Proposal presentation. at ufh 2018 alicepptx

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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 Proposal Presentation By Nasifu Kerebba Supervisor : Prof. O . Oyedeji Co-supervisors : Prof. A. Oyedeji Prof. R. Byamukama Dr. S. K. Kuria

2. Outline 2 Introduction and Literature review Background Approaches to isolation from crude Extract for Bioactive cpds Some Pesticidal activities of Tithonia diversifolia and Tephrosia vogelii Phytochemical Studies on T. diversifolia and T. vogelii Flavonoids’ participation in plants’ interaction with Insects Natural antioxidants and Antioxidant potential Research Problem Objectives Methodology Collection of Plant Materials and Extraction Bioassays Determining the Chemotypes and Rotenoid content Work done Acknowledgement

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. S ynthetic pesticides used as the conventional method of pest management. E ffective , Less distributed in rural areas, costly, toxic and pose a serious impact on food safety systems 2 , human health 3 . Okwute , S.K., 2012. Plants as Potential Sources of Pesticidal Agents: A Review . Chapter 9 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 . 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.

4. Calls for more research in less eco-toxic pesticides. Cont … 4 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 R epellence or anti- feedance 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

5. Cont … 5 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

6. Azadiracta indica Lantana camara Vernonia amygdalina Aloe ferox Nicotiana tabacum Source: World agroforestry center: Species database. 2015. Accessed April, 2017 6

7. 7 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

8. Some phytochemicals are known for insecticidal and antioxidant actions: 8 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 materials 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 .

9. Approaches to isolation from crude Extracts for Bioactive Cpds 9 Classic Bioassay-Guided Fractionation I nvolves targeted isolation of bioactive compound 12 . P reliminary extraction, drying, dissolving, precipitating and filtering then multiple chromatographic steps. Meanwhile the activity of separated fractions is evaluated to ‘weed out’ the nonactive ones. Large Sample required identify a single compound. Whole extract analysis U sing LC-MS and high field NMR , an efficient HPLC fractionation of crude extract with the higher pressures and control of solvent composition. RP-chromatography using C18 and a polar liquid. Effluents from column is directly fed into MS for ionization (ESI). very little sample materials and can detect thousands of metabolites. 12. Pieters , L. and Vlietnik , A.J., 2005. Bioguided isolation of pharmacologically active plant components, still a valuable strategy for the finding of new lead compounds? Journal of Ethnopharmacology ; 100: 57-60 .

10. Pesticidal Activities of T. diversifolia and T.vogelii 10 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 .

11. Phytochemical Studies and isolated compounds of T. diversifolia 11 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.

12. Phytochemical studies and isolated bioactive C pds of T.vogelii 12 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.

13. 13 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.

14. Natural antioxidants and antioxidant Potential 14 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 .

15. Research Problem 15 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 .

16. Hypotheses 16 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 will result from combined effect of extracts of T. diversifolia and T. vogelii against F rench bean pest Rotenoids content will vary with season and some T. vogelii plant material may be non- pesticidal 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

17. Justification and significance 17 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

18. Objectives 18 Main objective To determine the chemistry underlying pesticidal and antioxidant activities of T.diversifolia and assess the potential of T. vogelii from Eastern Uganda in pest control . Specific objectives i ) Evaluation of antioxidant properties in Essential oils and solvent extracts of T.diversifolia . ii) Whole extract analysis of T. diversifolia methanol extracts against Maize weevil, Sitophilus zeamais ( Motsch .): Anti-oviposition, adult emergency, and mortality .

19. Cont … 19 iii) Sesquiterpene lactones and diterpenoids of T.diversifolia ; Evaluation for Deterrence, Repellency, and Toxicity to S. zeamais . iv ) Chemotype , Rotenoid content and dose-response curves of Rotenoids of T. vogelii against Rhipicephalus ( Boophilus ) decoloratus . v) Evaluation of Pesticidal activity of essential oils of T.diversifolia and T.vogelii against S. zeamais . vi) Evaluating effects of combined extracts of T.diversifolia and T.vogelii extracts on storage pest in French beans Phaseolus vulgaris ; callosobruchus chinesis

20. Methods and Materials Botanical identification and Collection of plant materials 20 P esticidal 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)

21. 21 Research Approach

22. Biological Studies 22 Evaluation of Antioxidant activities DPPH Quantitative and Ferric reducing antioxidant power (FRAP) Assays determination will be used in evaluation of extracts, fractions and isolates. GC-MS will be used to identify antioxidant compounds in solvent extracts, fractions, isolates and essential oils. The active fractions will be purified using HPLC or Sephadex and the pure compound identified using UV, FTIR, MS and NMR . Evaluation for Pesticidal action Sitophilus zeamais , Rhipicephalus decoloratus and. callosobruchus chinesis will be reared in a temperature controlled room ( e.g 28 ± 1 °C, 55% RH 65–70% Relative Humidity (RH) and 12:12 hours (light: darkness).

