 3

4. Introduction and History
Botany
Nutritional composition and bioactive compounds
Health Benefits & Case studies
Safety
Formulations & products
Conclusion
CONTENTS
8 November 2023 Department of PSM&AC 4

5. Stevia – Honey leaf
• Botanical Name : Stevia rebaudiana Bertoni
• Family : Asteraceae
• Chromosome No : 2n=2x=22
• Origin : North-Eastern Paraguay
• Commonly known as Honey leaf, sweet leaf, sugar leaf, or Sweet herb
• Distribution : Tropical and South America, East Asia, South East Asia
8 November 2023 Department of PSM&AC 5

6. Stevia eupatoria
Hierba del Borrego
Diuretic and anti-malerial
Stevia salicifolia
Hierba del aire
Rheumatism, cathartic,
purgative, fever and cold
Stevia ovata
Round-leaved candyleaf and
Texas candyleaf
Steviaa serrata
Uriki, Chapo and Roniino
To make washes and
poultice for open wounds
For gastrointestinal disorders
Stevia plummaerae
Ronino, Plummer’s candyleaf
To make washes and
poultice for open wounds
Stevia trifida
Manzanilla de agua
Dysentery treatment
Other well-known species of the Stevia genus
8 November 2023 Department of PSM&AC 6

7. Figure 1: World-wide distribution of Stevia rebaudiana (Bertoni) plant [Inset, major Stevia growing states in India]
Gantait et al., 2017
8 November 2023 Department of PSM&AC 7

8. History of stevia
Moisés Santiago Bertoni Dr. Ovidio Rebaudi M. Bridel and R. Lavielle isolated the compounds
8 November 2023 Department of PSM&AC 8

9. Figure 2: The market value of stevia worldwide from 2017 to 2022 (in million U.S. dollars)
© Statista 2023
490.08
536.64
587.62
643.44
704.57
771.51
0
100
200
300
400
500
600
700
800
900
2017 2018 2019 2020 2021 2022*
Market value in million USD
8 November 2023 Department of PSM&AC 9

10. • Slender perennial herb grows up to a height of 60 -70 cm tall
• Stems - Erect and pubescent & produces secondary shoots
• Leaves - Sessile, opposite to oblanceolate, serrated
• Flowers – Small, white and arranged in an irregular cyme
• Fruit – Spindle-shaped achene with persistent pappus, 3.5-
4.0 mm long seeds
Botanical description of plant
Rossi et al., 2018
8 November 2023 Department of PSM&AC
10

11. Table 1. Nutritional composition of stevia leaves
Sl No. Components Content on a dry weight basis (g/100g)
1. Carbohydrates 53.00 - 66.50
2. Fat 1.90 - 5.60
3. Protein 9.63 - 16.00
4. Ash 3.08 – 15.50
5. Dietary fiber 6.80 -17.12
Minerals (mg/100g) Vitamins (mg/100g)
1. Sodium 29.4 1. Vitamin-C 14.97
2. Potassium 2195.3 2. Vitamin-B2 0.43
3. Iron 24.29 3. Vitamin B6 1.90
4. Phosphorus 372.1 4. Folic acid (Vit B9) 52.18
5. Magnesium 286.2 5. Niacin (Vit B3) 194
6. Zinc 1.423 6. Thiamine (Vit B1) 18.8
Khalid et al., 2021
8 November 2023 Department of PSM&AC 11

12. Table 2. Bioactive compounds and their sweetness
Singh et al., 2019
Bio-active
compounds
Steviol
glycosides
Steviol
Triterpenes
Flavonoids
Essential
oil
Phenolic
compounds
8 November 2023 Department of PSM&AC 12

13. Chemical configuration of stevioside along with its associated compounds
Chatsudthipong and Muanprasat 2009
8 November 2023 Department of PSM&AC 13

