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1. CHEMICAL AND BIOLOGICAL EVALUATION OF TITHONIA DIVERSIFOLIA AND TEPHROSIA VOGELII FROM UGANDA AS SOURCES OF ANTI-OXIDANT AND PESTICIDE PhD Progress Presentation By Nasifu Kerebba Supervisor : Prof. O . Oyedeji Co-supervisors : Prof. A. Oyedeji Prof. R. Byamukama Dr. S. K. Kuria 2019/08/31 1

2. Outline 2 Introduction and Literature review Background Flavonoids’ participation in plants’ interaction with Insects Some Pesticidal activities of Tithonia diversifolia and Tephrosia vogelii Phytochemical Studies on T. diversifolia and T. vogelii Research Problem Objectives Methodology Collection of Plant Materials and Extraction C ompare the pesticidal potential of the essential oils of T.diversifolia and T.vogelii against S. zeamais . Investigate the chemical variation in components and composition of T. vogelii essential oils and implications in control against S. zeamais Chemical evaluation of feeding deterrence activity of T. diversifolia non volatile and volatile substances against S. zeamais Evaluation of antioxidant properties of non-volatile samples of T.diversifolia . Acknowledgement 2019/08/31 2

3. Introduction and Literature review 3 Background Botanical plants used in Medicinal, Biological, Chemical and Agricultural. In Agric., e ffects of Pests and Diseases on food production leading to loss on farm & market inspired their use. Pests and diseases cause 35 % Loss of Agric. Crops on the field and 14% in storage 1 ≈ 50% a total loss every year. Losses in the field occur with field pests while in storage by grain storage pests. Pest is a destructive insect or any animal that attacks crops, food, livestock e.t.c. 2 Okwute , S.K., 2012. Plants as Potential Sources of Pesticidal Agents: A Review . Chapter 9 Alcorn ,G., Rutherford, P., 2005 Environment Protection Authority, “EPA Guidelines for: Responsible Pesticide Use, pp. 1–74 . 2019/08/31 3

4. During their attack they tend to destroy food crops and damage the livestock . As a result pest control is a fundamental aspect for food security 2 . Figure 2 : Pest damaged crops Source : http://theselfsufficientliving.com/15-garden-pests-natural-ways-control/ 2019/08/31 4

5. Synthetic pesticides used as the conventional method of pest management. Effective, Less distributed in rural areas, costly, toxic and pose a serious impact on food safety systems 2 , human health 3 . 2. Malhat , F.M., Haggag , M.N. and Loutfy , N.M., 2015. Residues of Organochlorines and Synthetic Pyrethroid Pesticides in Honey, an Indicator of Ambient Environment, a Pilot Study. Chemosphere ; 120: 457-461. 3. Roca , M., Miralles -Marco, A. and Ferré , J., 2014. Biomonitoring Exposure Assessment to Contemporary Pesticides in a School Children Population of Spain. Environmental Research , 131: 77-85. 2019/08/31 5

6. Calls for more research in less eco-toxic pesticides. Cont … 6 Reports in Africa indicate that extracts of local plants can be effective as crop protectants against pre-harvest and post-harvest pests 4 Compounds rapidly decompose, environmental friendly 5 Naturally at low levels with a diverse active ingredients Repellency or anti- feedant mode of action 6 More than 2000 plant species contain toxic principles; effective against insects. In Africa; Tephrosia vogelii , Lantana camara , Tagetes spp., Cypressus spp., Nicotiana tabacum , Musa spp., Moringa oleifera , lippia javanica , Tithonia diversifolia , Phytolacca dodecandra , Azadirachta indica , Aloe spp., Eucalyptus spp., Vernonia amygdalina , Capsicum frutescens , Warbugia stuhlmannii 5,6 have shown efficacy against pests in addition to other traditional uses 4. Khater , H.F., 2012. Prospects of botanical biopesticides in insect pest management. Pharmacologia 3(12): 641-656 5. Grzywacz , D., Stevenson, P.C., Mushobozi , W.L., Belmain , S. and Wilson, K., 2014. The Use of Indigenous Ecological Resources for Pest Control in Africa. Food Security , 6:71-86 6. Mkenda , P., Mwanauta , R., Stevenson, P.C., Ndakidemi , P., Mtei , K. and Belmain , S.R., 2015.Extracts from field margin weeds provide economically viable and environmentally benign pest control compared to synthetic pesticides, PLoS ONE ; 10e0143530 2019/08/31 6

7. Azadiracta indica Lantana camara Vernonia amygdalina Aloe ferox Nicotiana tabacum Source: World agroforestry center: Species database. 2015. Accessed April, 2017 7 2019/08/31 7

8. Cont … 8 In some parts of Uganda, (Victoria basin), smallholder farmers use: T. diversifolia , T. vogelii , L . camara , Aloe spp., N. tabacum , V. amygdalina , A. indica , Tagetes spp., Musa spp., Capsicum frutescens ,, M . oleifera , Cypressus spp., P. dodecandra , Eucalyptus spp ., 7 Tithonia diversifolia , 8 Tephrosia vogelii 8 7. Mugisha-Kamatenesi , M., Deng, A. L., Ogendo , J. O., Omolo , E. O., Mihale , M. J., Otim , M., Buyungo , J. P. and Bett , P. K., 2008. Indigenous Knowledge of Field Insect Pests and Their Management around Lake Victoria Basin in Uganda. African Journal of Environmental Science and Technology ; 2: 342-348. 8 . World agroforestry center: Species database. 2015. Accessed April 2017 2019/08/31 8

9. 9 T. diversifolia ; known as wild sunflower or tree marigold in the Asteraceae family mainly used i n Uganda for field and for storage pest mgt 9 T. vogelii ( Family ; Leguminosae ) commonly known as fish poison, largely used to control field pests rather than storage pests. Need to understand their phytochemical composition during their biological and Agricultural uses 10 such as pesticides, Phytotoxins et.c Gaps in the chemistry of biological activity may occur when efficacy is considered while using crude extracts of the botanicals instead of the real bioactive compound e.g. T. diversifolia Secondly when plant exhibits Chemical variability 9 . Mwine J, Damme Pvan , Kamoga G, Kudamba , Nasuuna M. and Jumba F, 2011. Ethnobotanical survey of pesticidal plants used in South Uganda: case study of Masaka district. Journal of Medicinal Plants Research, 5(7): 1155-1163 10. Bisht S.S. and Kamal, R., 1994. Garlic extract: An antifungal treatment for the control of storage of apple. Proceeding of the National Academy of Sciences India 64:  233-234 2019/08/31 9

10. Some phytochemicals are known for insecticidal and antioxidant actions: 10 toxic principles against pests such as isoflavonoids . imposing behavioral responses to insect pests: repellence, feeding deterrence, growth-regulating potentials and oviposition deterrence 11 ROS-detoxifying capacities- flavonoids and sesquiterpene lactones, giving them antioxidant potentials. Moreover flavonoids participate in plants’ interaction with animals (insects ) due to strong antioxidant activities. Calls for research to establish specific compounds; thus s tudy focusses on the: chemical basis of the previously reported biological activities (pesticidal and anti-oxidant) for T. diversifolia and a sses the potential T. vogelii essential oils in pest control. 11. Torres, P.J., Avila, G., de Vivar , A.R., Garcıa, A.M., Marín , J.C., Aranda, E. and Céspedes , C.L., 2003. Antioxidant and insect growth regulatory activities of stilbenes and extracts from Yucca periculosa . Phytochemistry ; 64: 463–473 . 2019/08/31 10

