Genotypic variability in colour pungency and antioxidant activity

Genotypic variability in colour, pungency and antioxidant activity among chilli genotypes Dr. V. K. Rao Indian Institute of Horticultural Research, Bangalore, India

Introduction 1. It is a major spice crop in India and some other countries 2. It is used for its pungency, flavour and colour. 3. India – worlds largest producer, consumer and exporter of chillies 4. Apart from India, other major countries producing chillies are South Africa, China, Pakistan and Mexico. 5. It is grown in almost all parts of India under various climatic conditions. 6. Different types of chillies are grown to be used for different purposes. High pungent, low pungent, high colour value ones etc. 7. India exports chillies in the form of dry chillies, dry powder, pickled chillies and chilli oleoresins.

Usage 1. It is used in fresh green state, fermented state, dry red powder. 2. Other products include pickles, sauce and paste 3. Paprika color – natural red colour - colouring substance in food industry 4. Oleoresin – For flavour and colour in food industry

Variability in Chillies Size, colour, shape, pungency, flavour, texture etc.

Objectives 1. To study the extent of variability in pungency, colour, antioxidant activity, vitamin C and total phenols among hybrids / varieties developed at IIHR as well as in different varieties collected from other places. 2. To know the relation between pungency and other parameters in chilli hybrids / varieties. 3. To know the extent of contribution of each parameter towards variability through principal component analysis. 4. To know the extent of variability among the samples analyzed through hierarchical clustering.

Plant Material 1 2 3 4 Arka Khyati Arka Meghana Arka Swetha Arka Haritha 5 6 7 Arka Abhir Arka Suphal Arka Lohit 8 Andaman collection 9 10 Bhut Jalokia Local Toom 11 Kerala Collection 12 Naga Chilli 13 Habanero Hybrid OP variety Natural cross of chinense vs frutescence ----do--- wastern ghats collection chinence grown in Nagaland exotic variety

Parameters studied 1. Pungency – by estimating capsaicinoids by HPLC (SHU units obtained by multiplication with 16. 1 million) 2. ASTA colour value – Spectrophometric method 3. Vitamin C – Titrimetric method by using 2, 6 -dichlorophenol indophenol 4. Total phenols - spectrophotometric assay 5. DPPH radical scavenging assay – spectrophotometric assay 6. FRAP antioxidant capacity assay - spectrophotometric assay

HPLC method for capsaicinoids analysis 1. Column : Onyx monolithic C 18, 100 x 4. 6 mm 2. Method: Gradient 3. Mobile phase: Water : Acetonitrile 4. Detection: 205 nm 5. Flow: 1. 8 ml/min 6. Injection: 20 µl 7. Std levels: 7 8. LOD: 0. 01 µg 9. LOQ: 0. 03 µg S. No. Time Acetonitrile % 1 0 – 8 minutes 30 % to 78 % 2 8. 01 – 12 minutes 78 %

Chromatogram of capsaicinoid standards

Capsaicinoids – pungency compounds Capsaicin, Dihydrocapsaicin – account for 90 -96 % of capsaicinoids

Total Capsaicinoids in Chilli Varieties S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 Variety Total SHU Capsaicin Dihydro CP/DC capsaicinoids % capsaicin mg/g % Arka Khyati 0. 1959 3295 66. 6 34. 4 1. 9 Arka Meghana 2. 111 33988 66 34 1. 94 Arka Swetha 1. 362 21928 65. 8 34. 2 1. 92 Arka Haritha 4. 1708 67151 61. 1 38. 9 1. 57 Arka Abhir 0. 1926 3101 60. 4 39. 6 1. 52 Arka Suphal 0. 4742 7636 66. 5 33. 5 1. 98 Arka Lohit 1. 9225 30954 62. 4 37. 6 1. 66 Andaman collection 40. 9646 941901 73. 9 26. 1 2. 83 Bhut Jalokia 27. 7312 493330 84. 1 15. 9 5. 29 Local Toom 6. 4686 104145 76. 9 23. 1 3. 32 Kerala Collection 8. 5754 141821 73. 1 26. 9 2. 72 Naga Chilli 29. 0245 623269 77. 8 22. 4 3. 51 Habanero 11. 6403 243932 74. 9 25. 1 2. 98 Large variation in pungency levels (3101 to 9. 4 million SHU) was observed. CP/DC ratio also varied from 1. 52 to 5. 29.

Variation in CP/DC ratio with pungency level CP/DC 6 5 R 2 = 0. 3656 CP/DC 4 3 CP/DC 2 1 0 0 100000 200000 300000 400000 500000 SHU 600000 700000 800000 900000 1000000

Total phenols and pungency S. No Variety Pungency (SHU) Total phenols (mg/g) 1 2 3 4 5 6 7 8 Arka Khyati Arka Meghana Arka Swetha Arka Haritha Arka Abhir Arka Suphal Arka Lohit Andaman collection 3295 33988 21928 67151 3101 7636 30954 941901 8. 4 9 8. 7 7. 2 9. 3 33 9 10 11 Bhut Jalokia Local Toom Kerala Collection 493330 104145 141821 23 14 11 12 13 Naga Chilli Habanero 623269 243932 22 19

Total phenols and pungency 35 R 2 = 0. 9464 Total phenols mg/g 30 25 20 15 10 5 0 0 200000 400000 SHU 600000 800000 1000000

Free radicals in human body

Function of dietary antioxidants Phytochemicals / antioxidants in diet Neutralize free radicals Prevent metabolic disorders / delay the onset of symptoms