14. Factors influencing the steviol glycosides synthesis in stevia
HIM-Stevia, RSIT 94-751, 94-1306, 95-166-13,
CIM-Madhu, CIM-Mithi, Morita, Acc 1 & 2
Gibberellin and cytokinin balance
Older ones - higher SGs & Colder months – low SGs
Temp – 20-30℃, Photoperiodic
condition, pH- 6.5-7.5, Rainfall
Drought & Salt stress
Irrigation – 1.2 IW/CPE, plant geometry 30 x 20 cm
Deflowering & PBZ @ 12ppm – high SGs
1. Genetics & cultivar
2. Environmental conditions
3. Age of the plant & season
4. Stress factors
5. Hormonal Balance
6. Agricultural practices
Sharma et al., 2015
8 November 2023 Department of PSM&AC 14

15. Table 3. Summary of influence of agro-techniques on herbage and SGs yield
Factor Influence over growth/ Biosynthesis of SGs Reference
Early transplanting (long duration),
wider spacing
High biomass yield and Steviol glycosides content Taleie et al., 2012
Kumar et al., 2013
Long day conditions (16/8h), high
temperature & light intensity
High phenolic compounds, anti-oxidant capability
and SGs, Vigorous vegetative growth and higher
biomass yield
Andrade et al., 2021
Maheshwar, 2005
Growth regulators (PBZ @ 12 ppm,
GA3 @50 ppm)
Improved stevia growth and antioxidant activity Karim et al., 2014
Stefanani and Rodriques,
1999
Extending the day by artificial
lighting/ midnight interruption by red
LED
Prevent flowering, increase in biomass and glycoside
content
Ceunen et al., 2012
Ceunen and Genus, 2012,
Ceunen and Genus, 2013
Salt-stress (NaCl) Moderately tolerant and decreased the RbA and St
content but their ratio is increased (1.1-1.4 times)
Zeng et al., 2013
Shade levels (50 & 75%) Early flowering and decreased SGs Kumar et al., 2014 (CSIR-
IHBT, Palmpur)
Decapitation of apical buds Increase in yield by 15-20% Pal et al., 2013
8 November 2023 Department of PSM&AC 15

16. Advantages of stevia over other sweeteners
250 X
sweeter
than sugar
100%
Natural &
Calorie free
Heat and pH
stable
Non-
fermentable
Highly Safe
for
diabetics
and caries
Not
metabolized
& easily
excreted Goyal et al., 2010
8 November 2023 Department of PSM&AC 16

17. Figure 3: Mean blood glucose response after ingestion of sucrose, 5 mg/mL stevia, and 10 mg/mL
stevia over a 2 h period
5.15
6.7
5.81
5.07
5.09
5.22
5.14
5.21
5.18
5.15
5.04
5
5.07
5.02
5.07
0 min 30 min 60 min 90 min 120 min
Blood glucose level (mmol/L)
Sucrose (0.2mg/ml) Stevia (5 mg/ml) Stevia (10 mg/ml)
Al-Sultan et al., 2021
8 November 2023 Department of PSM&AC 17

18. Figure 4: Steviol glycoside (SGs) metabolism in humans. LG., large; SM., small
StGs is undigested in
the upper
gastrointestinal tract
Hydrolysis of
StGs in the
colon
Glucuronidated
steviol is excreted via
Urine
© 2017 Global Stevia Institute
8 November 2023 Department of PSM&AC 18

19. F
Traditional uses and potential health benefits
Anti-inflammatory
Wound healing
Anti-diabetic
Anti-oxidant
Anti-hypertension
Anti-cariogenic
Blood pressure
Functional component
for food Industry
Anti-cancer
Anti-microbial
Polyphenols from stevia, neutralize
free radicals and chelate transition
metal ions
Regulates blood glucose by
enhancing insulin secretion
and insulin utilization in insulin-
deficient rat
Lowers mean arterial
blood pressure
Stevioside causes
vasorelaxation through an
inhibition of Ca influx in to
blood vessel
Anti-plaque and inhibit microbial growth and
reproduction in the oral cavity that causes gum
diseases and tooth decay
St could inhibit DNA synthesis and induce the
death of cancer cells via the mitochondrial
apoptotic pathway
Polyphenolic constituents showed an inhibitory
effect on tumor initiation & promotion.
Inhibit microbial growth
and reproduction – SGs,
flavonoids and
polyphenols
Guarani Indians – Herbal tea
(Ka-he-e), taste of bitter
medicine
Low-calorie drinks, baked
products, yogurt, smoothies,
coffee
EtOH extract shown a
reduction in wound area –
Reepithelization and epithelial
formation
MtOH extract - Inhibition
of the inflammatory activity
8 November 2023 Department of PSM&AC 19