11. 11 Flavonoids’ participation in plants’ interaction with I nsects The strong antioxidant potential of flavonoids makes them possess many biological activities including the insecticidal/ pesticidal activities P lant interact with other organism owing to the diverse chemical structures of flavonoids and varieties. Flavonoids are one of the chemicals released by plants for protection against natural predators by regulating the ovipositing and feeding behaviour of the predator/ insects. For example Naringenin , hesperetin-7-O-rutinoside and quercetin-3-O-rutinoside, induce oviposition in citrus feeding swallowtail butterfly Papilio xuthus 23 which have exhibited strong antioxidant potentials. 23. Ohsugi , T.; Nishida, R.; Fukami , H. Oviposition stimulant of Papilio xuthus , a Citrus feeding swallowtail butterfly. Agric. Biol. Chem. 1985, 49, 1897–1900. 2019/08/31 11

12. Natural antioxidants and antioxidant Potential 12 Two categories of natural anti-oxidants: water-soluble and lipid soluble anti-oxidants. Water soluble includes vitamin C (e.g. ascorbic acid) and phenolic compounds such as flavonoids, tannins, hydroxycinnamate esters and lignin . V itamin E and carotenoids are Lipid soluble antioxidants. The antioxidant capacity of phenolics is due to their ability to scavenge reactive oxygen species (ROS) as result of their electron donating properties and reaction as hydrogen donors 24 In vitro studies have shown that phenolic compounds demonstrate higher antioxidant activity than the tocopherols and ascorbate 25 24. Rice-Evans , C., Sampson, J., Bramley, P. M., and Holloway, D. E., 1997.Why do we expect carotenoids to be antioxidants in vivo. Free Radical Research, 26, 381–398. 25. Re , R., Pellegrini, N., Proteggente , A., Pannala , A., Yang, M., & Rice- Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 26(9/10), 1231–1237 . 2019/08/31 12

13. Pesticidal Activities of T. diversifolia and T.vogelii 13 Used on aphids and beetles 6 , red spider mite Oligonychus coffeae , 13 Aphis sp ., Crocidolomia binotalis , Ophiomyia phaseoli and a borer species 14 , S. zeamais adults in corn grains 15 , cowpea seed bruchid 16 T.vogelii has been used; controlling bruchids in beans and cowpeas 17 , Megalurothrips sjostedti and Apion varium on cowpeas 18 , aphids ( Brevicoryne brassicae L .) 19 , ticks and worms 20 . 13. Radhakrishnan , B. and Prabhakaran , P., 2014. Biocidal activity of certain indigenous plant extracts against red spider mite, Oligonychus coffeae ( Nietner ) infesting tea, Journal of Biopesticide ; 7: 29-34. 14. Moreno R. G., 1991. Utilization of wild sunflower ( Tithonia diversifolia A. Gray) leaf extract as an insecticide against selected vegetable insect pests. 15 Tavares , W. S., Faroni , L R D., Freitas, S. S., Ribeiro, P. E. A., Fouad H.A. and Zanuncio J.C., 2014. Effects of Astilbin from Dimorphandra mollis ( Fabaceae ) Flowers and Brazilian Plant Extracts on Sitophilus zeamais ( Coleoptera : Curculionidae ). Florida Entomologist ; 97(3): 892-901 16. Adedire C. O. and Akinneye , J. O., 2004. Biological activity of tree Marigold, Tithonia diversifolia , on cowpea seed bruchid , Callosobruchus maculatus ( Coleoptera : Bruchidae ). Annals of Applied Biology ; 144: 185–189 . 17. Adebayo , T. A, Olanira , A.O. and Akambi , W.B., 2007. Control of insect of cowpea in the field allelochems of Tephrosia vogelii and Petiveria alliaceae in South Guinea savannah of Nigeria. Agricultural Journal ; 2(3): 365-369 . 18. Alao F, Adebayo T. and Olaniran O., 2012. On-farm evaluation of natural toxicants from Tephrosia vogelii and Petiveria alliacea on Megalurothrips sjostedti and Apion varium of cowpea ( Vigna Unguiculata (L) Walp ). Bangladesh Journal of Agricultural Research ; 36(4):575-582 . 19. Mudzingua , S., Muzemu , S. and Chitamba , J., 2013. Pesticidal efficacy of crude aqueous extracts of Tephrosia vogelii L., Allium sativum L. and Solanum incanum in controlling aphids ( Brevicaryne brassicae L.) in rape ( Brassica napus L.). Journal of Research in Agriculture ; 2(1):157-163 . 20. Matovu , H. and Olila , D., 2007. Acaricidal activity of Tephrosia vogelii extracts on nymph and adult ticks. International Journal of Tropical Medicine ; 2: 83-88 . 2019/08/31 13

14. Phytochemical Studies and isolated compounds of T. diversifolia 14 S esquiterpenoids , diterpenoids , flavonoids and chlorogenic acids derivatives 21 Sesquiterpene lactones, the most abundant terpenoids and some isolated: Tirotundin-3- O -methyl ether ( 1 ), 1 β -hydroxytirotundin-3- O -methyl ether ( 2 ), tagitinin A ( 3 ), deacetylvguiestin ( 4 ), 1 β -hydroxydiversifolin-3- O -methyl ether ( 5 ), 1 β -hydroxytirotundin-1,3- O -dimethyl ether ( 6 ), tagitinin F-3- O -methyl ether ( 7 ), tagitinin F ( 8 ), tagitinin C ( 9 ), tagitinin F-3- O -methyl ether ( 10 ), 3-methoxytirotundin ( 11 ), 8 β - O -(2-methylbutyroyl)- tirotundin ( 12 ), 8 β - O -( isovaleroyl )- tirotundin ( 13 ) 21. Chagas-Paula , D. A.; Oliveira, R. B.; Rocha, B. A. and Da Costa, F.B., 2012. Ethnobotany, chemistry, and biological activities of the genus Tithonia ( Asteraceae ). Chemistry and Biodiversity, 9(2):210-235. 2019/08/31 14

15. Phytochemical studies and isolated bioactive C pds of T.vogelii 15 F lavonoid glycosides and flavonoid aglycones detected including rotenoids , Revealing two chemical varieties of T. vogelii 22 22. Stevenson , P.C., Kite, G.C. and Lewis, G.P., 2012. Distinct Chemotypes of Tephrosia Vogelii and Implications for Their Use in Pest Control and Soil Enrichment. Phytochemistry , 78: 135-146. 2019/08/31 15

16. 2019/08/31 16

17. Research Problem 17 Lack of specific information about specific compounds responsible for pesticidal and anti-oxidant activities of T. diversifolia limits its adoption for use in biological activities. Since m ost bioassays have been conducted using its crude extracts and a few of its fractions Little progress in developing new products. Chemical variability of T.vogeli i species can compromise its efficacy as a botanical pesticide resulting in farmers reporting no pesticidal activity . 2019/08/31 17

18. Previous research shows existence of chemotypes with in non-volatile extracts of T. vogelii leaf materials which have serious implications on pesticidal use but there is no such report that shows existence of chemical varieties within the components of the essential oils of T. vogelii and thereof implications on pesticidal use 2019/08/31 18

19. Hypotheses 19 E ssential oils components e.g monoterpenoids would deter oviposition and cause mortality of maize weevil T. diversifolia extract, fractions , essential oils and isolated compounds possess antioxidant potentials Essential oils of T. diversifolia and T. vogelii are good insecticidals against weevils Synergetic effect would occur in extracts of T. diversifolia thus extracts would possess more biological activities than pure isolated compounds Some Sesquiterpene lactones and diterpenoids of T.diversifolia ; would deter, repel and cause toxicity to Sitophilus zeamais Motschulsky Essential oils and extracts of T. diversifolia are good antifeedants against maize weevil Essential oils of T. diversifolia and T. vogelii would differ in their modes of action against the maize weevil 2019/08/31 19

20. Justification and significance 20 C hemical variability study is critical in assessing the potential of plants for pest control and avoiding variable efficacy For example absence of deguelin and rotenone among 25% of sampled T.vogelii materials from 13 different locations in Malawi resulted in two distinct chemotypes being proposed i.e. pesticidal and non pesticidal 26 In T. diversifolia , active compounds responsible for pesticidal and anti-oxidant activities enables exploitation of the plant Lastly , it is necessary to find out whether biological activities are due to multiple components in botanical extracts . Study of antioxidant and pesticide activities is motivated by ability of some phytochemicals to participate in plants interaction with animals/ pests 2019/08/31 20

