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1. 1 PhD Progress Presentation By Nasifu Kerebba Supervisor: Prof . O. Oyedeji Co-supervisors: Prof . A. Oyedeji Prof . R. Byamukama Dr. S. K. Kuria November, 2019 1 C hemical and Biological evaluation of T ithonia diversifolia and T ephrosia vogelii from U ganda as sources of anti-oxidant and pesticide

2. Outline Introduction and Literature review Background Phytochemical Studies on T. diversifolia and T. vogelii Research Problem Objectives Methodology Collection of Plant Materials and Extraction . Investigate the chemical variation in components and composition of T. vogelii essential oils and implications in control against S. zeamais C omparison of the pesticidal potential of the essential oils of T . diversifolia and T . vogelii 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 . References Acknowledgement 2 2

3. 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, human health [1] Reports in Africa indicate that extracts of local plants can be effective as crop protectants against pre-harvest and post-harvest pests[2] Compounds rapidly decompose ; environmental friendly Naturally at low levels with a diverse active ingredients Repellency or anti- feedant mode of action Introduction 3 Figure1 ( a &b ): Synthetic pesticide applications a b 3

4. 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 ., [3] 4 Tephrosia vogelii (Hook f .)and Tithonia diversifolia Commonly called fish bean plant Pesticidal plant, native to Africa and well-distributed across the tropics[4] Used in pre- and post-harvest pest management and ecto -parasite control on livestock T. diversifolia ; known as wild sunflower or tree marigold in the Asteraceae family mainly used in Uganda for field and for storage pest mgt 4

5. Some phytochemicals are known for insecticidal and antioxidant actions: toxic principles against pests such as isoflavonoids . imposing behavioral responses to insect pests: repellence, feeding deterrence, growth-regulating potentials and oviposition deterrence[5] 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. 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 e.g Naringenin , hesperetin-7-O-rutinoside and quercetin-3-O-rutinoside, induce oviposition in citrus feeding swallowtail butterfly Papilio xuthus [6] which have exhibited strong antioxidant potentials . 5 5

6. Phytochemical Studies of T. diversifolia S esquiterpenoids , diterpenoids , flavonoids and chlorogenic acids derivatives [7] 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 ) 6 6

7. Phytochemical studies of T.vogelii 7 F lavonoid glycosides and flavonoid aglycones detected including rotenoids , Revealing two chemical varieties of T. vogelii [8] 7 7

8. 8 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 Chemical variability of T. vogelii species can compromise its efficacy as a botanical pesticide resulting in farmers reporting no pesticidal activity . chemical basis of the previously reported biological activities ( pesticidal and anti-oxidant) for T. diversifolia and asses the potential T. vogelii essential oils in pest control . H ealth-related aspects of bio-markers of biological activity from the crude extracts 8

9. Research Problem 9 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. 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 9 9

10. Justification and significance 10 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 [8] 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 could trigger a new course of further research especially in healthy related aspects of biomarker i.e the fate of break down metabolites. 10 10

11. Aims To evaluate chemically the pesticidal and antioxidant activities of T.diversifolia and investigate chemical variation in T. vogelii essential oils from Uganda Specific objectives To investigate the chemical variation in components and composition of T. vogelii essential oils and pesticidal implications against S. zeamais To evaluate and compare the pesticidal potential of the essential oils of T.diversifolia and T.vogelii 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 . 11 11

12. Methods and Materials Botanical identification and Collection of plant materials 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) 12 12

13. 13 Research Approach 13 13

14. 14 Extraction and analysis of Essential oils 14 General experiment on essential oils

15. Rearing of insects Initial insect stocks were obtained from infected maize from the market. Plastic containers (flasks) were used to bleed colonies of the maize weevils at 30 ± 1 °C, 60 ± 5% RH and a photo period of 12:12 dark : light . The old adult insects were then removed from the flasks after 5 days to allow emergence of the new generation. After 7-14 days, the first generation of weevils of unsexed adult weevils were considered for the bioassay . Repellency and toxicity bioassays were carried out against reared S. zeamais Bioassays for Pesticidal evaluation 15 15

16. Repellence was done using the area preference method [9] Fumigant toxicity was done following the protocol set by [ 10]. Contact toxicity was done following procedure by [11] For repellence test, the concentrations used were 0,1 , 5 and 10 μ L while for contact and fumigant toxicity the test concentrations were:0, 10, 20 and 40 μ L 16 Figure 4 : Set up of Pesticidal bioassays (a) repellence (b) fumigant toxicity (c) Contact toxicity b a c 16

