Antiplasmodial and Acute Toxicity Studies of Fractions and Cassane-Type Diterpenoids from the Stem Bark of Caesalpinia pulcherrima (L.) Sw

Majority of the human race, especially from developing countries depend on the traditional system of medicine for the management of various diseases. Thousands of plant species are used medicinally, chiefly as herbal preparations in the indigenous systems of medicine in different countries and are sources of safe, cheap and potent drugs which have continued to be effective, successful, popular and modern chemistry has not been able to substitute a good number of them .Plants are natural reservoirs of medicinal agents which are widely assumed to be safe, but many are potentially toxic. In spite of the millions of chemical compounds currently synthesized in the laboratory, and available for screening for the action of therapeutic value, natural products,

particularly of plants origin remain the most important sources of new drugs. 3In recent years, a significant renewal of interest in natural products as potential sources for new medicines has been observed by scientists as well as pharmaceutical companies 4 .Thus, searches for new drugs with better and cheaper substitutes from plant origin are a natural choice.The medicinal value of these plants lies in some chemical substances that produce a definite physiological action on the human body. 5n the face of considerable progress made in the treatment of parasitic diseases, malaria remains a major public health challenge in prevalent areas of the world, predominantly because of the widespread resistance of malaria parasites to currently existing anti-malarial agents, the resistance of the mosquito vectors to currently obtainable insecticides, the limited success in the development of malarial vaccines and the devastating and undesirable reactions of conventional anti-malarial drugs. 1 These developments and the intricacy of producing efficient vaccines underscore the imperative need for new antimalarials.In developing countries, especially in Africa, accessible treatments against malaria are mainly based on the use of traditional herbal medicine.Indeed, local plants play a significant role in treatment of many infectious diseases and a good number of them rely on herbal remedies 1 .C. pulcherrima (CP) is a flowering plant in the legume family fabaceae.They are normally grown as ornamental flowers in tropical gardens 6 .The plant is known to be a rich source of cassane-type diterpenoids, lupeol, lupeol acetate, carotenoids, quercetin, rutin, beta sitosterol, glycosides, phenols and steroids 7,[8][9][10] .Different parts of the plant have been known to possess great medicinal value globally.The aerial parts are used as an abortifacient, emmenagogue, purgative, stimulant and emollient among the Nigerian communities. 3The fruits, flowers, leaves, and stem barks are used as regular remedies for the treatment of a number of disorders including pyrexia, menoxenia, wheezing, bronchitis and malarial infection among the Chinese communities 11 .The sap from the leaves is also said to be antipyretic, the sap from the flower is known to treat sores and the seeds are said to heal a bad cough, breathing difficulty and chest ache.Extracts from the root are also said to be abortifacient. 6,12Different workers, have reported the antiplasmodial activities of the leaves and stem bark of CP, 13,14 in these studies, the leaves of CP were shown to exhibit moderate antiplasmodial activity against Plasmodium berghei (PB), while the stem bark showed significant antiplasmodial effects with P˂0.005 and 14.6 ± 1.3 μg/mL 15 against PB.In spite of these recognized arrays of reports on the therapeutic and pharmacological potentials of this plant, there is yet a dearth of information on the in vitro antiplasmodial activity on the fractions and isolated compounds from the stem bark of C. pulcherrima.Therefore, this study aims to investigate the in vivo acute toxicity, cell cytotoxicity, and in vitro antiplasmodial potentials of the fractions and isolated compounds from the stem bark of C. pulcherrima, in order to validate its ethnomedicinal use in the treatment of human malaria infection in the tropics.

General experimental
The NMR spectra were determined on Bruker Avance AV-300 and AV-400 spectrometers in C3D6O.Chemical shifts are expressed in parts per million (ppm) using TMS as the internal standard.EI-MS was recorded at an ionizing voltage of 70 eV (direct probe) on a double focusing magnetic sector mass analyzer (JEOL JMS-600H).

Collection and preparation of C. pulcherrima stem bark
The

Extraction of Crude powdered sample
The powdered stem bark of C. pulcherrima (2.5 kg) was extracted with 7 L of methanol by maceration at room temperature for 96 hours.The extract was concentrated to dryness using a rotary evaporator at reduced pressure.The concentrated extract was weighed and the percentage yield calculated based on the initial weight of the crude powdered sample.The extract was stored in an air-tight container and kept in a refrigerator at 4 o C until further analysis.

