1. Pulcherrin A and I from the Stem Bark of Caesalpinia pulcherrima with their Activity against Chloroquine Sensitive and Resistant Strains of Plasmodium falciparum
Ogbeide, O.K. , Imieje, V., Erharuyi, O., Oseghale, I., Fasinu, P., Owolabi, J. B., Falodun, A., Yousuf, S. and Choudhary, M. I.

2. Natural Products Malaria Who is at Risk? Introduction
Key: Pink Band represents the Tropical Regions of the World where malaria is endemic
Sky blue band represents the Sub tropical, Temperate and Polar Regions

3. Artemisinin
Quinine Chloroquine
Primaquine
Piperaquine
Fig. 1: Structures of Some Commercial Antimalarial Drugs

4. Caesalpinia pulcherrima
Caesalpinia pulcherrima is an evergreen, low- branching and fast growing perennial shrub that can grow up to 4 m tall.
It is an ornamental plant, widely grown in household and community gardens and has an attractive inflorescence in orange, yellow and red. (Frisch et al., 2005)

5. Common Names
It is universally known as ‘Pride of Barbados’, ‘Peacock Flower’ , ‘Red Bird of Paradise’ and so on
It is locally known in Nigeria as Eko-omode by Yorubas, (Londa, 2004). It is called Akasibieka in Bini. (Patil et al., 1997; Roach et al., 2003).
Medicinal Uses
The plant has been used in traditional medicine as a stimulant, emenagogue, and abortificient (Srinivas, et al., 2003; Chiang et al., 2003).

6. Pharmacological Activities of Caesalpinia pulcherrima
Activity
Plant Part tested
References
Antiplasmodial
Leaves, stem bark
Venkatesalu et al., 2012; Ogu et al., 2012
Cytotoxic
Wood, aerial part, pod
Pawar et al., 2009; Chanda and Baravalia, 2011; Promsawan et al., 2003; Kumbhare et al., 2012; Akter et al., 2014
Analgesic
Flowers, leaves
Puratchikody and Nagalakshmi, 2005; Patel et al., 2010; Ramakrishna et al., 2011
Anti-inflammatory
Aerial part, leaves
Roach et al., 2003; Rao et al., 2005; Puratchikody and Nagalakshmi, 2005; Sharma and Rajani, 2011; Patel et al., 2010; Ramakrishna et al., 2011
Antimicrobial
Fruits, seeds, flowers, leaves, stem and root
Sudhakar et al., 2006; Chiang et al., 2003; Promsawan et al., 2003; Ragasa et al., 2002; Dhaked, 2011; Sowjanya et al., 2012; Vivek et al., 2013; Bungihan and Matias, 2013
Antiulcer
Bark, flower
Sharma and Rajani, 2011; Takawale et al., 2011; Ali et al., 2013
Antioxidant
Pods, flowers, stem bark, leaves
Hsu et al., 2012; Kumbhare et al., 2012;Bungihan and Matias, 2013; Yamuna and Padma, 2013; Vivek et al., 2013;Md. Asadujjamanet al., 2013; Zhipare et al., 2014
Hypolipidaemic
Leaves
Chichioco-Hernandez and Leonido, 2011
Anticonvulsant
Kumar et al., 2010
Antidiabetic
Flower
Balasubramanian et al., 2012

7. Plate 2: C. Pulcherrima in its Natural Habitat

8. Pulcherrin I
Pulcherrin J
Pulcherrin H
Pulcherrimin C
Pulcherrimin E
Pulcherrin B
Pulcherrimin A
Beta sitosterol
Pulcherrin A
Yodsaoue et al., 2011, Erharuyi et al.,2017, Pranithanchai et al., 2009, Patil et al., 1997,
Roach et al., 2003.
Fig. 4: Structures of Some Compounds Isolated From C. Pulcherrima

9. Justification for the Study
C. pulcherrima is being used for the treatment of malaria, but to the best of our knowledge, isolation and characterization of the active principles in it has not been scientifically reported.
It is therefore important to further evaluate the efficacy of the active agents in C. pulcherrima, in treating and managing malaria infections.

10. Aim and Objectives of the Study
The Aim of the Study is as Follows:
to isolation and characterise the antimalarial active compounds in the stem bark of Caesalpinia pulcherrima

11. Methodology Sample Collection and Preparation
The fresh stem bark of C.pulcherrima was collected from Ugbowo Campus, University of Benin, Benin City, Nigeria.
The plant sample was identified and prepared by the method described by Ogbeide et al., 2018

12. In Vitro Antimalarial Assay
The in vitro antimalarial assay procedure utilized an adaptation reported by Makler et al., 1993.
The assay was performed in a 96-well microplate and included two P. falciparum clones [Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine- resistant)].
In the primary screening, the crude plant extract was tested, in duplicate, at a single concentration 16µg/mL, only on the chloroquine sensitive (D6) strain of P. falciparum.

13. The extract showing greater than 50% growth
The extract showing greater than 50% growth inhibition of the parasite was subjected to the secondary screening.
The standard antimalarial agents chloroquine and artemisinin were used as positive controls, with dimethylsulfoxide (DMSO) (0.25%) as the negative control.