20. Effect of aspartame, monk fruit, stevia and sucrose-
sweetened beverages on postprandial glucose, insulin and
energy intake
Objective: To compare the effects of consuming NNS (artificial versus natural) and
sucrose (65g) on energy intake, blood glucose and insulin responses.
Clinical Nutrition Research Centre, Singapore Institute for Clinical
Sciences, Agency for Science, Technology and Research, 14
Medical Drive, Singapore 117599, Singapore
International Journal of Obesity
Tey et al., 2017
Impact Factor – 4.9
8 November 2023 Department of PSM&AC 20

21. Subjects and methods
• 34 male participants (21-50 years)
• Randomised crossover study with four treatments
(Double-blind)
• Test beverages (in 500 ml water) were as follows:
(i) 0.44 g aspartame,
(ii) 0.63 g monk fruit extract (50% Mogroside V),
(iii) 0.33 g stevia (Steviol Glycoside, Rebaudioside A),
(iv) 65 g sucrose
• Blood test – At 15, 30, 45, 60, 90, 120, 150 and 180 min
after baseline  Glucose and insulin analysis
Tey et al., 2017
8 November 2023 Department of PSM&AC 21

22. Figure 5. Appetite ratings (mean ± s.e; n = 30). *Linear mixed models showed statistically significant
differences in these appetite measures between the treatments at those time points, P <0.05
Rating of appetite and mood
states on a 100-mm visual
analogous scale anchored with
'not at all’ - rating (0 mm) and
'extremely’ - rating (100 mm)
a. Desire to eat
b. Hunger
c. Prospective consumption
d. Fullness
8 November 2023 Department of PSM&AC 22

23. Figure 6. Energy intake consumed at each meal on aspartame, monk fruit, stevia and sucrose test days
(mean ± s.e.; n = 30). A linear mixed model showed no significant difference in total daily energy intake
between the treatments (P = 0.831)
73%
107%
98%
8 November 2023 Department of PSM&AC 23

24. Figure 7. Temporal curves of the blood glucose response for the test beverages (mean ± s.e.; n = 30).
*Linear mixed models showed statistically significantly differences in blood glucose between the
treatments at those time points, P >0.05
8 November 2023 Department of PSM&AC 24

25. Figure 8. Temporal curves of the blood insulin response for the test beverages (mean ± s.e.;n = 10)
*Linear mixed models showed statistically significantly differences in blood insulin between the
treatments at those time points, P >0.05
Inference
8 November 2023 Department of PSM&AC 25

26. Antioxidant ability, total phenolic and flavonoid contents
of leaf extract of Stevia rebaudiana Bertoni Cultivated in
Morocco
Objective: To evaluate the contribution made through the free radical scavenging
capability of leaves of Stevia rebaudiana cultivated in different areas in Morocco
National Institute of Agricultural Research (INRA),
CRRA-Rabat, Rabat - Morocco
International Journal of Scientific & Engineering Research
Khiraui et al., 2018
Impact Factor – 4.2
8 November 2023 Department of PSM&AC 26

27. • Stevia leaves collection from six different locations
• Air-dried leaves powdered
• Extraction – Aqueous, methanol and ethanol
• Determination of
Total phenolic content
Flavonoid content
DPPH radicle scavenging activity
• Statistical analysis – Data reported in mean ± SD
using one-way ANOVA (F-test)
Materials and methods
Anti-oxidant activity
Flavonoid content
Total phenolic content
Khiraui et al., 2018
8 November 2023 Department of PSM&AC 27