17. 17 Chemical variation and implications on Pesticidal activity of Tephrosia vogelii (Hook f.) Essential oils against Sitophilus zeamais Motschulsky Study Area and plant materials collection Butaleja district (00 56’ N, 33 57’ E), eastern Uganda T. vogelii plant species were 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) 17

18. 18     Location     Village (sample) Flower color Latitude North Longitude East Sampling date Altitude Muyago (TV1 muya ) White 0°84′20″ 34°03′15″ 14/05/2017 1080m KyadongoB ( TV1 kya ) White 0°90′14″ 34°08′30″ 1/06/2017 1098m Kyadongo B (TV1 kyb) White 0°90′30″ 34°09′45″ 1/06/2017 1098m Kyadongo B (TV1 kyc) White 0°80′10″ 34°08′40″ 1/06/2017 1098m Muyago (TV2 muya) white 0°84′14.9″ 34°03′10″ 15/08/2017 1080m Kyadongo B (TV2 kya) white 0°70′30″ 34°07′35″ 15/08/2017 1098m Kyadongo B (TV2 kyb) white 0°90′14″ 34°08′30″ 15/08/2017 1098m Kyadongo B (TV2 kyc ) white 0°90′20″ 34°09′40″ 15/08/2017 1098m Kyadongo B (TV2 kyd) white 0°90′35″ 34°08′45″ 15/08/2017 1098m Muyago (TV3 muya) white 0°84′00″ 34°02′45″ 01/03/2018 1090m Muyago (TV3 muyb) white 0°84′14″ 34°03′09″ 01/03/2018 1090m Muyago (TV3 muyc) white 0°84′14.9″ 34°03′10″ 01/03/2018 1090m Kyadongo B (TV3 kya) white 0°90′14″ 34°08′30″ 01/03/2018 1098m Kyadongo B (TV4 kya) white 0°70′30″ 34°07′35″ 10/01/2019 1098m Kyadongo B (TV4 kyb) white 0°90′14″ 34°08′30″ 10/01/2019 1098m Kyadongo B (TV4 kyc) white 0°90′20″ 34°09′40″ 10/01/2019 1098m Muyago (TV4 muya) white 0°84′00″ 34°02′45″ 10/01/2019 1090m Muyago (TV4 muyb) white 0°84′14″ 34°03′09″ 10/01/2019 1090m Muyago (TV4 muyc) white 0°84′14.9″ 34°03′10″ 10/01/2019 1090m 18 Collection of sample material

19. 19 Evaluation for chemical varieties Principal component analysis (PCA) and Agglomerative hierarchical clustering (AHC) were performed on the data to group into components and clusters respectively PCA is a statistical tool that aims to represent the variation present in the data. It allows similarities and differences between data to be seen easily. PCA was performed on 19 samples × 23 variables Loadings matrix were obtained through the transformation of data from correlated to new uncorrelated variables called principal components 19

20. 20 AHC is an algorithm that brings together related objects into  distinctive clusters based on their similarities. The objects with in a cluster are thus very similar; clustering makes it easier to see the correlations. AHC was used to analyze seasonal and geographical influence on the yield and composition through classification. Classification of samples gave a d endrogram of major chemical constituents . Classification of major constituents gave a dendrogram of samples of T . vogelii that would enable analysis of effect of seasonal variation on composition 20

21. 21 Results and discussion Chemical constituents and composition of essential oils 21 Figure 6 : Chemical composition and constituents of oils from Muyago samples

22. 22 Chemical constituents and composition of the oils 22 Figure 7 : Chemical composition and constituents of oils from Kyadongo sample

23. 23 Figure 8 . 3D scatter plot of different correlations of chemical components using PCA 3 chemical groupings revealed based on the profiles of compounds from farnesene family: Farnesol ( Fnso ) type ( Chemotype 1). Ethanol, 2-butoxy-(E2B)and D-(+)-Alpha- pinene (αP) Chemotype 2: springene compounds (β- Springene (βS)and α- Springene (α Sp )) and the β- Farnesene (βF) Other compounds; (E)- Nerolidol ( EN) Cis-p-metha-1(7)-8-dien-2-ol( zMD ), D-limonene( Dl), 1,4-dihydroxy-p-menth-2-ene ( Dm ) 5,9-undecadien-2-one,6,10-dimethyl ( UD), and Farnesol (E)- methylether ( FnsoEm ) Chemotype3 was mixed chemotype , mixture of above two Classification of chemical compounds a) Chemical grouping using PCA 23