21. Objectives 21 Main objective To evaluate chemically the pesticidal and antioxidant activities of T.diversifolia and investigate chemotypes in T . vogelii essential oils from Eastern Uganda and thus implication on pest control . Specific objectives 2019/08/31 21

22. Cont … 22 To evaluate and compare the pesticidal potential of the essential oils of T.diversifolia and T.vogelii against S. zeamais . To investigate the chemical variation in components and composition of T. vogelii essential oils and implications in control against S. zeamais iii ) Chemical evaluation of feeding deterrence activity of T. diversifolia non volatile and volatile substances against S. zeamais iv) Evaluation of antioxidant properties of non-volatile samples of T.diversifolia . 2019/08/31 22

23. Methods and Materials Botanical identification and Collection of plant materials 23 Plants were collected from Butaleja and Kampala districts in Uganda Identification by a senior Botanist Rwaburindori Protase at the Department of Botany, Makerere University. The voucher specimens were deposited at Makerere University Herbalium and the voucher numbers are: Kerebba N. No 1- Tephrosia vogelii Hook. f. ( Leguminosae )(MHU 50735), Kerebba N. No 2- Tithonia diversifolia . (Hems) (MHU 50733) 2019/08/31 23

24. 24 Research Approach 2019/08/31 24

25. Compararative toxicity and repellent activities of the essential oils of T.diversifolia and T.vogelii against S. zeamais . Experimental scheme: 2019/08/31 25

26. Rearing of insects The insects to be used in the bioassays were obtained from infected maize from the market. Plastic containers (flasks) were used to bleed colonies of the maize weevils. In each container containing maize ( Zea mays L.), were added adult maize weevil to lay eggs at 30 ± 1 °C, 60 ± 5% RH and a photo period of 12:12 dark : light. Biological Studies 2019/08/31 26

27. To avoid any previous infestation, the grain were washed and dried in a stove at 25 °C for 12 h and then frozen at 4.0 ± 1 °C for 48 h prior to its use. The old adult insects were then removed from the flasks after 5 days to allow emergence of the new generation. After 7 days, the first generation of weevils of the same age were considered for the bioassay. Unsexed adult weevils that were used in all the experiments will be about one to two weeks old. 2019/08/31 27

28. The contact toxicity, fumigant toxicity and repellence bioassays were carried out against the reared maize weevils. The experiment was set up in a completely radomised design (CRD) which enabled replicates to be obtained at the same environmental conditions and time Figure 5: Set up of a repellence bioassay Biological Studies 2019/08/31 28

29. Oils of concentration of 1, 5,10, 20 and 4 μ L dissolved in 1mL of Hexane was used to determine the effects For repellence test, the concentrations used were 1, 5 and 10 μ L while for contact and fumigant toxicity the test concentrations were 10, 20 and 40 μ L Figure 6 : Set up of a contact toxicity bioassay 2019/08/31 29

30. Figure 7 : Set up of fumigant toxicity bioassay 2019/08/31 30

31. Extraction Hydro distillation of essential oils yielded yellow distillate of 0.18mL/kg dry weight for T. diversifolia and 0.3mL/kg dry weight of T. vogelii . Bioassays : Repellency , Contact toxicity and fumigant toxicity Results and Discussion 2019/08/31 31 Weevils generally significantly preferred the control treatment in a choice repellence bioassay (P ˂ 0.05,Fisher’s LSD test ). T. vogelii whose effect was dose-dependent was more repellent at 10 μ L/mL of essential oil (0.31 μ L/cm3); showing Class2 repellence (Percentage repellency (PR) = (20.1- 40%).

32. 2019/08/31 32 Class 0: PR = 0.01-0.1%; Class 1: PR = 0.1-20%; Class 2: PR = 20.1-40%; Class 3: PR - 40.1-60%; Class 4: PR = 60.1-80%, and Class 5: PR = 80.1-100% 23 G. Juliana and H.C.F. Su, Laboratory studies on several plant materials as insect repellents for protection of cereal grains. Journal of Economic Entomology, 76, 154-157 (1983).

33. 1 μL /mL essential oil treatment of T. vogelii (TV1) exhibited class 1 repellence, while the same treatment level for T. diversifolia (TD1) was of class 2 . A 5μL/mL T. vogelii (TV5) treatment showed class 1 just like that of T. diversifolia (TD5). Lastly, 10μL/mL T. vogelii treatment (TV10) showed class 2 whereas the same concentration of the oil for T. diversifolia (TD10) was class 1 T. vogelii whose effect was dose-dependent was more repellent at 10 μ L/mL of essential oil (0.31 μ L/cm3); showing Class2 repellence (Percentage repellency (PR) = (20.1- 40%). 2019/08/31 33

34. 2019/08/31 34 Preference index of T. diversifolia essential oils against S. zeamais Preference index of T. vogelii essential oils against S. zeamais The preference index (PI) for T. vogelii was entirely repellent in all treatments while that of T. diversifolia would be either repellent or attractive thus T. vogelii essential oil is a better repellent of the two plants

35. 2. Contact toxicity Time( hr ) of exposure   10μL/mL 0.3μL/g 20μL/mL 0.5 μL /g 40μL/mL 1.0 μL /g Control 0.0 μL /g Regression equation LC 50 μL /g LC 95 μL /g 48 TD 1.1±1.9a 3.3±1.9a 4.4±1.1a 0.0±0.0a y= 1.8+x 2.3×10 3 1.2×10 5 TV 4.4±1.1a 4.4±2.9a 3.3±1.9a   y= 3.5-0.2x 1.0×10 -7 4.8×10 -15 60 TD 1.1±1.1a 3.3±1.9a 4.4±1.1a 1.1±1.1a y= 1.8+x 2.3×10 3 1.2×10 5 TV 5.6±1.1a 4.4±2.9a 3.3±1.9a   y= 4.0-0.6x 1.4×10 -2 1.5×10 -5 72 TD 2.2±1.9a 6.7±1.9ab 5.6±2.2a 1.1±1.1a y= 2.2+0.8x 2.2×10 3 2.1×10 5 TV 6.7±1.9ab 4.4±2.9a 3.3±1.9a   y= 4.2-0.7x 5.3×10 -2 1.8×10 -4 84 TD 2.2±1.9a 6.7±1.9ab 5.6±2.2a 1.1±1.1a y= 2.2+0.8x 2.2×10 3 2.1×10 5 TV 6.7±1.9ab 5.6±4.0a 3.3±1.9a   y= 4.2-0.7x 6.8×10 -2 2.2×10 -4 96 TD 5.6±2.9a 8.9±1.1b 7.8±2.2b 1.1±1.1a y= 3.3+0.2x 3.7×10 7 5.0×10 14 TV 7.8±2.9b 6.7±5.1ab 4.4±1.1a   y= 4.2-0.6x 2.9×10 1 5.0×10 -5       P ˃ F   < 0,0001             Sig.   Yes       2019/08/31 35