14. UV, IR ,MS, 1HNMR, 13CNMR and 2D-NMR Invitro anti-malarial activity
Schematic Representation of the Extraction, Fractionation and Isolation Procedure (Ogbeide et al., 2018)
Plant Materials (2.5 kg)
Maceration, methanol
Crude Extracts (200 g)
EF, SiO2 13x150cm
HE (0.45g)
HEEA (1:1) (38.5g)
EA (25g)
EAME (127g)
ME (4g)
RCC
RCC
Compound 1-11
Compound 12 & 13
UV, IR ,MS, 1HNMR, 13CNMR and 2D-NMR
Key:
HE= n-Hexane 100%
EA=Ethyl acetate 100%
ME=Methanol 100%
HEEA=Hexane:Ethyl acetate50%
EAME=Ethyl acetate:Methanol 50%
EF= Exhaustive Fractionation
RCC= Repeated Column Chromatography
Invitro anti-malarial activity

15. Results and Discussion

16. Characterisation of The Isolated Compounds
Fig. 8: Chemical Structures of Cassane and Vouacapane Skeleton

17. Physical Constants and Spectra Data of Compound 1 from C
Physical Constants and Spectra Data of Compound 1 from C. pulcherrima Stem Bark
State: white solid
HR EI-MS m/z: : C29H36O5
yield: 125.5mg
DBE or DoU: 12
mp: OC
Rf : 0.60 (4x9.5cm); Hex: EtOAc(8.5:1.5)
UV: 222, 278nm (λmax)

18. Fig. 9 : UV spectral of Compound 1

19. Fig. 10 : IR Spectral of Compound 1
C---H
Stretch of SP2
Ar-H bending
C=C
stretch
=C-H bending
O---H
Stretch
C---H
Stretch of SP3
O---C
stretch
C=O
stretch
Fig. 10 : IR Spectral of Compound 1

20. HR EI-MS m/z: : C29H36O5 M+
iii
Fig. 11: EI-MS m/z: of Compound 1

21. Fig. 13 : 1HNMR of Compound 1

22. Fig : 1HNMR of Compound 1

23. Fig : 1HNMR of Compound 1

24. Aromatic signals Fig. 15 : 13C NMR of Compound 1 C-16 C-15 C-7 C-2’

25. Pulcherrin A
IUPAC Name: 6β-hydroxy-7β-cinnamoyloxyvouacapen-5α-ol

26. Rf : 0.716 Hex:EtOAc (8:2) (4x6cm) UV: 222, 230nm (λmax)
Compound 2
State: white powder
yield: 12.5mg
HR EI-MS m/z: C27H34O4 :
DBE: 11
Mp: OC
Rf : Hex:EtOAc (8:2) (4x6cm)
UV: 222, 230nm (λmax)

27. Fig. 18 : IR Spectral of Compound 2

28. M+
Fig. 19 : EI-MS of Compound 2

29. Compound 2
Fig. 20 : 1HNMR of Compound 2

30. Pulcherrin I:
IUPAC Name: 6β-benzoyloxyvouacapen-5α-ol

31. P. falciparum (D6) P. falciparum (W2) IC50 (µM) SI IC50 (µM) VERO IC50
Table 1: Secondary Antiplasmodial Screening against P. falciparum D6 and W2 Clones:
P. falciparum (D6)
P. falciparum (W2)
IC50 (µM) SI
IC50
(µM)
VERO IC50
Compound 1
7.84
1.30
5.36
1.91
10.25
Compound 2
11.27
1.00
Artemisinin
0.0937
9.0
0.1062
16.86
Chloroquine
0.4698
1.4
14.88

32. Conclusion
The study has demostrated that cassane diterpenoids from the stem bark of Caesalpinia pulcherrima could be a potential source of lead bioactive compounds for use as chemotherapeutic agents against D6 and W2 P. falciparum, with selectivity indices lower than the standard antimalarial drugs.
The study also validates the ethnomedicinal use of C. pulcherrima in the management of malarial

33. References
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Chin, Y., Balunas, M. J., Chai, H. B. and Kinghorn, A. D. (2006). Drug Discovery from Natural Sources. The AAPS Journal, Vol. 8(2), p
Patel, S. S., Verma, N. K., Chatterjee, C., Gauthaman, K. (2010). Screening of Caesalpinia pulcherrima Linn Flowers for Analgesic and Anti-inflammatory Activities. International Journal of Applied Research in Natural Products 3(3), p.1-5
Tan, R. X., Zheng, W. F., Tang H. Q. (1998) Biologically active substances from the genus Artemisia. Planta Med., 64, p
Kovács, P., Csaba, G., Pállinger, E., Czaker, R. (2007). Effects of taxol treatment on the microtubular system and mitochondria of Tetrahymena. Cell Biol Int., 31(7), p
Sarker, S.D., Latif, Z. & Gray A.I. (2006). Natural Products Isolation: An Overview; (Second Edition), Humana Press, New Jersey, United States of America,

34. Recommendation for Further Studies
The structure activity relationship of the pure compounds should be carried out as this could lead to the synthesis of novel, less toxic and less expensive bioactive agents for antimalarial activity
More toxicity studies should be done on the isolated compounds to establish their toxicity level

35. ACKNOWLEDGEMENTS

42. Special thanks to TWAS and ICCBS for the Ph. D
Special thanks to TWAS and ICCBS for the Ph.D. Postgraduate Fellowship Award which gave part of the financial support for this research.
This work was also partly supported by USAID/HED grant to Prof. A. Falodun to conduct malaria research
I also appreciate the National Center for Natural Product Research (NCNPR), Institute of Pharmaceutical Sciences, University of Mississippi for the use of their laboratory for the antiplasmodial study.
I greatly appreciate the assistance of Dr. Mrs. Nuzhat, Dr. Habibab, Dr. Mujeeb, Dr. Adhikari and Mr. Naviv, during the process of isolation and characterization at the H.E.J. Research Institute of Chemistry, ICCBS, Pakistan.

43. HE THAT HAS HIS NUT CRACKED BY BENEVOLENCE, GRACE AND PRIVILEGE SHOULD NOT FORGET TO BE GRATEFUL AND HUMBLE!

44. THANKS FOR LISTENING