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![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](https://image.slidesharecdn.com/stevianatureswonderherb-231108105922-ec407637/85/Stevia-Nature-s-Wonder-Herb-pptx-7-320.jpg)
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Stevia – Nature's Wonder Herb.pptx
The document discusses the ancient sweetener honey and the emergence of stevia as a natural substitute due to health concerns surrounding sugar consumption. It details the botanical characteristics, nutritional composition, and health benefits of stevia, including its anti-diabetic and antimicrobial properties. The discussion includes various studies on stevia's metabolic effects, market trends, and agro-techniques for enhancing its growth and glycoside yield.
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Stevia – Nature's Wonder Herb.pptx
- 1. 8 November 2023Department 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: HoneyIn 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 objectof 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 Nutritionalcomposition and bioactive compounds Health Benefits & Case studies Safety Formulations & products Conclusion CONTENTS 8 November 2023 Department of PSM&AC 4
- 5. Stevia – Honeyleaf • 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 delBorrego 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-widedistribution 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 MoisésSantiago Bertoni Dr. Ovidio Rebaudi M. Bridel and R. Lavielle isolated the compounds 8 November 2023 Department of PSM&AC 8
- 9. Figure 2: Themarket 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 perennialherb 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. Nutritionalcomposition 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. Bioactivecompounds 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 ofstevioside along with its associated compounds Chatsudthipong and Muanprasat 2009 8 November 2023 Department of PSM&AC 13
- 14. Factors influencing thesteviol 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. Summaryof 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 steviaover 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: Meanblood 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: Steviolglycoside (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 andpotential 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. Appetiteratings (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. Energyintake 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. Temporalcurves 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. Temporalcurves 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, totalphenolic 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 leavescollection 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. Totalphenolic 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. Scavengingactivities 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 ofextracts 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) Microbialinhibition 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: Antibacterialactivity of the Stevia rebaudiana Bertoni leaf extracts against the 16 bacterial strains Inference 8 November 2023 Department of PSM&AC 33
- 34. Antidiabetic property ofaqueous 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. Feedintake (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 Week1 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 Week1 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. Effectof 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. Effectof 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. Effectof 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 ofstevia 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 ofethanolic 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.noGroup 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 mgSt-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 mgSt-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. Effectof 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-2.08St-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 twentyanimals 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. Averagedaily 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) 8 November 2023 Department of PSM&AC 51
- 52. Forms of steviapreparations • Fresh/ dried leaves • Stevia extract • Liquid concentrate • Refined Steviol glycosides 8 November 2023 Department of PSM&AC 52
- 53. Whole leaf steviaextract 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 2023Department of PSM&AC 55
Editor's Notes
- #3 Honey used to be the main sweetener in the human diet in ancient civilisation However, in the 18th century, the process of extracting sucrose from sugar beet and sugar cane grew exponentially and clearly assumed preponderance. Nowadays, sucrose, or common table sugar, remains the most traditionally used sweetener and is available in a variety of refined forms. In 2022-23, global sucrose consumption came to 176 million metric tons. In the last few years, sugar overconsumption has become a pandemic, with serious consequences in public health terms. There is clear evidence for an association between eating too much sugar and being at higher risk for type II diabetes, obesity, dental caries and cardiovascular diseases, among other non-communicable diseases. Given this scenario, sweeteners in food products have spread, and this product is major a target of much interest for the industrial and scientific communities