24. 24 Objects Similarity Farnesol , β- Springene , α- Springene , β- Farnesene could form separate clusters alkylbenzenes and farnesol ; Pearson correlation, r ˃0.4, p˂0.05, for all alkylbenzene ) β- Springene and β- Farnesene (Pearson correlation, r=0.3 ) α- Springene and β- Farnesene (Pearson correlation r=0.2 ). α- Springene and farnesol (Pearson correlation, r ˃0.4, p˂0.05 ) N o correlation between either β- Springene or β- Farnesene with farnesol Figure 9 : Dendrogram of major components in the oils of T. vogelii Chemical groupings using AHC 24

25. Effect of seasonal variation on the percentage yield of the oils The yield of the A yellow distillate ranged between 0.18±0.01% to 0.22±0.01%(w/w) d.w for samples from Kyadongho B and 0.16±0.00% to 0.22±0.01%(w/w) d.w for Muyago samples Yield during rainy season (green) and dry season (red ) 25 Figure 10 : Percentage yield of oils of T. vogelii

26. 26 Rainy season: Cluster 1(Tv1muya, Tv2muya, Tv3kya ) cluster 2-Tv3muyb and Tv3muyc) Cluster 4-Tv2kya and Tv2kyb Tv3muya for cluster 6 D ry and rainy season: Cluster 3(Tv2kyd) and 5(Tv1kyc and Tv1kya ) D ry season (January ): cluster 7 (Tv4muya and Tv4muyc ) cluster 8 (Tv4kyc and Tv4muyb ) cluster 9 (Tv4kyb) and cluster 10 (Tv4kya) Similarities with in samples in same clusters but with several clusters formed indicate m ajor seasonal effects on the composition of the major constituents Figure 11 : Dendrogram of samples due to classification based on their major composition Effect of seasonal variation on the composition of the major constituents 26

27. 27 Chemotaxanomic significance of chemical varieties Three chemical varieties: Farnesol variety and springene type, and the mixed variety delimit the taxonomic status of this plant. Farnesol is an oxygenated sesquiterpene of various isomers; ( E,Z )- Farnesol , ( Z,E )- Farnesol and ( E,E )- Farnesol Springene is an isoprenoid hydrocarbon of diterpene nature . ( E,E )-β- springene is a diterpene homolog of ( E )-β- farnesene Both farnesol and springene , belong to the farnesene family Figure 12 : Structures of compounds with chemotaxonomic significance identified from T. vogelii 27

28. 28 Repellence and fumigant toxicity of the chemotypes of the volatile constituents of T. vogelii against S. zeamais No much difference in repellency effect of chemotype 1 and 2 C hemotype3 (mixed) showed lower effect composition of farnesol and springene was very low, partly explain the less repellency activity U nderlines the vital role that both farnesol and springene play in the repellency potential 28 Figure 13 : Mean percentage repellence against S. zeamais

29. 29 29 Figure 14 : P ercentage repellence of farnesol against S. zeamais Farnesol is effective at higher concentration as a repellent against S. zeamais lower dose (0.03μL) of farnesol had a very limited repellent effect against S. zeamais . P reference index evaluated for 0.31μL of farnesol per cm 3 air was 0.0 to -0.4 and 0.6 t0 -0.2 for lower dose R epellency activity increased with an increase in the amount of farnesol in the oil. Repellency effect of f arnesol against S. zeamais

30. Fumigant toxicity 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   30 30

31.   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 ) 31 A t a lower dose, Springene chemotype could exhibit a higher fumigant effect At a higher concentration, the effect is not so different The farnesol type could potentially exhibit a higher fumigant toxicity against S. zeamais . 31

32. 32 Conclusion Three chemical varieties based on the profiles of farnesene compounds; one that possesses the farnesol , and the other that has the springene and β- Farnesene type; all from the farnesene family and a mixed chemotype of the two V arieties within the volatile constituents of the oil thus delimits the chemotaxonomy of this plant species G eographical and seasonal variation could not significantly affect the yield of the oil significantly however; the composition and the constituents of the oils were affected by the harvest period. C hemotype 1 and 2 were similarly active repellents but more than chemotype 3, against S. zeamais . The difference in the toxicity of the oils (fumigant toxicity) against S. zeamais could undermine the efficacy of this plant 32