36. Time( hr )   10 μL /mL (20 μL /L ) 20 μL /mL (40 μL /L) 40 μL /mL (80 μL /L) Control (0 μL/L) Regression equation LC50 ( μL /L) 6 TD 17.8±2.9c 30.0±1.9c 46.7±7.7ab 0.0±0.0d y = 2.7 +1.4x 45.4 TV 4.4±4.4c 10.0±6.9c 1.1±1.1d y = 0.5 –1.2x 2.2×10 -4 12 TD 30.0±2.9c 57.8±10.6ab 70.0±15.8ab 1.1±1.1d y = 2.8 +1.7x 18.2 TV 5.6±4.0c 24.4±4.8c 16.7±1.9c y = -2.2 +1.7x 1.5×10 -4 24 TD 37.8±9.5b 63.3±9.6ab 75.6±12.2a 1.1±1.1d y = 3.0 +1.7x 14.5 TV 10±5.1c 48.9±4.0ab 42.2±13.1ab y = 2.2 +1.8x 38.6 36 TD 44.9±10.4b 84.2±8.1a 91.0±4.9a 1.1±1.1d y = 2.6 +2.4x 10.0 TV 14.4±6.8c 60.0±3.3ab 51.1±15.7ab y = 2.3 +1.8x 27.9 48 TD 50.6±14.4ab 87.6±6.8a 95.5±2.9a 1.1±1.1d y =2.3 +2.9x 9.0 TV 15.6±5.9c 64.4±4.0ab 55.6±14.4ab y = 2.4 +1.9x 24.6 60 TD 58.4±14.8ab 93.3±3.8a 98.9±1.1a 1.1±1.1d y = 1.7 +3.5x 8.5 TV 17.8±6.2c 66.7±3.3ab 57.8±12.2ab y = 2.5 +1.9x 22.6 72 TD 65.3±14.7ab 95.5±2.9a 98.9±1.1a 1.1±1.1d y = 2.3 +3.2x 6.9 TV 20.0±8.4c 70.0±3.8ab 57.8±12.2ab y = 2.7 +1.7x 21.1 84 TD 68.7±14.5ab 95.5±2.9a 98.9±1.1a 2.3±1.1d y = 2.6 +3.0x 6.3 TV 29.2±19.1b 77.8±4.0a 73.3±15.0a y = 2.8 +1.9x 14.3 96 TD 72.1±16.5ab 95.5±2.9a 98.9±1.1a 6.9±5.3c y = 2.8 +2.9x 5.8   TV 36.8±21.6b 81.0±3.9a 78.9±14.5a   y = 3.0 +1.9x 11.6       P ˃ F < 0,0001             Sig yes       Fumigant toxicity 2019/08/31 36

37. 2019/08/31 37 Probit analysis showed higher fumigant efficacy of T. diversifolia against S. zeamais LC 50 : 5.84 μ L/L versus LC 50 11.57 μ L/L for T. vogelii ; with nearly 100% mortality on the 60th hour against 57.8±21.2% for T. vogelii . T . vogelii , exhibited the highest contact toxicity, LC 50 28.6 μ L /g compared to LC 50 3.67×107 μ L/g for T. diversifolia at 96th hr

38. Group T. diversifolia T. vogelii Monoterpene hydrocarbons 76.2 2.7 Oxygenated monoterpenes 0.3 3.0 Sesquiterpene hydrocarbons 9.0 28.4 Oxygenated sesquiterpenes 5.3 8.1 Diterpene 0.5 0.7 Other hydrocarbons 7.9 47.5 Total 99.2 90.2 Gas chromatography (GC)–mass spectrometry and GC analysis indicated that monoterpenoids (76.5% composition) were the most predominant chemical constituents from T. diversifolia oil compared to xylene compounds in the T. vogelii essential oil. 2019/08/31 38

39. 2. Investigation of the chemical variation in components and composition of Tephrosia vogelii Essential oils and pesticidal activity against Sitophilus zeamais Study Area The study took place in Butaleja district in Eastern Uganda. Two study sites were considered for the study; Nampologoma Parish, Mazima sub-county , muyago village and Kyadongo parish ( between 33° 55′ to 34° 05′ E and 0° 50′ to 1° 00′ N), altitude -1050m to 1100m above mean sea level, bimodal rain fall peaks are; March to May and August- September 24 2019/08/31 39

40. 2019/08/31 40 Plant Materials The fresh samples of Tephrosia vogelii plant species was collected from Muyago and Kyadongo villages To determine potential geographic and seasonal variations in the occurrence of T. vogelii chemotypes , plant material were collected with in two major seasonal rainfall patterns in the district (March -May, June- July and August- September 2017)

41. Sample Collection     Location     Village (sample) Flower color Latitude North Longitude East Sampling date Altitude Muyago (TVm 1a ) (TV1 muya) White 0°84′20″ 34°03′15″ 14/05/2017 1080m KyadongoB (TVk 1a ) ( TV1 kya) White 0°90′14″ 34°08′30″ 1/06/2017 1098m Kyadongo B(TVk 1b ) (TV1 kyb) White 0°90′30″ 34°09′45″ 1/06/2017 1098m Kyadongo B(TVk 1c ) (TV1 kyc) White 0°80′10″ 34°08′40″ 1/06/2017 1098m Muyago(TVm 2a ) (TV2 muya) white 0°84′14.9″ 34°03′10″ 15/08/2017 1080m Kyadongo B(TVk 2a ) (TV2 kya) white 0°70′30″ 34°07′35″ 15/08/2017 1098m Kyadongo B(TVk 2b ) (TV2 kyb ) white 0°90′14″ 34°08′30″ 15/08/2017 1098m Kyadongo B(TVk 2c ) (TV2 kyc) white 0°90′20″ 34°09′40″ 15/08/2017 1098m Kyadongo B(TV 2d ) (TV2 kyd) white 0°90′35″ 34°08′45″ 15/08/2017 1098m Muyago(TVm 3a ) (TV3 muya) white 0°84′00″ 34°02′45″ 01/03/2018 1090m Muyago(TVm 3b ) (TV3 muyb) white 0°84′14″ 34°03′09″ 01/03/2018 1090m Muyago(TVm 3c ) (TV3 muyc) white 0°84′14.9″ 34°03′10″ 01/03/2018 1090m Kyadongo B(TVk 3a ) (TV3 kya) white 0°90′14″ 34°08′30″ 01/03/2018 1098m Kyadongo B(TVk 4a ) (TV4 kya) white 0°70′30″ 34°07′35″ 10/01/2019 1098m Kyadongo B(TV 4b ) (TV4 kyb) white 0°90′14″ 34°08′30″ 10/01/2019 1098m Kyadongo B(TV 4c ) (TV4 kyc) white 0°90′20″ 34°09′40″ 10/01/2019 1098m Muyago(TVm 4a ) (TV4 muya) white 0°84′00″ 34°02′45″ 10/01/2019 1090m Muyago(TVm 4b ) (TV4 muyb) white 0°84′14″ 34°03′09″ 10/01/2019 1090m Muyago(TVm 4c ) (TV4 muyc) white 0°84′14.9″ 34°03′10″ 10/01/2019 1090m Subscript; 1&2 is rain season sampling while 3&4 is dry season, TV = Tephrosia vogelii 2019/08/31 41

42. Extraction and analysis of essential oils Plant leaf materials (20g) was subjected to hydro-distillation set-up using Clevenger apparatus and I μ L of oil samples and standards were injected in the GC-MS. Synthetic standards were used in quantification: Myrcene , (±)-linalool, ethylbenzene, 2-undecanone, o-xylene, (-)-linalool, sabinene hydrate, p-cymene and R-(+)- limonene and a mixture of farnesol isomers, n- decane Cyclohexanone α - Terpeneol A mixture of xylene isomers α- pinene 2019/08/31 42

43. Results and discussion Analysis of the composition and comonents of various samples, shows two chemotypes being revealed: Some samples have farnesol / farnesal or farnesol isomer; Chemotype 1 (TV1kyb, TV1kyc, TV1 muya , TV2muya, TV3muyb, TV3muyc, TV4kyc) O thers were predominant with springene compounds (either β- springene or α- springene ; TV1kya, TV2kya, TV2kyb , TV2kyb, TV2kyc,TV3muya , TV4muyA,TV4muyb , TV3muyc, TV3kya ) Chemotype 2. β- springene was the most represented in this category. All the samples however had the aromatic hydrocarbons; ethylbenze and xylene isomers as the predominant compounds with no any clear 2019/08/31 43