- #4 Thus, a low-calorie sweetener, saccharine, became available in the 1879. As this sweetener was so popular, others followed, including cyclamates, aspartame and acesulfame K, which are the most widespread. The FDA has approved five artificial sweeteners: saccharin, acesulfame, aspartame, neotame, and sucralose. It has also approved one natural low-calorie sweetener, stevia Hence the need for natural substitutes is crucial. Natural sweeteners and synthetic sweeteners have the same purpose: to act as a sweet flavour while fewer or no calories at all to the diet Sweeteners have long since been the object of controversies and conflicts over the years, which have included allegations of liver and bladder toxicity, carcinogenicity, fetus malformations, along with other dangers
- #6 Distribution - North-Eastern Paraguay, USA, Brazil, Japan, Korea, Taiwan and South East Asia
- #7 The traditional uses of 29 species from Central and South America are available. Some of the most common popular uses of Stevia species are related to antidiarrheal, antiinflammatory, antimalarial uses, as a febrifuge, a diuretic, a diaphoretic, to treat heart diseases, stomachaches, and skin conditions
- #8 Semi-humid sub-tropical climate (20-30℃)
- #9 The Guarani Indians had known for centuries about the unique advantages of kaa he-he (a native term which translates as “sweet herb”) — long before the invaders from the Old World were lured by the treasures of the New. it was an important ingredient in the indigenous diet. Historians noted that these indigenous peoples had been sweetening herbal teas with stevia leaves "since ancient times.“ STEVIA were first researched by Spanish botanist and physician Petrus Jacobus Stevus (Pedro Jaime Esteve 1500–1556), from whose surname originates the Latinized word stevia. The plant has a long history of medicinal use in Paraguay and Brazil. The leaves have been traditionally used for hundreds of years in Paraguay and Brazil to sweeten local teas, medicines and as a "sweet treat“ In 1899, the Swiss botanist Moisés Santiago Bertoni, during his research in eastern Paraguay, first described the plant and the sweet taste in detail.Bertoni named the “new” variety of the Stevia genus in honor of a Paraguayan chemist named Rebaudi who subsequently became the first to extract the plant’s sweet constituent. “In placing in the mouth the smallest particle of any portion of the leaf or twig,” Bertoni wrote, “one is surprised at the strange and extreme sweetness contained therein Dr. Ovidio Rebaudi conducts the first complete study of stevia leaves. Neighboring countries begin cultivating stevia crops. French chemists M. Bridel and R. Lavielle isolate the compounds that give the plant its sweet taste. The glycosides are named stevioside and rebaudioside. England explores the possibility of using stevia when sugarcane supplies from the Caribbean are cut off during WWII. Japan begins cultivating stevia and extracting the various steviosides to be used in place of sugar. Following extensive safety tests, the steviosides are approved as a sweetener by the government of Japan Stevioside, the combination of sweet glycosides extracted from stevia leaves, achieves a 40% market share as a commercial sweetener in Japan M. Bridel and R. Lavielle, “The Sweet Principle of Kaahe-e (Stevia rebaudiana),” Pharmaceutical Chemistry Journal, Vol. 14, 1931, pp. 99-154 Ancient times (Guarani Indians) – ‘Ka-a He-e’ Sweet grass, used savor bitter drinks (Mate) 16th century Reports on Spaniards knowns about this herb 19th Century Europe, Dr. Moies Santigo Bertoni 1901- Bertoni (Few leaves of stevia grass were enough to sweeten large cup of tea) 1920 Stevia began to be cultivated in large quantities on plantations in Brazil and Paraguay In 1931 French chemists Briedel and Lavieille isolated the glycoside which provide Stevia with its sweet taste. This compound was named stevioside. During World War II, Stevia began to be actively consumed in the United Kingdom due to sugar shortages and rationing of its consumption In the 1970 Introduced to Japan and research was started to evaluate its beneficial potential for human health. Since then, the Japanese actively use this sweetener in a variety of foods and Japan is one of the major producers of Stevia now In 2013, the Coca-Cola Company started production of drinks containing Stevia instead of sugar and having 30 % less calories The Paraguayan chemist Ovidio Rebaudi published in the Argentine Journal of Pharmaceutical Chemistry (1990), the first scientific article about the process of obtaining the sweetening principles from Stevia plants, arousing the interest of European countries such as England, France, and Germany.
