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1. New hydroxycinnamic acid derivatives from Tithonia diversifolia ( Hemsl .) A. Gray and anti-oxidant activity Nasifu Kerebba Department of Chemistry Makerere University, Uganda Nov, 11-12, 2021 1

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3. Abstract 3 Fig. 1 : Graphical abstract

4. 7 new hydroxycinnamic acid derivatives identified from T. diversifolia using UPLC-ESI-MS/MS and 1D NMR. In total, 55 phenolic compounds from aqueous fractions of T. diversifolia have been annotated including: 4 hydroxybenzoic acids (HB), 19 hydroxycinnamic acids (HC), 22 flavonoids, 1 saponin triterpenoid (ST) and 9 coumarins and furanocoumarins . This study further showed that DPPH radical scavenging effect and reducing power antioxidant activity of tested samples of T. diversifolia showed moderate correlation with the phenolic content present in these samples. Highlights 4

5. 2 approaches to isolation of bioactive from a biological crude extract: C lassic bioassay-guided isolation W hole extract analysis using either NMR or LC–MS spectroscopic techniques ( Wheat, 2013 ). The challenges of isolating and characterizing complex crude extracts that comprise a multitude of compounds are solved by hyphenating high-performance liquid chromatography and mass spectrometry(LC–MS ). The LC–MS provides the user with a multitude of technical options and applications. Why UPLC-ESI-MS/MS for characterisation of biological crude extract? 5

6. 6 Why UPLC-ESI-MS/MS on T. diversifolia extract for phenolic compound identification? Phenolic acids and their derivatives have for long exhibited several biological activities ( Adlercreutz , 2002) S tudies on T. diversifolia anti-oxidant potential have linked it directly to its TPC and TFC in the crude extracts ( Ojo et al., 2018, Pantoja Pulido et al., 2017) The studies, however, do not satisfactorily demonstrate a detailed phytochemical analysis that reveals numerous phenolic compounds and other compounds believed to possess the anti-oxidant potential and other biological activities.

7. 7 Preparation of fractions and extraction Air-dried leaves of T. diversifolia (59.5g) were extracted in methanol for 1month after deffating in petroleum ether for 24hrs Resuspension in different percentages of aqueous methanol to give six fractions i.e. 80%, 70%, 60%, 50%, 0% MeOH /H 2 O for fractions; fraction 1 (F1) (4.1g), fraction 2 (F2) (0.7g), fraction 3 (F3) (0.2g), fraction 4 (F4) (0.5g), fraction 5 (F5) (0.3g) and then redistributed with petroleum ether which constituted fraction 6 (F6) (151.1g )> Isolation to give NK1F4

8. 8 UHPLC-ESI-MS base peak chromatogram Fig. 2

9. In general 3 criteria are used in annotation with LC-MS/MS: A ccurate mass match which is automatically searched, using METLIN ( http://metlin.scripps.edu/metabo_search.php ) and other libraries including PubChem ( http://pubchem.ncbi.nlm.nih.gov/ ) with all compounds whose accurate mass error (AME) > 5 ppm, were considered unidentified. Secondly , mass fragmentation patterns searched in the above databases, if available. A few phenolic compound standards were spiked under similar UPLC-ESI-MS/MS conditions and fragmentation patterns were compared to identify a given compound based on retention time, mass fragmentation and ionisation modes MS-MS of the compounds in the sample fragmented to corresponding product ion mass spectra . Characterisation of phenolic compounds from T. diversifolia with UPLCESIMS/MS 9

10. 10 And lastly, the number of carbon atoms in the peak was calculated if isotope abundances were available. The predicted number of carbon atoms in the putatively identified compound was used to reduce false annotations . In combination with 1D 1 H and 13 C NMR for NK1F4 , UV Fig. 3

11. 11 Fig. 4: Ms/ ms and uv for NK1F4

12. However, the above MS/MS spectra do not contain fragment series associated with terminal and nonterminal epoxide fragmentation patterns. In nonterminal epoxides trans annular cleavage with H transfer and elimination of an alkenyl radical to generate C n H 2n+1 O fragments ( m/z 45, 59, 73 …) occurs or γ-cleavage to generate C n H 2n O fragments ( m/z 44, 58, 72 …) and alkene (C n H 2n; 28, 42, 56...) series for terminal epoxides ( Badertscher et al., 2009). The compounds are therefore not epoxide derivatives of these new caffeic acids 12

13. 13 Fig. 5: MS/MS for novel compounds A, B, C, and D

14. 4- O -(1- O -caffeoylglucosyl) quinic acid (A) 4- O -caffeoyl-2-hydroxy-3-methylbutanoic acid(B) 14 Fig. 6: Proposed fragmentation scheme for cps A and B

15. 4- O -caffeoyl-2-hydroxybutanoic acid (C) 3,4-dihydroxyphenyl-4- O -caffeoyl-2-hydroxybutanoate (D) 15 Fig. 7: Proposed fragmentation scheme for cps c and D

16. 16 Fig. 8 : MS/MS for novel compounds E, F and G

17. The alkene (C n H 2n; 28, 42, 56...) series has been detected at m/z 84, 99, 113. supports the existence of the vinylic group in the compound ( Badertscher et al., (2009) 4-vinylphenyl 4- O -caffeoyl-2-hydroxy-3-methylbutanoate(F) 3-(4-hydroxy-3-methoxyphenyl) acryloyloxy ) methyl-4- O -caffeoyl-2-hydroxybutanoate (E) 17 MS 2 fragments m/z 102, 193, 282, 296, 327, 375 and m/z 383 Fig. 9 : Proposed fragmentation scheme for cps E and F