28. Figure 9a. Total phenolic content of S. rebaudiana leaves is equivalent to gallic acid of dry weight. 9b. Flavonoid
compounds content of S. rebaudiana leaves equivalent to rutine of dry weight from different geographical areasin
Morocco. Values are expressed as the mean ± SE (n=3). Bars carrying different letters are significantly different at
P<0.05 among regions
9b
9a
8 November 2023 Department of PSM&AC 28

29. Figure 10a. Scavenging activities of different leaf extracts of S. rebaudiana leaves against DPPH radical of dry
weight from different geographical areas in Morocco. 10b. Percentage of antioxidant activity of ethanolic leaf
extract of S. rebaudiana and standards antioxidants by DPPH radical scavenging method. Values are expressed
as the mean ± SE (n=3). Bars carrying different letters are significantly different at P<0.05 among regions
Agadir Berkane Larache Marrakech Rabat Sefrou
90
80
70
60
50
40
30
20
10
0
Scavenging
activity
of
DPPH
radical
(%
inhibition)
10a 10b
Khiraui et al., 2018
Inference
8 November 2023 Department of PSM&AC 29

30. Antimicrobial potential of extracts from stevia rebaudiana
leaves against bacteria of importance in dental caries
Objective: To evaluate the antibacterial activity of Stevia rebaudiana Bertoni leaf extracts
against cariogenic bacteria
Department of Microbiology and Dental Research
Centre, Pontifia Universidad Javeriana, Bogota,
Colombia
Acta Odontologicia Latinoamericana
Gamboa and Chaves, 2012
h5–index 10
8 November 2023 Department of PSM&AC 30

31. Material and Methods
• Stevia leaves extraction using – Ethanol,
Methanol, Ethyle acetate, Chloroform and
Hexane  Cold soaking technique
• Bacteria – 16 oral bacteria strains form genera
Streptococcus (n= 12) and Lactobacillus (n= 4)
• Antimicrobial assay  Well diffusion method
• Each assay was performed in triplicate and the
average reported in mm
8 November 2023 Department of PSM&AC 31

32. 0
2
4
6
8
10
12
14
Hexane (30
mg/ml)
Methanol
(120mg/ml)
Ethanol
(120mg/ml)
Ethyl aceate
(60mg/ml)
Chlorofarm
(60mg/ml)
Microbial inhibition zone (mm)
Figure 11. Inhibition zone by different extracts of Stevia rebaudiana Bertoni on cariogenic bacterial
strains
8 November 2023 Department of PSM&AC 32

33. Table 4: Antibacterial activity of the Stevia rebaudiana Bertoni leaf extracts against the 16
bacterial strains
Inference
8 November 2023 Department of PSM&AC 33

34. Antidiabetic property of aqueous extract of Stevia
rebaudiana Bertoni leaves in STZ-induced diabetes in
albino rats
Objective: To explore the anti-diabetic effect of aqueous extract of Stevia
rebaudiana leaves in albino rats
Department of Food Science, Nutrition & Home
Econimics, Government College University,
Faisalabad, 38000, Pakistan
Complementary and Alternative medicine
Ahmad and Ahmad, 2018
Impact Factor – 4.13
8 November 2023 Department of PSM&AC 34

35. • In-vivo study, 60 healthy adult male albino rats… 6 groups of 10 rats
• Diabetes induction .. 40mg/kg intravenous streptozotocin (STZ) injection
• Blood samples… glucose levels tested at every week interval up to 8 weeks
Material and methods
Sl.no Group Description of treatment
1 N0: Non-diabetic rats (control) Basal diet + Distilled water
2 D0: STZ-induced diabetic rats (diabetic control) Basal diet + Distilled water
3 D1: STZ-induced rats Basal diet + Distilled water with aqueous extract of Stevia (AES) – 200 ppm/kg
4 D2: STZ-induced rats Basal diet + Distilled water with AES – 300 ppm/Kg
5 D3: STZ-induced rats Basal diet + Distilled water with AES – 400 ppm/Kg
6 D4: STZ-induced rats Basal diet + Distilled water with AES – 500 ppm/Kg
Stevia leaves aqueous extract
Blood glucose level
Glycosylated hemoglobin (HbA1c) level
Insulin level (ELISA)
Liver glycogen
8 November 2023 35
Department of PSM&AC
Streptozotocin 40mg/Kg