44. distinction between their compositions. Farnesol or farnesal is an oxygenated sesquiterpene with various isomers ; (E,Z)-Farnesol, (Z,E)-Farnesol and (E,E)-Farnesol. Springene is an isoprenoid hydrocarbon of diterpene nature β- springene and α- springene ( E,E)-β-springene, is a diterpene homologue of (E)-β-farnesene (3). 2019/08/31 44

45. P esticidal Evaluation of the chemotypes of the oils of T.vogelii Time( hr ) control Sample 10µl/ml 20µl/ml 40µl/ml   6 0.0±0.0b TV4 Kyc 4.4±4.4ef 5.67±6.9j 1.1±1.1de       TV4 muya 0.0±0.0f 7.41±1.4j 3.4±0.0de   12 1.1±1.1ab TV4 Kyc 5.53±4.0def 21.60±4.8i 16.7±1.9de       TV4 muya 11.5±3.0cdef 31.98±4.2hi 34.5±0.0cd   24 1.1±1.1ab TV4 Kyc 10±5.1def 48.9±4.0fg 42.2±13.1bcd       TV4 muya 18.4±5.0cdef 41.4±2.8gh 37.9±0.0bcd   36 1.1±1.1ab TV4 Kyc 14.4±6.8cdef 60.0±3.3def 51.1±15.7abc       TV4 muya 23.0±8.0bcdef 50.0±7.0fg 44.8±2.2abcd   48 1.1±1.1ab TV4 Kyc 15.6±5.9cdef 64.4±4.0de 55.6±14.4abc       TV4 muya 31.0±11.9abcdef 51.7±8.4fg 44.8±2.2abcd   60 1.1±1.1ab TV4 Kyc 17.8±5.9cdef 66.7±3.3bcd 57.8±12.2abc       TV4 muya 37.9±13.9abcd 53.4±7.0efg 67.2±1.4abc   72 1.1±1.1ab TV4 Kyc 20.0±8.4cdef 70.0±3.8abcd 57.8±12.2abc       TV4 muya 43.7±14.9abc 60.3±1.4def 44.48±1.4bcd   84 2.3±1.1ab TV4 Kyc 29.2±19.1bcdef 77.8±4.0ab 73.3±15.0ab       TV4 muya 52.9±15.9ab 69.0±8.4cd 77.6±4.2ab   96 6.9±5.3a TV4 Kyc 36.8±21.6abcde 81.0±3.9a 78.9±14.5a       TV4 muya 63.2±18.5a 75.0±6.9abc 87.9±8.6a   2019/08/31 45

46.   control 10µl/ml 20µl/ml 40µl/ml P ˃ F 0.49   0.017 ˂0.0001 0.001 F 0.98   2.31 29.86 3.55 Sig. No   Yes Yes Yes LSD 6.00   36.70 11.33 34.21 R 2 (model) 0.30   0.52 0.95 0.68 Fumigant toxicity(mortality) due to TV4 Kyc ( Farnesol chemotype ) and TV4 muya ( Springene chemotype ) 2019/08/31 46

47. 3. Feeding deterrence activity of Tithonia diversifolia non volatile and volatile substances against S.zeamais Methodology a) Extraction, fractionation and isolation of non-volatile substances The leaf materials of T.diversifolia was extracted in methanol after defatting in Petroleum ether. The Petroleum ether fraction was then re-dissolved in ethanol to extract the terpenoids and added to the methanol extract of the extracted black syrup. The syrup will be re-suspended in different percentages of aqueous methanol to give several fractions i.e. 80%, 70%, 60%, 100%, 0% MeOH /H2O for fractions F1, F2, F3, F4, F5 and then be distributed with Petroleum ether which constitute fraction F6. 2019/08/31 47

48. Dissolving F5 in 100% and filtering on whatman paper, gave a pure compound NK1F5 (10.83g ) after recrystallizing in methanol after solvent evaporation under reduced pressure. F3 was purified on a secondary column to give NK1F3. F4 was partitioned in a column over dichloromethane: methanol (5:0, 5:1, 5:2, 5:4, 4:6, 2:8, 1:9, 0:1) to give compounds NK1F4, NK2F4, NK4F4 and NK5F4 and F4 1 ; purified on a column using Chloroform:ethylacetate (100:0, 75:25, 55:45) to give compound NK3F4 . The active fractions and compounds are to be identified using FTIR, MS and NMR. 2019/08/31 48

49. b) Extraction of volatile substances Plant leaf materials were subjected to hydro-distillation using Clevenger apparatus set up Preparation of discs for volatile assays Flour disks were prepared using the method by Xie et al 25 40g of flour was dissolved in 200ml of water. Disks were formed by dropping the suspension onto petri dishes using a micro pipette (200 μ l) and then allowed to dry over night. Figure 7 . Flour disks 2019/08/31 49 25. Xie , Y.S., Bodnaryk , R.P. and Fields, P.G., 1996. A rapid and simple flour-disk bioassay for testing substances active against stored-product insects. The Canadian Entomologist, 128(5), pp.865-875

50. Flour disks were treated with n-hexane solutions (5ml) containing different concentrations (0µl for controls, 5 µl, 10 µl and 20 µl) of T. diversifolia oil. After the solvents had dried the disks were placed on glass vials with 2.5cm diameter and 5.5cm dimension The disks with the vials will be weighed before the weevils are added and immediately after the weevils have been added. The reverse method of weighing of the glass vials with its contents will be finally done after 3 days. 2019/08/31 50

51. Feeding deterrence index (FDI) =[(C-T)/C] * 100, C and T are control and treated disc weight consumed by the insect respectively Preparation of discs to be used for non- volatile assays Disc were prepared following protocols set by Xie et al 25 10% w/w solutions will be prepared by dissolving 100mg of a non-volatile substance and a gram of flour in 5ml of water for all six fractions and isolated compounds. The suspensions will be then dropped onto petri dishes using micro pipette to form disks 2019/08/31 51

52. The formed disks were left in a fume hood to dry for a night. After the disks had dried they were placed in petri dishes of know mass and weighed with the petri dishes, after which 25 weevils were added and weighed again immediately. After 72hr, the weighing was repeated in the reverse fashion of the weighing at 0hr. The nutritional indices: Relative growth rate (RGR), Relative consumption rate(RCR), Efficiency of conversion of ingested food(ECI) and Feeding deterrence index (FDI ) 2019/08/31 52

53. Figure 9 : Feeding deterrence setup for the essential oils of T.diversifolia Figure 8 : Feeding deterrence setup for the non volatile substances of T.diversifolia 2019/08/31 53

54. RGR=( X-Y)/(Y ×t), X-Y= change in insect weight RCR=D /( Y×t ), ECI (%)=(RGR/RCR)* 100. FDI =[( C-T)/C] * 100 X = weight of live insects on the third day (mg)/no. of live insects on the third day, Y = original weight of insects (mg)/original no. of insects; weight of consumed disc, D=biomass ingested (mg)/no. of live insects on the third day, C and T are control and treated disc weight consumed by the insect respectively. t= feeding period (days), t=3 days 2019/08/31 54

55. Results and Discussion Test sample % w/w D(mg) X(mg) Y(mg) (RGR) mg/mg mean weight day-1 RCR mg/mg mean weight day-1 ECI% FDI*100 Control 0.61±0.17cdefg 1.96±0.18abcdefg 2.20±0.18cdefghi -0.04±0.01abcde 0.10±0.03cde -35.95±6.34abcdefg   Crude 10 0.10±0.02ghij 1.42±0.12hi 2.09±0.04defghi -0.11±0.02ef 0.01±0.00f - 22.73±40.91abcdefgh 5 0.46±0.05cdefghij 2.27±0.17a 2.53±0.09abcde -0.03±abc 0.06±def -6.28abcde 31.80±3.89abcdefgh 1 0.05±0.01hij 1.78±0.12abcdefghi 2.28±0.15cdefghi -0.07±bcdef 0.01±f - 92.52±1.62a   0.5 0.03±0.00ij 1.71±0.02cdefghi 2.07±0.10 defghi -0.06±0.02bcdefg 0.01±0.00f - 86.11±0.00ab F₁ 10 0.03±0.02ij 1.47±0.11hi 2.03±0.03defghi -0.09±0.01def 0.01±0.00f - 54.55±36.36abcdefg 5 0.35±0.08defghij 2.16±0.03abc 2.40±0.08bcdef -0.04±abcd 0.05±def -40.13bcdefgh 48.41±12.01abcdefg 1 1.87±0.02 a 2.02±0.10abcdefg 2.00±0.11defghi 0.00±ab 0.31±a 1.51abc - 15.08±43.72 defghij   0.5 0.48±0.08cdefghij 1.72±0.07cdefghi 2.04±0.04defghi -0.05±0.01abcde 0.08±0.01def -69.08±17.22fghi - 2019/08/31 55