18. 3-Hydroxypropyl-4-(3-(4- O -caffeoyl-2-(3-(3-(2,3-dihydroxyphenyl) acryloyloxy ) propanoyloxy ) butanoyloxy )-2-methoxyphenyl)but-2enoat 18 [ M+H] + ion; m/z 764.4482 MS 2 fragments ; m/z 742, 663, 477, 304, 282, 296, 102, 193, 680 Fig.10 : Proposed fragmentation scheme for G

19. 19 Fig. 11 : Proposed fragmentation scheme for byakangelicin Other compounds Other HC Coumarin and furanocoumarins HB Flavonoids ST Mechanisms of f ragmentation Simple inductive cleavage Retro Dields Alder (RDA) cleavage elimination [M+H] + ; 335.1832 MS/MS fragment ions m/z 179, 225, 173, 149, 147, 103, 299, 581 265, 221, 215, 205 and 133

20. 20 Total of 77 peaks could be detected with 55 better identified phenolic compounds. These include: 4 HB, 19 HC and their derivatives, 12 flavonols , 5 flavones, 2 flavanones , four flavanols , 1ST and 9 coumarins and furanocoumarins . The other peaks were unknown: 4 HC derivatives, 2 flavonoids, 1coumarin metabolite, 4other hydroxyl compounds that included quinic acids and glucaric acids, 1 fatty acid and 9 unknown peaks.

21. UPLCESIMS/MS helped in studying the distributions of phenolic compounds thus the anti-oxidant potential of the T. diversifolia samples . Fractions with kaempferol and quercetin derivatives such as kaempferol-dihexoside , quercetin-glucosylpentoside , quercetin-rhamnosylglucoside , quercetin-3-O-rhamnoside and kaempferol-rhamnosyl glucoside . Had higher antioxidant potentials Quercetin and kaempferol ( flavonols ) are the best anti-oxidant compounds ( Agati et al., 2011; Tattini et al., 2004). 21

22. Total phenol content, total flavonoid content and the anti-oxidant capacity of T. diversifolia 22 Fig. 12: A graph of the proportion of percentage DPPH scavenging activity of tested samples

23. Classification of associations: small =0.1-0.29, moderate 0.3-0.49, large ˃0.5. Table 1: Total antioxidant activity, total phenol content and total flavonoid content of the tested samples of T. diversifolia 23 Table 2 : Correlations of different assays

24. 24 All these findings reveal that the antioxidant potential of extracts or fractions cannot be predicted on the basis of their total phenolic content. This is because the anti-oxidant properties of phenolic compounds are limited by their chemical structures (Rice-Evans et al., 1996 ). Flavones and catechins are, in general, better anti-oxidants compared to other polyphenols Additionally, the number of hydroxyl groups and the extent of glycosylation play an additional role in the anti-oxidant activity.

25. Among flavonoids, the antioxidant properties have been enhanced by the presence of a 2, 3 double bond neighbouring the carbonyl group in the C ring, a free hydroxyl group at the 3 position on the C ring and the presence of hydroxyl groups in the 5 and 7 positions on the A ring (Rice-Evans et al., 1996; Pietta , 2000). Flavonoids with these 3-OH groups have displayed maximum radical scavenging activity. Eg , quercetin 3- O glucoside has exhibited an IC 50 (DPPH) of 39.3 μM while the IC 50 of kaempferol 3- O glucoside is greater than 500 μM ( Tattini et al., 2004) 25

26. Conclusion The UPLC-ESI-MS/MS revealed seven previously non-described compounds in nature from T. diversifolia through deductions from 1D NMR of related caffeic acid derivatives, in addition to several other phenolic compounds characterised. The DPPH scavenging capacities and reducing power of T. diversifolia were moderately attributed to its phenolic content in this study . 26

27. 27 References 1. Badertscher , M., Bühlmann , P. and Pretsch , E., 2009. Structure determination of organic compounds. Springer Berlin Heidelberg pg 369- 370 2. Baruah , N.C., Sharma, R.P., Madhusudanan , K.P., Thyagarajan , G., Herz , W. and Murari , R., 1979. Sesquiterpene lactones of Tithonia diversifolia . Stereochemistry of the tagitinins and related compounds. The Journal of Organic Chemistry 44(11), 1831-1835. 3. dos Santos, M.D., Martins, P.R., dos Santos, P.A., Bortocan , R., Iamamoto , Y., and Lopes, N.P., 2005. Oxidative metabolism of 5-O-caffeoylquinic acid ( chlorogenic acid), a bioactive natural product , by metalloporphyrin and rat liver mitochondria. European Journal of Pharmaceutical Sciences 26(1), 62-70. 4. Ojo , O.A., Ojo , A.B., Ajiboye , B.O., Olaiya , O., Mary Abiola Okesola , M.A., Boligond , A.A., de Campos , M.M.A., Oyinloyea , B.E. and Kappo , A.P., 2018. HPLC-DAD fingerprinting analysis, antioxidant activities of Tithonia diversifolia ( Hemsl .) A. Gray leaves and its inhibition of key enzymes linked to Alzheimer’s disease. Toxicology Reports 5, 585-592. 5. Pantoja Pulido , K.D., Colmenares Dulcey , A.J. and Isaza Martínez , J.H., 2017. New caffeic acid derivative from Tithonia diversifolia ( Hemsl .) A. Gray butanolic extract and its antioxidant activity . Food and Chemical Toxicology 109, 1079-1085.