36. Figure 12a. Feed intake (g) 12b. Water intake in normal and diabetic rats during 8 weeks (rat/week). Results are
expressed as amount of feed/water intake levels of diabetic and non-diabetic rats (mean ± standard deviation (SD). n
= 10). The feed/water intake of diabetic rats (D1, D2, D3 and D4) received stevia aqueous extract in different
concentrations (200, 300, 400 and 500 ppm) respectively significantly (P < 0.05) decreased from non-diabetic (N0)
and diabetic (D0) control groups
8 November 2023 Department of PSM&AC 36

37. 100
110
120
130
140
150
160
170
180
190
200
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Body
weight
of
rates
(g)
Interval
N0 D0 D1 D2 D3 D4
Figure 13. Effect of Stevia aqueous extract on body weight of diabetic and non-diabetic rats
23.27% - N0
19.48%- D0
13.84 – 20.55%
8 November 2023 Department of PSM&AC 37

38. 0
50
100
150
200
250
300
350
400
450
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Random
blood
glucose
level
(mg/dL)
Interval
N0 D0 D1 D2 D3 D4
0
50
100
150
200
250
300
350
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Fasting
blood
glucose
level
(mg/dL)
Interval
N0 D0 D1 D2 D3 D4
D0 – 16.81%
D0 – 15.04%
64.87-66.09%
71.74-73.24%
Figure 14a. Random and 14b. Fasting blood glucose levels (mg/mL) of normal and diabetic rats
8 November 2023 Department of PSM&AC 38

39. Figure 15. Effect of Stevia aqueous extract on the glycosylated hemoglobin (HbA1c) level of the rats. Results are expressed as percentage
of HbA1c levels of diabetic and non-diabetic rats (mean ± standard deviation (SD). n = 10). a, b, c, d represent significant difference (P <
0.05) in HbA1c levels treatment wise. HbA1c levels of diabetic rats (D1, D2, D3 and D4) received stevia aqueous extract in different
concentrations (200, 300, 400 and 500 ppm) respectively significantly (P < 0.05) decreased as compared diabetic (D0) control groups and
near to N0
8 November 2023 Department of PSM&AC 39

40. Figure 16. Effect of Stevia aqueous extract on insulin levels of different groups of rats. Results are expressed as concentration of
insulin levels of diabetic and non-diabetic rats (mean ± standard deviation (SD). n = 10). a, b, c, d represent significant
difference (P < 0.05) in insulin levels treatment wise. The insulin levels of diabetic rats (D1, D2, D3 and D4) received stevia
aqueous extract in different concentrations (200, 300, 400 and 500 ppm) respectively significantly (P < 0.05) increased as
compared diabetic (D0) control groups and near to N0
8 November 2023 Department of PSM&AC 40

41. Figure 17. Effect of Stevia aqueous extract on the glycogen level of the rats. Results are expressed as concentration of glycogen
levels of diabetic and non-diabetic rats (mean ± standard deviation (SD). n = 10). a, b, c, d represent significant difference (P <
0.05) in insulin levels treatment wise. The glycogen levels of diabetic rats (D1, D2, D3 and D4) received stevia aqueous extract
in different concentrations (200, 300, 400 and 500 ppm) respectively significantly (P < 0.05) increased as compared diabetic (D0)
control groups and near to N0
Inference
8 November 2023 Department of PSM&AC 41

42. Global safety of stevia
1970s
Evaluated by the European
Commission’s Scientific Committee
on Food (SCF)
1999 2004
Joint FAO/WHO Expert
Committee on Food Additives
(JECFA), established tentative
purity specifications
2008
JECFA established a
permanent acceptable
daily intake (ADI) for
SGs of 4 mg steviol eq.
kg−1 body weight/day
and validated its safety
for use as a food and
beverage sweetener
2009
US-FDA issued safe
(GRAS) status to
SGs (St and Rb-A)
for use in food
2011
EFSA declared
SGs as safe and
established ADI
same as JECFA
2015
FSSAI, India in 2015
approved the use of
SGs as an sweetener
in various foods
• Currently in over 150 countries approved Stevia as a sweetener and food additive
US-FEMA issued GRAS
level for natural stevia
flavours
1985
Global Stevia Institute, 2023
China, Japan, S-Korea
have permitted use of
Stevia
8 November 2023 Department of PSM&AC 42