- #10 Stevia industry - statistics & facts Stevia is categorized as a natural sweetener which can be derived from the leaves of the Stevia rebaudiana plant and is used as a sugar substitute. The active parts of stevia are steviol glycosides which are estimated to be 200 to 300 times sweeter than sugar, supply no calories or carbohydrates, and have no influence on the glycemic index. Stevia’s global market value was forecast to reach over 770 million U.S. dollars by 2022. This upward trend is also shown by the increasing number of global food and beverage product launches that contain stevia. In a regional comparison, Chile, the United States and France had the highest consumer awareness of stevia. Global stevia market by region – Europe>N-America> East-Asia>South Asia> Latin America> MEA>Oceania The usage of stevia in the American food industry is regulated by the U.S. Food and Drug Administration (FDA). Whole leave stevia or crude stevia extracts have not received approval for commercial use in the United States, but the highly refined sweetener compounds such as stevioside or rebaudioside A were approved as Generally Recognized As Safe (GRAS). These ingredients are used among various food categories and as a base for table-top sweeteners. Splenda was the best-selling stevia-based table-top sweetener brand in the United States, followed by Truvia and Sweet'N Low
- #11 Stems - Erect and pubescent, produces secondary shoots, dies off and is renewed annually Leaves – 22-25mm long Flowers – Capitulam/ head, Small, white and arranged in an irregular cyme Fruit is a five ribbed spindle shaped achene with persistent pappus (cypsela). Seed 3.5-4.0 mm long
- #12 Other biologically active compounds Proteins (11.2-16.0%), 61.9% CHO, 1.9-3.73% lipids, 6.8-15.2 g dietary fibre (Dry leaves) Chemical composition in preparations from stevia differs in fresh and dried leaves and depends on method of processing
- #13 Dulcoside A & C and minor glycosides, including flavonoid glycosides, coumarins, cinnamic acids, phenylpropanoids, and some essential oils are also present. Rebaudiside B-F, Steviolmonoside, Rubusoside, Dulcoside –A, Steviol bioside, Penta-glucoside rebaudiside D and Rebaudiside M – 0.2 & 0.1% respectively Rebaudiside M is commercialized by PureCirrcle Limited and The Coca-Cola Company for food and beverages Minor glycosides reb D and M are exciting interest because they have very low side tastes and a much cleaner sweetness than other glycosides or blends Rebaudiside U, T, R and S Stevia has become a potential alternative source by replacing artificial sweeteners like Saccharin, Aspartame, Asulfam-k, etc.
- #14 Steviol diterpene glycosides- stevioside and Rebaudioside A Sweetner compounds (>30 compounds) 1931 – 1st isolated sativoside form Stevia (diterpene glycoside consisting of three molecules of glucose and a glucone moiety – steviol) 4-13% of all glycosides in stevia Rebaudioside A is another steviol diterpene glycoside The percentage of its sweetness fluctuates from 30 % to 40% that is ~ 180-400 times sweeter than sugar Stevioside – 6.5-9.1%, Rebaudiside A – 2.3- 3.8%, Rebaudiside B-F, Steviolmonoside, Rubusoside, Dulcoside –A, Steviol bioside, Penta-glucoside rebaudiside D and Rebaudiside M – 0.2 & 0.1% respectively Rebaudiside M is commercialized by PureCirrcle Limited and The Coca-Cola Company for food and beverages Rebaudiside U, T, R and S Steviol and Isosteviol are the hydrolysis products of Stevioside – Therapautic properties Most of these have same chemical backbone structure (Steviol) but differ in the residues of CHO at positions C13 and C19 Other biologically active compounds Benefits Anti-diabetic, antihypertensive, antitumor, anti-carcinogenic, anti-inflammatory, and bactericidal activities Protective effect of stevia on the digestive system and skin disorders and complications associated with the metabolic syndromes It should be noted, that not only Stevia contains stevial glycosides. Rubus suavissimus also known as sweet tea, contains steviol monoside, rebaudioside A, B, C, D, F, M, stevioside, steviolbioside, and rubusoside (Uhler and Yang, 2018).