33. 33 The complementary part of all other compounds found in the same oils plays a crucial role in the overall repellent and insecticidal effect of this oil M ore study is needed that aims to optimize and standardize the chemical varieties and harvesting period needed for recommendation to smallhold farmers especially under field conditions before it can be adopted more widely 33

34. Extraction Hydro distillation of essential oils yielded light yellow distillate of 0.2±0.0% (w/ w,N =4) dry weight for T. diversifolia and yellow oil of 0.2±0.0%(w/ w,N =4) dry weight of T. vogelii . Bioassays : Repellency , Contact toxicity and fumigant toxicity Results and Discussion 34 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%). Comparative toxicity and repellency effects of the essential oils of Tephrosia vogelii (Hook f.) and Tithonia diversifolia ( Hemsl .) A. Gray against Sitophilus zeamais Motschulsky 34

35. 35 Repellency class: 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 This implies that at higher dosage, T. vogelii exhibited higher repellency efficacy especially over exposure period of 72hr from 12hrs. 35

36. 36 Preference index (PI) of T. diversifolia essential oils against S. zeamais Preference index (PI) of T. vogelii essential oils against S. zeamais F or T. vogelii , PI 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 36

37. 37 Contact toxicity At lower concetration (0.25 μ g/L), the mortalities due to T. vogelii was greater than that due to T. diversifolia against S. zeamais . At higher doses (0.5 μ g/L and 1.0 μ g/L), T. diversifolia exhibited a more contact toxicity effect on S. zeamais compared to that of T. vogelii . S . zeamais are however less sensitive to these oils due to contact exposure, this could explain why the percentage mortalities were very low. The cumulative percentage mortality due to T. diversifolia was just only 7.8±2.2 % and 8.9±1.1 % while that of T. vogelii was 4.4±1.1% and 6.7±5.1 % for 1.0 μ g/L and 0.5 μ g/L dosages respectively after 96 th hour of exposure. 37

38. 38 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 Fumigant toxicity The cumulative mortalities shows that the oils of T. diversifolia , exhibited the highest fumigant efficacy against S. zeamais ; LC 50 5.84 μL /L versus LC 50 11.57 μL /L for T. vogelii after 96 hours 38

39. Group T. diversifolia T. vogelii Monoterpene hydrocarbons 44.9±5.6% 2.4±0.2 Oxygenated monoterpenes 1.7±0.2 13.1±0.2 Sesquiterpene hydrocarbons 2.4±0.2 1.4±0.1 Oxygenated sesquiterpenes 1.4±0.1 7.0±1.5 Diterpene 0.2±0.1 0.2±0.0 Other hydrocarbons 4.1±0.0 62.9±1.5% Total 54.6±5.2% 86.8±1.5% Analysis of the essential oils 39 39

40. 40 α- pinene at all concentrations, o-xylene and farnesol at higher dose of 0.3 μl /cm 3 of air could demonstrate repellent effect against S. zeamais from 48hr exposure partly explain the difference between the repellent toxicity of these two plants. The dose dependent nature of o-xylene and non-dose dependent α - pinene could explain the disparities in their fumigant potential . Figure 5: Log transformed cumulative mortalities of S. zeamais due to different doses of α- pinene and o-xylene 40

41. 41 The essential oils of T. vogelii and T. diversifolia both showed considerable pesticidal potential. However T. vogelii essential oil could be promoted more for repellent and contact toxicity effect especially when a mixed variety is used whereas T. diversifolia better suits fumigation effect against S. zeamais . The high amount of aromatic hydrocarbons (particularly the xylene isomers), linalool and α - terpineol in T. vogelii essential oils could be behind its contact toxicity and repellent advantage over T. diversifolia . Conversely , high amount of α - pinene , given its non-dose dependent and other monoterpenes give advantage to T. diversifolia fumigant toxicity against S. zeamais over that of T. vogelii . Conclusions 41