56. F₂ 10 0.02±0.02j 1.60±0.18defghi 2.06±0.02defghi -0.08±0.03cdef 0.00±f - 72.73±27.27abcde 5 0.68±0.16bcdef 2.00±0.12abcdefg 2.34±0.06cdefghi -0.05±abcde 0.10±def -54.69efghi -4.59±31.80bcdefghi 1 0.92±0.40bc 1.71±0.07cdefghi 1.78±0.29i 0.00±ab 0.20±bc 14.42a 17.76±46.73abcdefgh   0.5 0.13±0.07fghij 1.69±0.12cdefghi 2.05±0.12defghi -0.06±0.00abcdef 0.02±0.01ef - 48.61±26.39abcdefg F₃ 10 0.12±0.03fghij 1.48±0.15hi 1.94±0.08efghi -0.08±0.02cdef 0.02±0.00ef - -50.00±31.82hij 5 0.03±0.01ij 2.03±0.07abcde 2.40±0.20bcdefgh -0.04±abcd 0.00f - 95.5±0.10a 1 0.24±0.05efghij 1.76±0.10abcdefghi 2.09±0.06defghi -0.05±abcde 0.02±ef - 18.66±11.01abcdefgh   0.5 0.59±0.03cdefghi 1.80±0.04abcdefghi 2.08±0.02defghi -0.05±0.00abcdef 0.10±0.00def -47.65±5.62bcdefgh -81.94±0.00ij 2019/08/31 56

57. F₄ 10 0.14±0.12fghij 1.33±0.03i 2.05±0.08defghi -0.12±0.01f 0.02±0.02ef - 76.64±19.26bcdefghi 5 0.25±0.22efghij 1.88±0.05abcdefgh 2.71±0.06abc -0.10±0.01ef 0.03±ef - 30.55±20.77abcdefgh 1 0.19±0.02fghij 1.86±0.12abcdefgh 2.33±0.04cdefghi -0.07±0.01bcdef 0.03±0.03ef - 46.38±4.95abcdefg   0.5 0.36±0.16cdefghij 1.62± defghi 2.18cdefghi -0.09def 0.06±def - 16.67±16.67abcdefgh F₅ 10 0.25±0.19efghij 1.68±0.22cdefghi 1.98±0.08efghi -0.05±0.03abcde 0.04±0.03def - 27.27±0.00abcdefgh 5 0.39±0.02cdefghij 2.10±0.11abcdef 2.60±0.01abcd -0.07±bcdef 0.05±def - 43.11±1.06abcdefg 1 0.29±0.08efghij 1.82±0.03abcdefghi 2.33±0.05cdefghi -0.07±0.00bcdef 0.04±0.03def - 46.11±13.49abcdefg   0.5 0.52±0.18cdefghij 1.73±0.03bcdefghi 2.08±0.05 defghi -0.06±0.00abcdef 0.08±0.03def -77.83±30.97ghi -12.50±34.72defghi 2019/08/31 57

58. F₆ 10 0.15±0.02fghij 2.16±0.07abc 2.97±0.09ab -0.09±0.00def 0.02±0.00ef - -100.00±0.00j 5 0.20±0.06fghij 2.06±0.06abcdef 2.41±0.08bcdefgh -0.05±abcde 0.03±ef - 67.49±12.04abcdef 1 1.18±0.34b 1.87±0.15abcdefgh 1.89±0.15fghi 0.00±ab 0.21±b -6.67abcde -7.86±34.35cdefghij   0.5 0.14±0.12fghij 1.56±0.10fghi 1.87±0.20fghi -0.05±0.01abcde 0.03±0.02ef -81.48±0.00hi 72.22±22.22abcde F₁₁ 10 0.34±0.08defghij 1.90±0.22abcdefgh 3.12±1.15a -0.11±0.06ef 0.05±0.03def - -18.18±0.00efghij 5 0.24±0.14efghij 2.09±0.01abcde 2.61±0.05abcd -0.07±bcdef 0.03±ef -99.17i 66.08±21.26abcdef 1 0.60±0.57cdefgh 1.74±0.08bcdefghi 2.02±0.28defghi -0.03±abc 0.09±def -64.64fghi 8.88±85.51abcdefgh   0.5 0.61±0.07cdefg 1.76±0.16abcdefghi 2.07±0.06 defghi -0.05±0.02abcd 0.10±0.01def -50.93±9.07defghi -22.22±27.78fghij 2019/08/31 58

59. NK5F4 10 0.04±0.00ij 1.66±0.09cdefghi 2.28±0.03cdefghi -0.09±def 0.01±f - 91.58±a   5 0.27±0.08efghij 1.57±0.09efghi 2.07±0.07defghi -0.08±cdef 0.04±def - 51.82abcdefg NK1F3 10 0.25±0.10efghij 1.68±0.04cdefghi 2.03±0.05defghi -0.06±abcdef 0.04±def -36.35abcdefgh 52.64abcdefg   5 0.32±0.11defghij 1.60±0.06defghi 1.94±0.01efghi -0.06±abcdef 0.06±def -1.34abcd 41.06abcdefgh   1 0.20±0.10fghij 1.60±0.02defghi 2.05±0.01defghi -0.07±bcdef 0.03±ef -4.28abcde 42.03abcdefgh   0.5 0.33±0.13defghij 1.65±0.05cdefghi 2.03±0.07defghi -0.06±abcdef 0.05±def -26.90abcdefg 42.24abcdefgh NK1F4 10 0.22±0.09fghij 2.10±0.75abcd 2.39±0.62bcdefgh - 0.04±0.02 abcd 0.03±ef -33.51abcdefgh 67.88abcdef   5 0.01±0.01j 1.77±0.19abcdefghi 2.08±0.63defghi -0.03±0.06abc 0.00±f - 97.03a   1 0.20±0.02fghij 1.89±0.15abcdefgh 2.36±0.47bcdefghi -0.07±bcdef 0.03±ef -2.32abcd 46.38abcdefgh   0.5 0.30±0.10defghij 1.89±0.00abcdefgh 2.12±0.00cdefghi -0.04±abcd 0.05±def -0.87abcd 21.01abcdefgh NK2F4 5 0.41cdefghij 1.62±0.15defghi 1.88±0.10fghi -0.05±abcde 0.07±def -47.97cdefghi 63.37abcdef   1 0.42cdefghij 1.74±0.14bcdefghi 2.06±0.05defghi -0.05±abcde 0.07±def -18.75abcdef 16.17abcdefgh   0.5 0.37cdefghij 1.66±0.09cdefghi 2.14±0.07cdefghi -0.08±cdef 0.06±def - 42.44abcdefgh 2019/08/31 59