- #15 Genetics and cultivar S/R– HIM-Stevia (5.87/7.34%), CIM-Madhu (12.57/5.8%), Morita (1.1/13.2) Environmental conditions Temperature – 20-30 ℃ (affected under extreme cold and hot conditions) pH- 6.5 -7.5 Plant age – Higher concentration in older ones Seasonal changes – low in colder months Hormonal regulation – Gibberlins and cytokinin Stress factors – Drought/ excessive heat/pest Agricultural practices- Irrigation, plant geometry, Deflowering
- #16 Annual crop in sub-temperate to temperate regions and perennial in tropical regions Crop is sensitive to low temperature – must be harvested before onset of mansoon
- #17 It is 100% natural, having no (zero) calories, is 200-300 times sweeter than sugar, heat stable to 198C and over a range of pH 3-9, non-fermentable, a flavour enhancer, and is anti-plaque and anti-caries It is also non-calorific and nonfermentable and does not darken upon cooking. There have been no reports of adverse effects from the use of stevia products by humans. Hence, stevia has been named a ‘calorie-free bio-sweetener of high quality’. In addition, it is also used in the production of Gibberellic acids from the fungus Gibberella fujikuroi mutants.
- #19 The absorption, metabolism, and excretion of steviol glycosides have been extensively reviewed by multiple scientific authorities and experts Steviol glycosides are undigested in the upper gastrointestinal tract. They are hydrolyzed or degraded only when they come into contact with microbiota in the colon that cleave the glycosidic linkages, removing the sugar moieties, leaving behind the steviol backbone that is absorbed systemically, glucuronidated in the liver, and excreted via urine in humans and via feces in rats
- #22 Approved by The NHG - Domain Specific Review Board in Singapore Registered at Australian & New Zealand Clinical Trials Registry as ACTRN12615001321538 Participants are asked to attend 5 sessions, 1 screening and 4 test sessions
- #23 The subjects with sucrose treatment were felt the lower desire to eat
- #27 Free radicles are continuously formed in an organism as a result of metabolic process or exposure to stresses, may contribute a large number of human diseases, including cancer, obesity, diabetes, and neurodegenerative diseases. Under physiological conditions, the capacity of endogenous antioxidant defense is sufficient to neutralize free radicals and prevent oxidative damages, but aging and excessive caloric intake are accompanied by imbalance between the production and elimination of free radicals followed by chronic oxidative stress and systemic inflammation development Therefore, using bioactive phytochemicals having antioxidant and anti-inflammatory activities is considered as a promising therapeutic approach to combat aging and associated pathological conditions
- #30 Stevia leaves contain a number of phenolic compounds that are able to neutralize free radicals and chelate transition metal ions, thus preventing the involvement of the latter in free radical generation via the Fenton reaction Thus, the highest total content of phenols was observed in glycol-aqueous extracts from Stevia leaves, with lower phenol content in aqueous extracts and the lowest levels in ethanol extracts
- #31 Stevia derivative such as octa— acetylombuoside, ombuine and retusine were found to have antimicrobial action against few types of gram-positive bacteria Antibacterial activity of Stevia extracts in vitro against pathogenic bacteria such as Bacillus subtilis, Klebsiella pneumonia, Proteus vulgaris, Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas fluorescence. Six extracts from the leaves and three extracts from Stevia flowers obtained using of different solvents showed good antibacterial activity against all tested microorganisms
- #32 Gamboa and Chaves (2012) also studied the activity of different Stevia extracts against microorganisms that cause tooth decay. Extracts were obtained from dried S. rebaudiana leaves using hexane, methanol, ethanol, ethyl acetate or chloroform as solvents. The antimicrobial activity of these five extracts against 16 bacterial strains of the genera Streptococcus and Lactobacillus was evaluated by the well-known diffusion method.