42. Evaluation of Feeding deterrence activity of Tithonia diversifolia non volatile and volatile substances against S . zeamais Extraction, fractionation and isolation of non-volatile substances F ractions F1, F2, F3, F4, F5 and then be distributed with Petroleum ether which constitute fraction F6 . F3 was loaded 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, and F4 1 ; purified on a column using Chloroform:ethylacetate (100:0, 75:25, 55:45) to give compound NK3F4. Recrystallising F5 in 100 % MeOH and filtering on whatman paper, gave a pure compound NK1F5(10.83g ) 42 42

43. 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 nutritional indices: Relative growth rate (RGR), Relative consumption rate(RCR), Efficiency of conversion of ingested food(ECI) and Feeding deterrence index (FDI ) 43 Disc were prepared following protocols set by Xie et al [23] Preparation of discs to be used for non- volatile antifeedant assays 43

44. 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 44 Set up of feeding deterrence bioassays 44

45. 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 45 45

46. 46 The FDI evaluates the potential of a substance to induce the cessation on feeding when tasted by an insect, and whether it can continue to feed when provided with alternative food source . The feeding deterrence activity of tested substance was evaluated based on nutritional indices: FDI, RGR, RCR and ECI If a substance could reduce diet consumption, but does not show effect on food utilisation/ relative consumption and growth rate with respect to the that of the control, it is an indication of a behavioural effect- Antifeedant effect Fraction 3(F3) did not reduce the relative food consumption, but there was an increase in relative growth rate with reduced utilisation of food Results and discussion 46

47. 47 Similary F4 did not reduce diet consumption at all tested concentrations and there was inhibition in the growth rate of weevils with reduction in utilisation of food. This was an indication of a feeding deterrence effect F5 did not affect disk consumption. It though inhibited the relative growth rate of the weevils and reduced the utilisation of food except at a higher concentration A similar scenario with F6 was observed but in this case at 1%w/w concentration treatment NK1F3, NK1F4, NK3F4 and NK1F5 did not affect the amount of the disk consumed and the utilisation of diet compared to that of the control . 47

48. 48 They generally inhibited the relative growth rate of weevils. Implying that these compounds could exhibit an antifeedant effect Fractions F3, F4 and partly F5 showed mean diety relative consumption below that of the control level Compound NK3F4 had the highest feeding deterrence index at 81.19±5.94% among the isolated compounds 48

49. 49 The essential oils did not have any significant effect (P > 0.05) (Table 5.2) on the nutritional indices and mortality suggesting that the oils did not have any antifeedant effect on the weevils in the concentration range of 0 to 20 μl of oil (0, 0.07, 0.15 and 0.29 μl /mg of flour disks) but the antifeedant effect is possible at higher concentrations . 49

50. Spectral studies of the isolated compounds 50 50

51. 51 51

52. 52 Proton NMR for NK1F3 52

53. 53 Proton NMR for NK1F4 54

54. 54 Proton NMR for NK3F4 54

55. 55 COSY for NK3F4 55

56. 56 NOESY for NK3F4 56

57. 57 C13 NMR for NK1F5 57

58. 58 FTIR, NMR and HR-ESI-MS DATA Compound NK1F3, a white crystalline solid. The 1H NMR (400 MHz, D2O) ( δ) alkene proton at 5.38 (s, 1H), 4.82, 4.80, 4.79, 4.76, 4.76, 4.70, 4.64, 4.64, 4.60, 4.59, 4.57, carbonyl/ lactone group at 4.25 (s, 1H), 3.65, 3.49-3.57(d, J=32Hz, 2H), 3.27 (s, 1H), 2.42 (s, 1H), 0.0-2.0 (m, J=80Hz). Compound NK1F4 was white crystalline solid with molecular formula was determined to be by an [MH] peak in the High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) at m/z 282.0741( Calcd for). NK1F4 HR-ESI-MS at m/z 301.1007 FTIR for NK1F4 suggested the presence of a para aromatic sp2 C-H(824 cm-1), an alkoxy C-O (1173 cm-1), an ester (1735 cm-1) and an a -methylene γ- lactone (1765, 1783 cm-1) group in the molecule. The 1H NMR spectrum gave the following signals: 1H NMR (400 MHz, D2O) δ 8.25 ( m, 1H), meta aromatic hydrogen 7.41 (m, 1H), alkene proton 5.38 (s, 1H), carbonyl ester/lactone group (3.27-4.25) [4.25 (s, 1H), 3.64 (s, 1H), 3.27 (d, 1H)], alkane protons 1.23-1.80) [2.42 (s, 1H), 2.14 (s, 1H), 1.83(d, J, 1H), 1.43 (s, 1H), 1.23 (m, J=84Hz, 6H)]. The 13C NMR (101 MHz, D2O) δ 140.90, 124.75, 121.40, 96.96, 49.36, 46.68 58