60. NK3F4 0.1 0.10±0.04ghij 1.67±0.01cdefghi 1.94±0.09efghi -0.05±abcde 0.01±f - 81.19abc NK4F4 10 0.14±0.10fghij 1.56±0.03fghi 1.82±0.05ghi -0.05±abcde 0.03±ef -64.37fghi 69.31abcdef   5 0.19±0.11fghij 1.52±0.07ghi 2.00±0.06defghi -0.08±cdef 0.02±ef - 76.73abcd   0.5 0.86±0.00bcd 1.61±0.11defghi 1.81±0.19hi -0.03±abc 0.08±def 11.47ab -78.22ij NK1F5 10 0.67±0.17bcdef 1.87±0.39abcdefgh 1.87fghi 0.01±a 0.14±bcd 1.97abc 43.30abcdefg   5 0.25±0.08efghij 1.68±0.22cdefghi 2.22±0.11cdefghi -0.08±cdef 0.04±def -2.53abcd 16.43abcdefgh   1 0.29±0.08efghij 1.82±0.05abcdefghi 2.33±0.03cdefghi -0.07±0.00bcdef 0.04±def -1.79abcd 16.43abcdefgh   0.5 0.48±0.42cdefghij 1.62±0.00defghi 1.85±0.00fghi -0.04±abcd 0.09±def -4.04abcde -35.51ghij P ˃ F   ˂0.0001 0.016 0.006 0.032 ˂0.0001 ˂0.0001 0.022 F   3.28 1.67 1.82 1.56 2.81 2.81 1.67 Sig.   Yes Yes Yes Yes Yes Yes Yes LSD   0.31 0.29 0.37 0.04 0.05 0.06 54.55 R 2   0.67 0.50 0.52 0.48 0.62 0.63 0.54 2019/08/31 60

61. 2019/08/31 61 Concn μl /ml D(mg) X(mg) Y(mg) (RGR) mg/mg day-1 RCR mg/mg day-1 ECI% FDI*100 %tage dead 21.77±6.91a 2.92±0.04a 3.08±0.10a -0.02±0.01a 2.32±0.70a -0.70±0.34a   4.00±2.31a 5 20.97±1.45a 3.01±0.05a 3.23±0.12a -0.02±0.01a 2.18±0.21a -1.14±0.49a -41.38±71.90a 2.67±1.33a 10 18.70±1.47a 2.98±0.10a 3.08±0.10a -0.01±0.00c 2.02±0.15a -0.58±3.60a -20.64±55.80a 2.67±1.33a 20 12.03±3.02a 2.83±0.09a 3.11±0.24a -0.03±0.03a 1.35±0.40a -4.91±5.26a 13.49±49.29a 4.23±2.5a P ˃ F 0.4 0.407 0.874 0.939 0.431 0.628 0.813 0.903 F 1.3 1.09 0.23 0.13 1.03 0.61 0.22 0.19 Sig. No No No No No No No No Feeding deterrency activity of the Essential oil

62. 2019/08/31 62

63. Spectral studies of the isolated compounds 2019/08/31 63

64. 2019/08/31 64

65. 2019/08/31 65

66. 2019/08/31 66

67. 2019/08/31 67 Proton NMR for NK1F3

68. 2019/08/31 68 Proton NMR for NK1F4

69. 2019/08/31 69 COSY for NK1F4

70. 2019/08/31 70 NOESY for NK1F4

71. 2019/08/31 71 Proton NMR for NK3F4

72. 2019/08/31 72 C13 NMR for NK3F4

73. 2019/08/31 73 COSYfor NK3F4

74. 2019/08/31 74 NOESY for NK3F4

75. 2019/08/31 75 NMR DATA NK1F5 : 1 H NMR (400 MHz, D 2 O) δ 11.29, 7.32, 5.37, 4.76, 4.76, 4.70, 4.64, 4.64, 4.61, 4.60, 4.57, 4.24, 3.64, 3.48, 3.18, 2.41, 1.83, 1.69, 1.42, 1.21, 1.03, -3.83.   NK1F4: 1 H NMR (400 MHz, D 2 O) δ 7.38, 5.38, 4.82, 4.77, 4.76, 4.70, 4.64, 4.62, 4.60, 4.58, 4.25, 3.64, 3.27, 3.18, 2.42, 2.14, 1.83, 1.72, 1.43, 1.22, 1.04, -3.83. NK1F3: 1 H NMR (400 MHz, D 2 O) δ 10.62, 5.38, 4.82, 4.80, 4.79, 4.76, 4.76, 4.70, 4.64, 4.64, 4.60, 4.59, 4.57, 4.25, 3.65, 3.49, 3.27, 2.42, 2.15, 2.00, 1.83, 1.72, 1.43, 1.23, 1.04, -3.83. NK2F4: 1 H NMR (400 MHz, D 2 O) δ 7.12, 5.36, 4.76, 4.76, 4.70, 4.64, 4.64, 4.63, 4.62, 4.58, 4.23, 3.62, 3.48, 3.26, 3.08, 2.39, 2.14, 1.99, 1.82, 1.69, 1.41, 1.20, 1.02, -3.83. NK3F4: 1 H NMR (400 MHz, CDCl 3 ) δ 9.85, 8.01, 7.54, 7.34, 7.28, 7.22, 7.02, 5.46, 5.38, 5.32, 5.20, 5.18, 5.17, 5.14, 5.13, 5.07, 5.04, 5.03, 5.01, 4.84, 4.70, 4.68, 4.37, 4.17, 4.15, 4.14, 3.98, 3.78, 3.74, 3.58, 3.56, 3.55, 3.52, 2.68, 2.51, 2.33, 2.30, 2.25, 2.20, 2.13, 2.07, 2.03, 2.02, 1.88, 1.86, 1.71, 1.69, 1.68, 1.58, 1.47, 1.45, 1.44, 1.41, 1.39, 1.32, 1.30, 1.28, 1.24, 1.21, 1.18, 1.16, 1.13, 1.11, 1.08, 1.05, 1.03, 1.02, 0.99, 0.95, 0.91, 0.90, 0.90, 0.89, 0.88, 0.87, 0.84, 0.83, 0.81, 0.74, 0.72, 0.70, 0.69, 0.67, 0.57, 0.56, 0.09.   13 C NMR (101 MHz, CDCl 3 ) δ 77.35, 77.03, 76.71.

76. 2019/08/31 76 4. Antioxidant Potential of Tithonia diversifolia non-volatile and volatile extracts Methodology Extraction , fractionation and isolation of non-volatile substances The leaf materials of T.diversifolia was extracted in methanol after defatting in Petroleum ether. The Petroleum ether fraction was then re-dissolved in ethanol to extract the terpenoids and added to the methanol extract of the extracted black syrup. The syrup will be re-suspended in different percentages of aqueous methanol to give several fractions i.e. 80%, 70%, 60%, 100%, 0 %

77. 2019/08/31 77 MeOH /H2O for fractions F1, F2, F3, F4, F5 and then be distributed with Petroleum ether which constitute fraction F6 Dissolving F5 in 100% and filtering on whatman paper, gave a pure compound NK1F5 (10.83g) after recrystallizing in methanol after solvent evaporation under reduced pressure. F3 was purified on a secondary column to give NK1F3. F4 was partitioned in a column over dichloromethane: methanol (5:0, 5:1, 5:2, 5:4, 4:6, 2:8, 1:9, 0:1) to give compounds NK1F4, NK2F4, NK4F4 and NK5F4 and F4 1 ; purified on a column using Chloroform:ethylacetate (100:0, 75:25, 55:45) to give compound NK3F4.

