Phyto pharmaceutical - TOCOPHEROLS AND TOCOTRIENOLS (Vitamin E )

Vitamin E (tocopherol) is a naturally occurring antioxidant. Biochemical functions of vitamin E. applications of vitamin E. symptoms of vitamin E deficiency. Global scenario of production and consumption of natural vitamin E and mixed tocopherols

1. PHYTO-PHARMACEUTICAL Submitted To: Dr. Sujani kamble Department of Pharmacognosy Government college of pharmacy Bangalore Presented by : Sudhindra Kini Department of Pharmacognosy Government college of pharmacy Bangalore Topic :- Tocopherol and Tocotrienols 1

2. Contents Introduction History Chemistry Properties Characteristic Feature Dietary sources Isolation of Tocopherol and Tocotrienols Estimation of Tocopherol and Tocotrienols Biochemical Use Health Benefit Pharmaceutical use Deficiency symptoms Current market scenario for vitamin E 2

3. Introduction Vitamin E is the name given to a group of tocopherols and tocotrienols. Vitamin E (tocopherol and tocotrienol) is a naturally occurring antioxidant. Greek : tokos -child birth; pheros -to bear; ol -alcohol It is essential for normal reproduction in many animals. Hence known as Fertility vitamin . 3

4. History Vitamin E was discovered by Herbert McLean Evans and Katharine Scott Bishop(1922). It was isolated first time in pure form by Gladys Anderson Emerson, (1935) at the University of California, Berkeley . Tocopherol was isolated from wheat germ in 1936 The first use for vitamin E was as a therapeutic agent by Widenbauer (1938). 4

5. Chemistry About eight vitamin E have been identified – α, β, ɤ, δ Tocopherol – α , β, ɤ, δ T ocotrienol The Basic Nucleus called the Chroman ( tocol ) nucleus . Chromane ring is attached to Phytyl tail Phytyl tail is composed of 3-units of isoprenoid . The position of methyl groups located on the chroman nucleus varies in tocopherols and tocotrienols. The antioxidant property is due to the –OH group of chromane ring. 5

6. Tocopherol Chemistry 6 α-Tocopherol --- R 1 =R 2 =R 3 = CH 3 β- Tocopherol --- R 1 =R 3 = CH 3 R 2 =H ɤ-Tocopherol --- R 1 =R 2 = CH 3 R 3 = H δ- Tocopherol --- R 1 =R 2 =R 3 = H

7. Tocotrienols Chemistry 7 α- Tocotrienols --- R 1 =R 2 =R 3 = CH 3 β- Tocotrienols --- R 1 =R 3 = CH 3 R 2 = H ɤ- Tocotrienols --- R 1 =R 2 = CH 3 R 3 = H δ- Tocotrienols--- R 1 =R 2 =R 3 = H R2

8. Properties Form :- Transparent , V iscous , Oily liquids. Odour :- Characteristic odour Colour :- Light yellow to reddish brown. Solubility :- Insoluble in water soluble in organic solvents Density :- 0.950 g/cu cm Molecular Formula: C 29 H 50 O 2 The colour may change gradually into dark brown when exposed to direct air or light. 8

9. Characteristic Features : Tocotrienols seem to cause inhibition of the growth of breast cancerous cells, whereas the tocopherols fail to do so . The biological functionalities of tocotrienols and tocopherols are quite different . Biological activity of tocopherols resembles to that of Vitamin E . Tocotrienols have explicitely shown their distinct cholesterol-lowering effect. 9

10. α-Tocopherol is most Bioactive compound. ɤ-Tocopherol are most abundant compounds in diet. The US-FDA considers only α-Tocopherol as Vitamin E. α- Tocotrienols are 50 times potent than Tocopherol in antioxidant activity. 10

11. Dietary sources Almond (Prunus amygdalus Batsch) Amaranth oil ( Amaranthus cruentus L.) Avocado (Persea americana Mill.) Canola (Brassica rapa L.) Cashew Nut (Anacardium occidentale L.) Castor (Ricinus communis L.) Coconut (Cocos nucifera L.) Corn or maize (Zea mays L.) Cotton seed (Gossypium herbaceum L.) Cumin (Cuminum cyminum L .) Flax seed (Linum usitatissimum 11 Groundnut (Arachis hypogea) Mustard (Brassica juncea L.) Neem (Azadirachta indica A. Juss.) Olive (Olea europaea L.) Palm (Elaeis guineensis Jacq.) Psyllium (Plantago ovata Forssk.) Pumpkin (Cucurbita pepo L.) Rice bran (Oryza sativa L.) Sesame (Sesamum indicum L.) Soybean (Glycine max L.) Sunflower (Helianthus annuus)