43. Toxicological evaluation of ethanolic extract from Stevia
rebaudiana Bertoni leaves: Genotoxicity and sub-chronic
oral toxicity
Objective: To evaluate the toxicity of ethanolic extract of Stevia rebaudiana Bertoni leaves through a
battery of in vitro and in vivo tests.
Key Laboratory of Food Safety Risk Assessment of Ministry of
Health, National Center for Food Safety Risk Assessment, Beijing
100021, China
Regulatory Toxicology and Pharmacology
Zhang et al., 2017
Impact Factor –
8 November 2023 Department of PSM&AC 43

44. Material and methods
S.no Group Description of treatment
1 Control Normal diet
2 Maximum dose Dietary concentration of 3.12%
3 Medium dose Dietary concentration of 2.08%
4 Low dose Dietary concentration of 1.04%
8 November 2023 44
Department of PSM&AC
Genotoxicity study
Bacterial reverse mutation assay (Ames test)
Mouse bone marrow micronucleus assay
Mouse sperm malformation assay
90-Day repeated dose study
40 male and 40 female Sprague-Dawley rats were randomly divided into 4 groups
8.3 mg/kg BW per day
Hematology and clinical assay  RBC, HG, WBC, PLT and leukocytes as well as ALT,
AST, ALP, TP, ALB, GLU, BUN, SCr, CHO and TG respectively
Pathology  Necroscopy was performed  Heart, kidney, liver, spleen, testes, thymus,
Stomach, Duodenum, adrenals and ovary  microscopic examination
Zhang et al., 2017

45. 0
500
1000
1500
2000
2500
3000
- S9 - S9 - S9 - S9
TA97 TA98 TA100 TA102
Bacterial
revert
mutation
Salmonella typhimurium strains
Solvent control SE-62 µg SE-185 µg SE-556 µg SE-1667 µg SE-5000 µg Positive control
Figure18. Effect of Stevia leaves extract on bacterial reverse mutation (Ames test)
8 November 2023 Department of PSM&AC 45

46. 42
44
46
48
50
52
54
56
0 St-2500 mg St-5000 mg St-10000 mg CPA, 40 mg 0 St-500 mg St-5000 mg St-10000 mg CPA-40 mg
Male Female
PCE/RBC
(%)
Treatment dosage
0
2
4
6
8
10
12
14
16
18
0 St-2500 mg St-5000 mg St-10000 mg CPA-40 mg 0 St-500 mg St-5000 mg St-10000 mg CPA-40 mg
Male Female
MN/PCE
(%)
Treatment dosage
Table 19. Effect of Stevia leaves extract on micronucleus induction in mouse bone marrow
8 November 2023 Department of PSM&AC 46

47. 0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 St-500 mg St-5000 mg St-10000 mg CPA-40 mg
Incidence
of
spermatozoa
malformation
(%)
Treatment
Figure 20. Effect of Stevia rebaudiana Bertoni leaves extract on sperm malformation in mouse
8 November 2023 Department of PSM&AC 47

48. Table 9. Effect of Stevia leaves extracts on hematology in rats on day 91
WBC, white blood cell (leukocyte) count; RBC, red blood cell (erythrocyte) count; PLT, platelet count; HB, hemoglobin. Data were analyzed with ANOVA followed by Dunnett's
test for multiple comparisons, all values are expressed as mean ± S.D. of each group. *P < 0.05, significantly different as compared with the controls
ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; BUN, urea nitrogen; SCr, serum creatinine; CHO,
cholesterol; TG, triglycerides; GLU, Glucose; TP, total protein; Alb, albumin. Data were analyzed with ANONA followed by Dunnett's test for multiple
comparison, all values are expressed as mean ± S.D. of each group. *P < 0.05, significantly different as compared with the controls.
Table 10. Effect of Stevia rebaudiana Bertoni leaves extract on clinical biochemistry in rats on day 91
8 November 2023 Department of PSM&AC 48