59. 59 Compound NK3F4 [MH] peak in the HR-ESI-MS at m/z 290.2330( Calcd for). FT-IR spectrum of NK3F4 suggested the presence of an aldehydic C-H stretch, broad peak (2936 cm-1), an alkoxy C-O (1056 cm-1) group, a germinal disubstituted alkene sp2C-H (971 cm-1) and a weak sp2 C-H stretch (1466cm-1) group in the molecule. The 1H NMR (400 MHz, CDCl3) signals ( δ) showed presence of aldehyde proton RC(=O)-H at 9.85 (s, H), aromatic protons; Ph -H at 7.54 (s,1H) and 7.02 (s,1H), ester proton CH-O at 5.46, proton at 5.38- 5.13 (m, 3H, J=24HZ), protons of at 4.84 (s, 1H), PhCH2OH at 4.37(s, 3H), group of lactone group at 4.18-4.13(d, 1J=6HZ), Proton of 3.62- 3.52(m, J=4HZ), 2.07(s, 1H), simple sp3 C-H 1.82(s, 5H), simple sp3 C-H (CH, CH2) at 1.58(s, 5H), 1.47, simple sp3 C-H(CH or CH2 or CH3) at 1.32-1.28 (m, J=8HZ,10H), simple sp3 C-H(CH or CH2 or CH3) at 1.08(s, 3H), simple sp3 C-H(CH or CH2 or CH3) at 1.05-1.02(t, J=2.0HZ, 3H), simple sp3 C-H(CH or CH2 or CH3) at 0.89- 0.87(d, J=4HZ, 7H), simple sp3 C-H(CH or CH2 or CH3) at 0.74- 0.69(t, J=2.0HZ, 7H), simple sp3 C-H(CH or CH2 or CH3) at 0.67(s,1H), simple sp3 C-H(CH or CH2 or CH3) at 0.57(s,1H), simple sp3 C-H(CH or CH2 or CH3) at 0.56(s,1H). Dept13C NMR (101 MHz, CDCl3) δ 158.65, 153.23, 151.66, 134.65, 127.66, 125.45, 124.98, 92.91, 90.43, 85.03, 77.20, 50.07, 43.35, 15.31, 9.70, 2.27. Compound NK1F5 was a colourless crystalline solid with molecular formula was determined to be by an [MH] peak in the High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) at m/z 247.1550.The IR spectrum of NK1F4 suggested the presence of a para aromatic sp2 C-H(824 cm-1), an alkoxy C-O (1173 cm-1), an ester (1735 cm-1) and an a -methylene γ- lactone (1765, 1783 cm-1) group in the molecule. The 1H NMR spectrum gave the following signals: 1H NMR (400 MHz, D2O) δ 8.25 ( m, 1H), meta aromatic hydrogen 7.41 (m, 1H), alkene proton 5.38 (s, 1H), carbonyl ester/lactone group (3.27-4.25) [4.25 (s, 1H), 3.64 (s, 1H), 3.27 (d, 1H)], alkane protons 1.23-1.80) [2.42 (s, 1H), 2.14 (s, 1H), 1.83(d, J, 1H), 1.43 (s, 1H), 1.23 (m, J=84Hz, 6H)]. 59

60. 60 Evaluation of Antioxidant Potential of Tithonia diversifolia non-volatile and volatile extracts Assays 1. 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). Percentage 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 60

61. 61 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 61

62. 62 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 62

63. 63 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 63

64. 64 Reducing power assay The higher the absorbance, the stronger reducing power of the sample. 64

65. 65 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. 65

66. 66 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 66

67. 67 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     NK1F5 -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     NK1F4 -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 67

68. 68 Total phenol calibration curve Total flavonoid calibration curve 68

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70. 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 1 article published 2 experimental manuscripts have been submitted to journals At least 5 publications is expected 70 70 70

71. Acknowledgement 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. Natural Products Research group. Samuel Zipho 71 71

72. THANK YOU FOR LISTENING 72 72