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.

Natural Products Malaria Who is at Risk? Introduction www.wikimedia.org/wikipedia/commons/1/1e/Lactitude_zones.png 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

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

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)

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).

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

Plate 2: C. Pulcherrima in its Natural Habitat

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

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.

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

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

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.

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.

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

Results and Discussion

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

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: 464.2563: C29H36O5 yield: 125.5mg DBE or DoU: 12 mp: 122-124 OC Rf : 0.60 (4x9.5cm); Hex: EtOAc(8.5:1.5) UV: 222, 278nm (λmax)

Fig. 9 : UV spectral of Compound 1

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

HR EI-MS m/z: 464.2563: C29H36O5 M+ iii Fig. 11: EI-MS m/z: of Compound 1

Fig. 13 : 1HNMR of Compound 1

Fig. 13.1 : 1HNMR of Compound 1

Fig. 13.2 : 1HNMR of Compound 1

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

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

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 : 422.2457 DBE: 11 Mp: 128-129OC Rf : 0.716 Hex:EtOAc (8:2) (4x6cm) UV: 222, 230nm (λmax)  

Fig. 18 : IR Spectral of Compound 2

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

Compound 2 Fig. 20 : 1HNMR of Compound 2

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

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

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

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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

ACKNOWLEDGEMENTS

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.

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