12. Dietary sources 12

13. Vitamin E content in oil and fats 13

14. Isolation of Tocopherol and Tocotrienol Physical distillation for concentration and purification of tocopherol Chemical process for concentration and purification of tocopherol Enzymatic reactions process for purification of tocopherol Isolation of tocopherols and tocotrienols from a variety of natural sources by semi-preparative high performance liquid chromatography Purification of tocopherol with super critical CO2 ( SC-CO2) extraction method 14

15. Physical method Step 1 :- Sample Autoclave Splitting ( 5 Kg ) 190- 250 C Step 2 :- Distillation Collect Bottom product (205-210 C) ( 1 torr vacuum) Step 3 :- Distillation Tocopherols and Tocotrienols Mixture (230-245 C) 15 Distillation HPLC or column chromatography

16. Chemical method :- 16 Sample (Soya bean) 0.8 Part of MeOH + 9% NaOH Pellets 10% H 2 SO 4 Reflux for 2 hrs @ 65 C Reflux for 2 hrs @ 65 C Wash with Hot water (pH – Neutral ) Distillate (Methyl esters) Dried at 80 -85 C Distillation (210 -220 C , 1Torr) Residue (Tocopherol & tocotrienols) Distillation (230 C , 100-200 Torr) Distillate (Phytosterols ) Residue Tocopherol and tocotrienols

17. Enzymatic Method Here enzyme and glycerine are used for isolation. Enzyme act as catalyst. Glycerine act as solvent. Phytosterols are convereted to sterol esters Free fatty acids are removed by distillation. Mainly BTSA Enzyme are used 17

18. Procedure :- Step 1 :- 300 gms + 5% BTSA + Glycerine Reaction Temperature 64- 65 C Sample Step 2 :- Filtration Filtrate Mother Liquor (Enzymes) wash (pH Neutral) Dried Material Step 3 :- Dried Material Distillation 18

19. Isolation by Semi-Preparative High Performance Liquid Chromatography Chemical, Apparatus and Reagent :- HPLC grade solvents 1. 40% V/V Tetrahydrofuran 2. 100%V/V hexane L-Ascorbic acid Sample For extraction Column :- 10 μ Alltech Econosil silica Guard column :- 40 μ supeleco Pellicular Silica Collection :- PTFE Bottles Detector :- Fluorescence Detector 19

20. Isolation 20 Filter 200 ml ethanol 5g Ascorbic acid 30ml of water Wash n-hexane layer with water Filter via Na2SO4 Rotary Evaporator Removes KOH 10ml methanol Separate n-hexane layer Sample in Erlenmeyer's Flask 50 ml n-Hexane (3x) Saponification (80 °C) 1.2 ml KOH, Mix 60°C 10 Min Cover the mouth Residue Vitamin E Evaporate 2X – 20 ml n-Hexane Filtrate + 20ml water Supernant liq is Filtered Centrifugation ( 20 ° C)

21. Purification Sample injection Mobile phase 1 . 40% V/V Tetrahydrofuran 2. 100%V/V hexane Flow rate – 8-9 ml Column :- 10 μ Alltech Econosil silica Detector : - Fluorescence Detector Order of Elution α-Tocopherol - α- Tocotrienols - β- Tocopherol- ɤ- Tocotrienols - ɤ-Tocopherol β- Tocotrienols - δ- Tocopherol - δ- Tocotrienols 21

22. 5. Super Critical Extraction Method Super critical carbon dioxide extraction was conducted at two pressure levels 200 bar and 220 bar and temperature 40 C and 60 C. The objective was to increase the concentration by removing maximum fatty acid methyl esters. Specific level of pressure and temperature of extraction caused the increase in the solubility Procedure The raw material was loaded in reactor vessel at room temperature. Extraction was started only when the temperature was applied. CO 2 was fed by adjusting the initial pressure . Different temperatures viz. 40 C, 60 C and 80 C were taken at various combinations of pressure at 150,180 and 200 bar. DOD was chemically esterified for the trials to convert all the available fatty acid to methyl esters. The reaction time was adjusted for 1 hr . . 22