78. 2019/08/31 78 F1 was chromatographed on silica gel 0.040–0.063 mm column using n-hexane/ EtOAc (in 10% increasing concentration of ethyl acetate from 0 to 100%) furnishing several fraction Active fractions contained 3 compounds ; CP8, CP 9 and CP 10 respectively. The active fractions will be purified using HPLC or Sephadex and the pure compound identified using FTIR, MS and NMR . The structural elucidation of the pure compounds are to be carried out by 1 H and 13 C NMR, 1D and 2D and the spectral data are compared with those from the literature

79. 2019/08/31 79

80. Biological Studies 80 Evaluation of Antioxidant activities DPPH qualitative and Quantitative assays DPPH scavenging ability (%) = [( Abscontrol – Abssamples )/ Abscontrol ] × 100 Abs control is absorbance of the control (freshly prepared DPPH solution in methanol). P ercentage of scavenging activity or inhibition was plotted against log concentration From graph, IC50 value was calculated by linear regression analysis. The extract concentration providing 50% inhibition (IC50) was calculated from the graph of scavenging effect percentage against extract concentration in the solution 2019/08/31 80

81. 2019/08/31 81 Reducing antioxidant power Assays determination The reducing property of test sample was standardized against ascorbic acid expressed as μg / mL. The reduing ability was calculated as percentage inhibition . Total phenol content (TPC) and Total flavonoid content (TFC) were used in evaluation of potential of extracts, fractions and isolates. TPC =(GAE×V×DF)/W Where GAE is Gallic acid equivalent concentration (mg/ml) in sample based on calibration curve V is volume of extract, DF is dilution factor, W is mass of sample used

82. 2019/08/31 82 TFC =(RFE×V×DF)/W Where RFE is Riboflavin equivalent concentration (mg/ml) in sample based on calibration curve V is volume of extract, DF is dilution factor, W is mass of sample used TPC and TFC to be correlated with total antioxidant potential in solvent extracts, fractions, isolates

83. 2019/08/31 83 Results and Discussion Sample Concentration Absorbances at 490 nm   %Scavanging Activity Crude 1.0mg/ml 0.054±0.001abcdef   58.96±13.48abcde   0.5mg/ml 0.065±0.003abcdef   43.43±18.56abcde   0.25mg/ml 0.06±0.005abcdef   51.89±3.43abcde Fraction 1 1.0mg/ml -0.003±0.005bcdef   100.90±2.81abcd   0.5mg/ml 0.008±0.003bcdef   92.46±5.18abcd   0.25mg/ml -0.019±0.010def   112.34±2.73abc Fraction 2 1.0mg/ml -0.024±0.002ef   119.51±7.38ab   0.5mg/ml -0.026±0.003ef   124.77±11.38a   0.25mg/ml -0.036±0.003ef   127.90±6.967a Fraction 3 1.0mg/ml 0.097±0.022abcdef   30.57±6.52bcde   0.5mg/ml 0.086±0.013abcdef   25.60±17.11cdef   0.25mg/ml 0.088±0.013abcdef   35.80±11.96abcde Fraction 4 1.0mg/ml 0.111±0.034abcdef   19.94±1.97cdef   0.5mg/ml 0.158±0.042abc   -29.24±13.94ef   0.25mg/ml 0.147±0.045abcd   -2.53±2.60ef 1 F5 1.0mg/ml 0.219±0.031a   -63.11±30.46f   0.5mg/ml 0.148±0.040abc   -22.41±13.11ef   0.25mg/ml 0.151±0.045abc   -4.70±3.50ef Fraction 6 1.0mg/ml 0.114±0.008abcdef   16.49±20.51def   0.5mg/ml 0.149±0.005abc   -33.61±45.14ef   0.25mg/ml 0.162±0.011ab   -20.35±31.31ef 1F4 1.0mg/ml 0.084±0.039abcdef   45.36±9.62abcde   0.5mg/ml 0.110±0.046abcdef   15.20±5.12def   0.25mg/ml 0.109±0.046abcdef   28.56±8.23bcdef Ascobic acid 1.0mg/ml -0.036±0.023ef   121.22±9.46ab   0.5mg/ml 0.004±0.004bcdef   94.53±4.96abcd   0.25mg/ml -0.045±0.021f   128.27±5.33a BHT 1.0mg/ml -0.037±0.034ef   119.87±17.72ab   0.5mg/ml -0.008±0.005cdef   106.06±1.57abcd   0.25mg/ml -0.003±0.043bcdef   92.80±31.78abcd

84. 2019/08/31 84 Reducing power assay The higher the absorbance, the stronger reducing power of the sample.

85. 2019/08/31 85 The reducing power of BHT was highest followed by ascorbic acid owing to higher absorbance. The samples exhibited concentration-dependent reducing power profile. The ferric reducing by the tested samples was in the decreasing order of BHT ˃ Ascobic acid ˃ 1F5 ˃ Fraction 4˃1F4˃Fraction1˃Fraction3˃ negativecontrol ˃Fraction2˃Crude ˃Fraction6 between 0.4mg/ml to about 1mg/ml.

86. 2019/08/31 86 The reducing power of Fraction 4 was highest, even more than that of the pure isolated compound 1F4 probably due to synergitic effects. The reducing power was expressed as ascorbic acid and BHT equivalents (1mg/ml).   Crude Fraction 1 Fraction 2 Fraction 3 Fraction 4 1F5 Fraction 6 1f4 Control Ferricyanide reducing power (AAE 1mg/ml) Abs Ascobic acid = -9.00E02+0.280*Concn, R 2 =0.959 0.41 (0.02) cd 0.59 (0.00) abc 0.23 (0.00) d 0.42 (0.04) cd 0.78 (0.05) a 0.69 (0.07) ab - 0.57 (0.05) bc 0.58 (0.12) bc Ferricyanide reducing power (BHT 1mg/ml) Abs BHT = -0.34+1,19*Concn, R 2 =0.952 0.30 (0.03) ab 0.36 (0.03) ab 0.22 (0.10) bc 0.31 (0.02) ab 0.39 (0.03) a 0.36 (0.03) a 0.18 (0.04) c 0.34 (0.03) ab 0.35 (0.04) ab

87. 2019/08/31 87 Sample Abs.(corrected) at 750nm TPC based on Calibration curve (mg/ml) TPC mg GAE/100g of extract/ compound Corrected Abs at 415nm TFC based on calibration curve TFC RFE/100g of sample/ compound Crude 0.024±0.020b 0.018±0.004a 353.33±67.66a 0.132± 0.015b     Fraction 1 -0.034±0.020d 0.008±0.004bc 153.33± 59.25bc 0.087 ±0.019b     Fraction 2 -0.007±0.021c 0.012±0.004abc 246.67± 65.66abc 0.133± 0.011b     Fraction 3 0.015±0.023b 0.016±0.004ab 326.67± 65.66ab 0.114 ±0.035b     Fraction 4 0.041±0.025a 0.021±0.005a 413.33± 70.55a 0.107 ±0.002b     Pure 1F5 -0.033±0.021d 0.008±0.004bc 160.00±52.92bc 0.097± 0.009b     Fraction 6 0.018±0.026b 0.017±0.005ab 333.33± 70.55ab 0.297± 0.024a     Pure 1F4 -0.063±0.004e 0.004±0.001c 80.00± 34.64c 0.097± 0.023b     P ˃ F ˂0.0001 0.016 0.016 < 0.0001     F 101.036 3.577 3.577 19.056     Sig. Yes Yes Yes Yes     LSD 0.011 0.009 185.618 4.896     R 2 (model) 0.978 0.610 0.610 0.893     Abs. is absorbance, Total phenol content (TPC) calibration curve, Y = -0.076+5.617× X, Y= Galic acid Absorbance , X= Concentration) Determination of Total phenol and Total Flavonoid Content

88. 2019/08/31 88 Total phenol calibration curve Total flavonoid calibration curve

89. Statistical analysis ANOVA followed by post hoc tests were performed using XLSTAT software, 2018 Results were expressed as the mean ± SEM (standard error of the mean). P values lower than 0.05 were considered significant Conclusion It is therefore, anticipated that this study will lead to isolation of lead molecules to facilitate future isolation for commercial purposes At least 5 publications will be made with 4 publications before April 2020 89 2019/08/31 89

90. Acknowledgement 90 Special thanks to God Almighty Prof . O. O. Oyedeji , Prof. A . Oyedeji , Prof. R. Byamukama and Dr . S. K. Kuria NRF/TWAS, GMRDC;UFH and DRD; WSU for Funding. University of Fort Hare Walter Sisulu University Makerere University Natural Products Research group. 2019/08/31 90

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