49. 0
0.5
1
1.5
2
2.5
3
3.5
4
0 St-1.04 St-2.08 St-3.12 0 St-1.04 St-2.08 St-3.12
Female Male
Weight
of
the
organs
after
91
days
treatment
(%)
Dose (%)
Liver Kidney Spleen Thymus Heart Testes
Figure 21. Effect of Stevia rebaudiana Bertoni leaves extract on relative organ weight in rats
8 November 2023 Department of PSM&AC 49

50. Tissues from twenty animals were examined in each group
Table 12. Histopathology examinations on rats after Stevia rebaudiana Bertoni leaves extract treatment.
Inference
Microscopic lesions Dose (% diet)
0 (Control) 3.12
Liver Degenaration 0 0
Necrosis 1/20 1/20
Infalmmation 0 0
Cholangiolar changes 0 0
Kidney Glomular atropy 0 0
Inflammatory exudate 0 0
Papillary necrosis 0 0
Imterstial cell hyperplasia 0 0
Heart Myocardial necrosis 3/20 3/20
Degeneration 0 0
Cellular infiltration 0 0
Other tissues (Spleen, stomach, duodenum, thymus, adrenals, testes, ovary) 0 0
8 November 2023 Department of PSM&AC 50

51. Table 13. Average daily consumption in different countries as per regulatory authorities
S.No Country/region Samples ADI (mg/kg BW/day) Regulatory authority
1
Canada
Dietary exposure 50
Food standards
Fruits & table juices 1100
Tabletop sweetener 40000
2 China Food stuffs 170-10000
3 India Food stuffs 200-3500 FSSAI
4 South Africa Food stuffs 330 EFSA
5 European Union Food stuffs 20-3300 EFSA
JECFA established a permanent acceptable daily intake (ADI) for SGs of 4 mg steviol eq. kg−1 body
weight/day (Conversion factors- Stv-0.4 & RbA-0.33)
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52. Forms of stevia preparations
• Fresh/ dried leaves
• Stevia extract
• Liquid concentrate
• Refined Steviol glycosides
8 November 2023 Department of PSM&AC 52

53. Whole leaf stevia extract Vs Stevia extract
Whole Leaf Stevia Extract
• Dried leaves  Ground  Crude extract (Water,
ethanol, methanol, isopropyl alcohol)
• Retains a broader spectrum of compounds
(Fibres, Phytochemicals and sweeteners)
• Balanced sweetness – more palatable
• Potential health benefits – antioxidants and fibers
• Larger quantities are needed to meet the desired
level of sweetness
• Impacts the calorie count
Stevia Extract
• Dried leaves  Ground  Refining (Stevioside
& Rebaudioside A)
• Only sweet compounds are extracted
• Highly concentrated sweetness
• Negligible impact on blood sugar
• Little to no bitter aftertaste
• Consumed in a more diluted version
• Lack of potential health benefits of whole leaf
• Adultrants - maltodextrin, lactose, sucrose
8 November 2023 Department of PSM&AC 53

54. Ingradian Incorporated - American food and beverage ingredient
provider, Westchester
Tate & Lyle PLC - Global supplier of food beverages ingredients,
London
Archer Daniels Midland Company - Commonly known as ADM,
is an American multinational food processing and commodities
trading corporation, Chicago
Cargill, Incorporated, American global food corporation,
Minnetonka, Minnesota
HOWTIAN- Manufacturer of Inositol, Baicalin, Stevia (SoPure™)
and Pyrroloquinoline Quinone (PureQQ®) products, as well as
various plant-based ingredients for food & beverage, nutraceutical,
pharmaceutical, and personal care industries, China
World stevia market leaders
8 November 2023 Department of PSM&AC 54

55. Conclusion
8 November 2023 Department of PSM&AC 55