Antioxidant capacity S. No Variety DPPH scavenging FRAP antioxidant activity (mg/g capacity (mg/g AEAC units) 1 2 3 4 5 6 7 8 3. 74 4. 30 4. 93 3. 39 2. 76 4. 09 35. 15 4. 26 5. 88 6. 69 3. 90 3. 39 5. 12 51. 26 9 10 11 Arka Khyati Arka Meghana Arka Swetha Arka Haritha Arka Abhir Arka Suphal Arka Lohit Andaman collection Bhut Jalokia Local Toom Kerala Collection 19. 92 8. 88 6. 79 26. 75 11. 93 9. 24 12 13 Naga Chilli Habanero 24. 39 13. 35 32. 71 31. 86

Comparison of antioxidant levels among varieties 60. 00 Activity mg/g 50. 00 40. 00 30. 00 DPPH FRAP 20. 00 10. 00 1 2 3 4 5 6 7 varities 8 9 10 11 12 13

Carotenoid pigments in chillies A. Red carotenoids capsanthin capsorubin

B. Yellow carotenoids zeaxanthin antheraxanthin mutatoxanthin Β-cryptoxanthin

Distribution of red and yellow carotenoids S. No. 1 2 3 4 5 6 7 Variety Red yellow Total Ratio carotenoids corotenoids (Red/Yellow) (mg/100 g) mg/100 g 147. 0 119. 6 130. 8 80. 7 154. 8 125. 7 87. 3 109. 7 94. 9 145. 7 65. 5 113. 1 98. 7 38. 8 256. 7 214. 5 276. 4 146. 1 267. 9 227. 8 126. 2 1. 3 0. 9 1. 2 1. 4 1. 3 2. 2 8 9 10 Arka Khyati Arka Meghana Arka Swetha Arka Haritha Arka Abhir Arka Suphal Arka Lohit Andaman collection Bhut Jalokia Local Toom 45. 9 40. 8 28. 8 30. 0 94. 7 75. 9 135. 5 1. 6 1. 5 0. 4 11 12 13 Kerala Collection Naga Chilli Habanero 46. 2 56. 4 4. 1 36. 2 33. 3 2. 7 82. 4 89. 8 6. 9 1. 3 1. 7 1. 5

Comparative levels of red and yellow carotenoids 180. 0 160. 0 140. 0 Red carotenoids (mg/100 g) 120. 0 100. 0 yellow carotenoids (mg/100 g) 80. 0 60. 0 40. 0 20. 0 1 2 3 4 5 6 7 Varieties 8 9 10 11 12 13

Total carotenoids and pungency S. No. Variety Total ASTA colour Pungency corotenoids value (SHU) mg/100 g 1 2 Arka Khyati Arka Meghana 256. 74 214. 48 98 82 3295 33988 3 4 Arka Swetha Arka Haritha 276. 43 146. 12 106 56 21928 67151 5 Arka Abhir 267. 88 103 3101 6 Arka Suphal 227. 77 87 7636 7 Arka Lohit Andaman collection Bhut Jalokia Local Toom Kerala Collection Naga Chilli Habanero 126. 16 48 30954 74. 72 75. 88 135. 49 82. 37 89. 77 6. 87 30 30 53 32 35 3 941901 493330 104145 141821 623269 243932 8 9 10 11 12 13

Relation between total carotenoids and pungency 1000000 800000 600000 SHU 400000 Pungency (SHU) 200000 0 0 -200000 50 100 150 200 Total Carotenoids mg/100 g 250 300

Vitamin Chilli varieties S. No. Variety Vitamin C Pungency mg/100 g SHU FW 80000 1 2 3 4 5 6 7 Arka Khyati Arka Meghana Arka Swetha Arka Haritha Arka Abhir Arka Suphal Arka Lohit 77 3295 89. 5 113 100 172 94 179. 5 33988 21928 67151 3101 7636 30954 Pungency SHU 70000 60000 50000 40000 30000 20000 10000 0 0 50 100 Vitamin C mg / 100 g 150 Pungency VS vitamin C 200

Principal component Analysis Principal Component Plot 1. 5 1. 0 Andaman collection ASTA colour value A Swetha A Abhir A Khyati PCA 2 (10. 3%) 0. 5 Naga chilli 0. 0 DPPH Antioxidant activity mg/ Pungency (SHU) g Total phenols mg/ g DW FRAP Antioxidant activity mg/ g DW A Suphal A Meghana Bhut jalokia Local toom A Harita -0. 5 A Lohit -1. 0 kerala collection Habanero -1. 5 -6 -4 -2 0 PCA 1 (88. 6%) 2 4

Hierarchical Clustering Naga Chilli Bhut Jalokia Arka Lohit Arka Meghana Arka Swetha Arka Abhir Arka Khyati Arka Suphal Arka Haritha Local Toom Kerala Collection Habanero Andaman collection 0 Hierarchical Clustering 111685399953 Distance/Similarity Measure = Squared Euclidean Distance Cluster Method = Nearest Neighbour

CONCLUSIONS 1. Large variation in pungency levels (3101 to 9. 4 million SHU) was observed. CP/DC ratio also varied from 1. 52 to 5. 29 2. Total phenols correlated well with pungency levels. 3. Some of the varieties showed high antioxidant capacities as well as radical scavenging activities. Highly pungent varieties showed more antioxidant capacity. 4. Red capsaicinoids were more (up to 2. 2 times) when compared to yellow ones except in one variety (local toom). 5. ASTA colour values varied widely from 3 to 106 among the varieties. It appears that there is a negative correlation between colour values and pungency. 6. Some of the varieties possessed high vitamin C content (179 mg/100 g FW) 7. Three distinct groups have appeared in PCA analysis indicating their similarity. PCA biplot indicates that values of all the parameters are positively correlated to each other except colour value. 8. Heirarchical clustering indicated that andaman collection is chemically different from all other varieties.

THANK YOU