23. Callosobruchus chinensis (Adzuki bean weevil) Adult Sitophilus zeamais on maize grain Rhipicephalus ( Boophilus ) decoloratus – African blue tick Source (image) : United States Department of Agriculture, Agricultural Research Service 23

24. 24 The S. zeamais and C. chinesis insects are then allowed to lay eggs for 14 days following the procedure of Ogendo et al., (2003; 2005) 27-28 . Adults lay eggs on maize seed and French bean; at least 7 days for S. zeamais or 20-24 days for C . chinesis later the next generation of adults emerges from the maize or bean. The insects used for bioassays will be at least 7 or 20-24 days post-emergence for S. zeamais and C. chinesis respectively . The contact toxicity, fumigation (oils), anti- feedant and repellent Bioassays of extracts, fractions and isolates are performed against pests . 26. Nyirenda , S. P. and Stevenson, P. C., 2011. Farmers’ insect pest management practices and pesticidal plant use in the protection of stored maize and beans in Southern Africa. International Journal of Pest Management; 57: 41−49 . 27. Ogendo , J. O., Deng, A. L., Belmain , S. R., Walker, D. J. and Musandu , A. A. O., 2003.Comparison of toxic and repellent effects of lantana camara l. with tephrosia vogelii hook and a synthetic pesticide against sitophilus zeamais motschulsky ( coleoptera : curculionidae ) in stored maize grain. Insect Science Application ; 23(2): 127-135. 28. Ogendo , J. O., Belmain , S.R., Deng, A. L. and Walker and Walker, D. L., 2005. Efficancy of Lantana camara and Tephrosia vogelli Hook against Sitophilus zeamais ( Coleoptera : Curculionidae ) in stored maize grains. Journal of Food Technology in Africa . 9: 29-35 .

25. Determining the C hemotypes and flavonoid profile 25 Flavonoid profile of the leaf material of T.vogelii will be carried out by obtaining leaf materials from different collection sites, Then analyse for flavonoids using the LC-MS analysis and then compared . The proportion of T.vogelii chemotypes determined in each site gives an insight on the relevance in pest control . Determining Rotenoid content The isolation and analysis will be carried out using LC-MS/HPLC. The standards Rotenone, deguelin and tephrosin will be purchased and used in the study .

26. 26 To confirm identification, several HPLC and TLC conditions will be used in comparison the with reference standards. The rotenoid content will be determined by determining the content of rotenoid compounds (Rotenone, deguelin and tephrosin ) using Methodology described by Stevenson et al. 2012 22 Dose Response curve of rotenoids against R . decoloratus Tick collection Semi-engorged adults of the Rhipicephalus decoloratus are collected manually from infected cattle or purchased from University of Pretoria (Vet. Medicine)

27. Ticks are kept in plastic aerated cell culture flasks and identified at the species level . In vitro acaricidal activity of rotenoids against R.decoloratus is then evaluated Statistical analysis ANOVA followed by post hoc tests will be performed using Graph Pad Instat to compare the mortality and eggs laid among and between treatments at equivalent concentrations. Results will be expressed as the mean ± SEM (standard error of the mean). P values lower than 0.05 will be considered significant Conclusion It is therefore, anticipated that this study will to isolation of lead molecules to facilitate future isolation for commercial purposes 27

28. Timeline 28 Month 2017 - 2019 Activities (*) April 2017 Sept 2017 Feb 2018 Dec 2018 Feb 2019 May 2019 Jun 2019 July 2019 Aug 2019 Proposal Writing * * Lab Work * * * Write-up * * Submission of 1 st full Thesis draft * * * Submission of 2 nd full Thesis draft * Submission of final Thesis for Examination * *

29. Work done 29 Collection and identification of samples Confirmation and deposition of voucher specimen at Makerere University Herbarium Phytochemical screening Extraction and isolation (impure) of T.diversifolia for evaluation of antioxidant Field survey of T. vogelii Extraction of T. vogelii for flavonoid profile study to determine chemotypes available to farmers Hydrodistillation of fresh T.diversifolia samples at Makerere university Review Manuscript (chapter two) and introduction chapter (one)

30. Acknowledgement 30 Special thanks to God Almighty Prof . O. O. Oyedeji , Prof. A . Oyedeji , Prof. R. Byamukama and Dr . S. K. Kuria NRF/TWAS, GMRDC for Funding. University of Fort Hare Walter Sisulu University Makerere University Natural Products Research group.

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