23. UV Absorption Spectra :- a. For Tocopherols b . For Tocotrienols :- 23 Compound λ max α-Tocopherol 292 β- Tocopherol 296 ɤ-Tocopherol 298 δ- Tocopherol 298 Compound λmax α- Tocotrienols 292 β- Tocotrienols 294 ɤ- Tocotrienols 296 δ- Tocotrienols 297

24. UV Absorption Spectra :- 24

25. GC-MS- 25

26. 26

27. Estimation by HPLC :- INTRODUCTION The estimation of vitamins E helps in obtaining information as to the existence of an avitaminosis . Apparatus (a) Separation column, Shodex SIL-5B, 4.6 mm I.D. × 250 mm, or equivalent column (b) Column temperature - 30 °C (c) Mobile phase, n-hexane-isopropanol (100:1, v/v) (Both HPLC grade) (d) Flow rate, 1.0 mL/min (e) Detection, UV 300 nm 27

28. Preparation of standard solutions for calibration curve (1) Tocopherol standard stock solution 25 mg of α-, β-, γ-, and δ-tocopherols into a beaker and dissolve in n-hexane. Transfer it with n-hexane into a 100-mL brown volumetric flask. Dilute to volume with n-hexane. (2) Internal standard solution 25 mg of 2, 2, 5, 7, 8-pentametyl-6-hydroxychroman or 2-metyl-2-phytyl-6-hydroxychroman into a beaker and dissolve in n-hexane. Transfer it with n-hexane to a 100-mL brown volumetric flask. Dilute to volume with n-hexane. 28

29. Standard solutions for calibration curve Using whole pipets, add 1, 2, 3 and 5 mL of the tocopherol standard stock solution transfer to 20-mL glass stoppered-Erlenmeyer flask. To each of the flasks, add 1 mL of the internal standard solution Dilute with n-hexane to about 10 mL each Preparation of Test solution Accurately weigh a proper quantity of test sample into a 20-m L glass stoppered-Erlenmeyer flask. Add 1 mL of the internal standard solution to the flask using a whole pipette. Dilute with n-hexane to about 10 mL . Filter the solution through a membrane filter of a 0.45 μm pore-size. Use the filtrate as test solution for HPLC analysis. 29

30. Preparation of calibration curve Inject 20 μL each of the standard solutions prepared into HPLC. From the obtained chromatograms , measure the peak areas of the tocopherols and the internal standard substance. Draw the calibration curves of the tocopherols by plotting the weight ratio ( Wx / Ws ) of the tocopherols ( Wx ) to the internal standard substance ( Ws ) against the peak area ratio ( Ax /As) of the tocopherols ( Ax ) to the internal standard substance (As).   Determination of tocopherols in test solution Inject 20 μL of the test solution prepared in into HPLC. Calculate the peak area ratios of the tocopherols to the internal standard substance based on the peak areas of each substance from the obtained chromatogram, and convert them to weight ratios. Calculate the contents of each of the tocopherols in the test sample using the following formula: Round off fractions to the second decimal place. 30

31. % Each tocopherols = ( S x1000 Where - Wx / Ws : Weight ratio of a tocopherol to the internal standard substance Ms: weight of the internal standard substance contained in 1 mL of internal standard solution (mg ) S: Sample weight (g)   31 ×100

32. Absorption, transport and storage :- Vitamin E is absorbed along with fat in the small intestine. Bile salts are necessary for the absorption . In the liver, it is incorporated into lipoproteins (VLDL and LDL) and transported. Vitamin E is stored in adipose tissue, liver and muscle. The normal plasma level of tocopherol is less than 1 mg/dl. 32

33. The Biochemical functions of vitamin E E ssential for the membrane structure and integrity of the cell. Prevents the peroxidation of polyunsaturated fatty acids in various tissues and membranes. Protects RBC from haemolysis by oxidizing agents. Increases the synthesis of heme by enhancing the activity of enzymes δ- aminolevulinic acid (ALA) synthase and ALA dehydratase . R equired for cellular respiration through ETC . 33

34. Required for proper storage of creatine in skeletal muscle. Optimal absorption of amino acids from the intestine. Proper synthesis of nucleic acids . Vitamin E has been recommended for the prevention of chronic diseases such as cancer and heart diseases Protects liver from being damaged by toxic compounds 34