- #34 Stevia derivative such as octa— acetylombuoside, ombuine and retusine were found to have antimicrobial action against few types of gram-positive bacteria For the four Lactobacillus species, the inhibition zones obtained between 12.3 and 17.3 mm were somewhat larger with ethyl acetate and chloroform extracts, suggesting that these bacteria were the most susceptible microorganisms. In another study, four groups of rats were fed with stevioside, rebaudioside A or sucrose, which were added to the main diet. There were significant differences in the rates of sulcal caries and Streptococcus sobrinus count between a group fed diet with 30 % sucrose and the other 3 groups. There were no differences between groups fed stevioside or rebaudioside A and groups without additives. Thus, it can be argued that neither stevioside nor rebaudioside A are cariogenic (Das et al., 1992)
- #43 SCF-Scientific committee on food (European commission) JECFA- Joint FAO/WHO Expert committee on food Additives 4mg/kg steviol eq. This compares with 40 mg/ kg for aspartame, 5 mg/ kg for saccharin and 15 mg/ kg sucralose. 4mg stevio eq.= 12 mg rebaudisode A US-FDA –Food and Drug Administration (GRAS- Generally recognized as safe) EFSA-European food safety authority US-FEMA- FSSAI- Food Safety and Standards Authority of India
- #46 NaN3, Sodium azide; 2-AF, 2-aminofluorene; 4NOPD, 4-nitro-o-phenylenediamine; MMC, mitomycin; DHAQ, 8-dihydroxyanthraquinone. Data were analyzed with ANONA followed by Dunnett's test for multiple comparison, all values from triplicate plates are expressed as mean ± S.D. *P < 0.05, significantly different as compared with the controls.
- #47 CPA, cyclophosphamide; PCE, polychromatic erythrocyte; RBC, red blood cells; MN, micronucleus. Five animals were treated in each group; MN incidence (MN/PCE) was analyzed with Fisher's exact test. *P < 0.05, significantly different as compared with the controls.
- #48 Sperm malformation rate was analyzed with Fisher's exact test. *P < 0.05, significantly different as compared with the control
- #50 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.
- #52 It was first evaluated by the European Commission’s Scientific Committee on Food (SCF) in 1985, 1999 and then in 2004 by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), which first established tentative purity specifications that were later made permanent In 2008, JECFA established a permanent acceptable daily intake (ADI) for SGs of 4 mg kg−1 body weight/day and validated its safety for use as a food and beverage sweetener. In 2008 and 2009, the Food and Drug Administration issued safe (GRAS: Generally Recognized As Safe) status to SGs (St and Rb-A) for use in food Later, in 2010 and 2011, the European Food Safety Authority (EFSA) declared SGs safe and established an ADI consistent with that of JECFA. Rb-A and St dry powders are very stable when stored in polyethylene bags at ambient temperature and controlled humidity for at least 3 years and 2 years, respectively The Food Safety and Standard Authority of India (FSSAI) in 2012 approved the use of SGs as an sweetener in various foods Thus stevia is globally safe for use as a natural sweetener in various food and beverages
- #54 Crystalline powders Numerous process for refining patented by various countries Dissolution of sweetener in boiling water/ any preferred solvent Ion exchange separation Filtration by coagulation of precipitation Crystallization Drying [SGs – Extraction, Bioconversion, Glycosylation, Fermentation] Intially methanol was used as a solvent for better separation and efficiency
- #55 Ingredion Incorporated is an American food and beverage ingredient provider based in Westchester, Illinois,[3] producing mainly starches, non-GMO sweeteners, stevia, and pea protein. (1906) customers in >120 countries Tate & Lyle PLC is a British-headquartered, global supplier of food and beverage ingredients to industrial markets. Archer Daniels Midland Company -commonly known as ADM, is an American multinational food processing and commodities trading corporation, founded in 1902 and headquartered in Chicago, Illinois Cargill, Incorporated, is a private, American global food corporation, Minnetonka, Minnesota world’s largest producer of stevia and inositol