35. Medical Benefit   Vitamin E as an antioxidant. Vitamin E used in the treatment of cancer. Prevention of Heart Disease. Vitamin E as Natural Neuroprotective Vitamins. 35

36. Vitamin E as an antioxidant Antioxidants help in eliminating free radicals ( ROS & RNS). The antioxidant properties of this compound is responsible for promoting blood circulation to the scalp. Antioxidant plays a role in anti-inflammatory processes, inhibiting platelet aggregation and increasing immunity. 36

37. Vitamin E used in the treatment of cancer Vitamin E helps in protecting constituents of cells from the detrimental effects of free radicals , which is a major contributor in development of cancer. Initiate Apoptosis pathway by activation of caspases. A ffects on PI 3 K/PDK/ Akt Mitogenic Signaling . Vitamin E has antiproliferative activity by blocking EGF dependent mitogensis. Vitamin E also offers the potential of blocking the creation of carcinogenic nitrosamines. Vitamin E has property of DNA Polymerase Inhibition. 37

38. Prevention of Heart Disease 38

39. Lipid Peroxidation Antioxidants play an important role in slowing the process of atherosclerosis, especially by preventing oxidation of LDL. Stroke-Induced Injuries Oral supplementation of the tocotrienol complex acts on key molecular checkpoints 39

40. Vitamin E as Natural Neuroprotective Vitamins vitamin E is offering protection against accumulation of free radicals, contributing to cognitive decline and neurodegenerative diseases e.g. Alzheimer's disease. Maintenance of cognitive performance or slowing its decline in the process of normal aging. 40

41. Health Benefit Vitamin E in the Potential Treatment of Infectious Disease. Eg :- Chlamydia pneumonia Counter measure against Radiological Threat. vitamin E oil is beneficial for the skin’s healing process.   Vitamin E used in age-related muscular degeneration (AMD)- oxidative stress have a major role to play in these disorders Vitamin E used as a dietary supplement 41

42. Pharmaceutical use Vitamin E in its synthetic form is currently being utilized by many pharma companies in their multivitamin offerings. Natural forms of vitamin E as mixed tocopherols can be utilized in an array of cosmetic formulations for checking oxidation. 42

43. Tocopherol Use 43

44. 44 Tocopherol Use

45. 45 Tocotrienols Use

46. 46

47. Deficiency symptoms The symptoms of vitamin E deficiency vary from one animal species to another. Sterility D egenerative changes in muscle M egaloblastic anaemia Increased fragility of erythrocytes M inor neurological symptoms. 47

48. Global scenario of production and consumption of vitamin E Vitamin E - 13.6 MT @ 2014 - 18.1 MT @ 2020 Value - 574.1 million $ @ 2012 - 1.05 Billion $ @ 2020 Animal feed - 18 MT @ 2012 - 30 MT @ 2020 Cosmetics - Beverages and Food - 48 7 .3% Annual Growth Rate 7.8% Annual Growth Rate 6.6% Annual Growth Rate 9.9% Annual Growth Rate 7.7% Annual Growth Rate

49. Reference Tocotrienols Vitamin E Beyond Tocopherols, Ronald Ross Watson Victor R. Preedy , CRC Press, 2 nd edition . Trease and Evans pharmacognosy ,16th edition, W.B saunders Edinburg New-York . Biochemistry, 3 rd Edition, Dr. U. Satyanarayana Dr. U. Chakrapani Elsevier Publication Textbook of pharmacognosy and phytochemistry by vinod D. rangarl part-1 career publication, Nasik India . Identification, isolation, P urification and Quantification of mixed tocopherols and natural vitamin E from various plant sources, Khamar , Ripal R, Department of Botany, Jasrai , Yogesh T, Saurashtra University. 49

50. Standardization of Botanicals by V. Rajpal . Isolation of four tocopherols and four tocotrienols from a variety of natural sources by semi-preparative high performance liquid chromatography, Tai-Sun Shin, J. Samuel Godber Department of Food Science, Louisiana Agricultural Experimental Station, Louisiana State University Agricultural Center . Sunflower Oils-Reactivity and Assay Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva . Quantitative Analysis of Vitamin E in Vegetable Oils, Japan Customs Analysis Methods, Updated in June 2003 (JCAM No.107-R3 ). Sangha , J. K. (2014): Nutraceuticals and functional foods - an innovative approach for management of lifestyle diseases. 50

51. Thank-You 51