Acute Toxicity Studies Animals
Twenty-Five Swiss albino mice of either sex with average weight of 24.5 g obtained from the Nigerian Institute of Medical Research (NIMR), Lagos, Nigeria, were used for this investigation.The mice were housed in standard environmental condition of ambient temperature and relative humidity, and 12 h light and 12 h dark cycles.They had free access to standard pellet diet and water according to the National Institutes of Health (NIH) Guide for the care and use of laboratory Animals. 19

Experimental
The acute toxicity study was performed by a method described by Igbe et al, 20 with modifications.The mice were divided into five separate groups of five mice each labeled 1-5.Groups 2-5 were the test groups, while group 1 was the control.Groups 2-5 received 1000, 2000, 4000, 8000 mg/kg of the methanol crude extract suspended in gum acacia respectively by oro-gastric syringe, while group 1 (control) received 10 % gum acacia solution by oral route.The animals were observed for common symptoms of toxicity and mortality within 24 hours, and the animals that survived after 24 hours were observed for any signs of delayed toxicity for 14 days.The median lethal dose (LD50) was calculated using Equation 1: LD50 = √a * b Where a = lowest lethal dose (where death of mice occurred) b = highest non-lethal dose (where no death of mice occurred) 20,21 Antiplasmodial/Parasite lactate dehydrogenase assay The in vitro antiplasmodial assay procedure employed was an adaptation of the parasite lactate dehydrogenase (pLDH) assay developed by Makler et al. 22 The assay was performed in a 96-well micro plate and included two P. falciparum clones.[Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine resistant)].In primary screening, the different fractions were tested in duplicate only on the chloroquine-sensitive (D6) strain of P. falciparum.The fractions and compounds showing ˃50% growth inhibition of the parasite were subjected to screening.The standard antimalarial agents chloroquine and artemisinin were used as positive controls, with DMSO (0.25 %) as negative control.The selectivity indexes were determined by measuring the cytotoxicity of samples on mammalian cells (VERO, monkey kidney fibroblast).All experiments were carried out in duplicate.

Results and Discussion
The percentage yield from crude extract and fractions are as shown in table 1 below: The phytochemical analysis of the methanol and aqueous crude extracts of the stem bark of C. pulcherrima revealed the presence of saponins, phenols, flavonoids terpenoids and tannins.These findings are in agreement with those of Sivasankari 25 and Sharma and Rajani 26 .Phytochemicals are compounds that act as free radical scavengers to help eradicate the highly charged oxygen molecules that are byproducts of metabolized oxygen 27 and are believed to provide several Ogbeide et al., 2018 health benefits. 28Saponins are known to exhibit anti-inflammatory activity and erythrocyte haemolysis. 29,30 lavonoids commonly found in fruits and vegetable have been linked to decreased risk of mortality from coronary heart diseases and many more. 31Alkaloids, Phenols and Tannins are also known for their antimicrobial, antidiarrhoeal and anthelmintic properties. 32n screening drugs, determination of LD50 is usually an initial step in the assessment and evaluation of the toxic properties of a substance. 33n this study, there was no sign of toxicity and change in behavioral pattern observed in the experimental animals treated with crude extract for doses up to 2000mg/kg.However, at doses 4000 to 8000 mg/kg body weight, there were obvious signs of toxicity: lethargic, anorexia, sleepiness and deaths.The LD50 was established at 5656.85 mg/kg.These results supported the findings of Sharma and Rajani on the aerial parts of C. pulcherrima 17 and the findings of Ogu et al., 34 on the aqueous extract of the stem bark of C. pulcherrima. 28'Based on Hodge and Sterner scale', a test drug administered orally is considered extremely toxic at ≤ 1 mg kg -1 , highly toxic at 1-50 mg kg -1 , moderately toxic at 50-500 mg kg -1 , slightly toxic at 500-5000 mg kg -1 , practically nontoxic at 5000-15,000 mg kg -1 and relatively harmless at ≥15,00 mg kg - 1 . 35However, it was reported 36 that any substance with LD50 ≥ 1000 mg/kg is considered to be low toxicity or relatively safe.8][39] The three fractions exhibited significant antiplasmodial activities however, the n-hexane:ethyl acetate fraction(1:1) (HEEA) exhibited the highest antiplasmodial activity against the chloroquine sensitive (D6) and chloroquine resistant (W2) P. falciparum clones at IC50 3.7 and 5.3µg/mL, respectively, while the methanol fraction (ME), exhibited the lowest antiplasmodial activities against the D6 and W2 P. falciparum clones at IC50 7.0 and 5.5 µg/mL, respectively.Due to the significant antiplasmodial activities exhibited by the three fractions, they were further assessed for their cytotoxicity activity in order to determine their selectivity index (see table 4).The significant antiplasmodial activity exhibited by these fractions against chloroquine sensitive and chloroquine resistant clones of P. falciparum could be due to the high presence of some active phytochemical components which may be acting in synergy with one another or singly to exert as implicated by Ogu et al. [31] ,in the in vivo antiplamodial activity of the aqueous extract of C. pulcherrima stem bark.The HEEA exhibited the highest antiplasmodial activity against both clones of P. falciparum and as a result, was further subjected to column Chromatography to obtain compounds 1 and 2. Compound 1 (CP13) was isolated as white crystalline solid and displayed a molecular ion at 448.2617(M + ) by HREIMS corresponding to a molecular formula C29H36O4, signifying twelve (12) degrees of unsaturation.The MS, 1 H-NMR and 13 C-NMR also revealed the presence of trans cinnamoyl moiety.The 1 H and 13 C NMR spectroscopic data (see Table 6) were characteristics of the cassane-type diterpenoids.The spectroscopic data was compared with that of literature and was found to be Pulcherrin J. 40 The observed HMBC correlations of H-6 resonating at δ 5.43(t, J=3.0) to the carbonyl carbon of the cinnamyl side chain at δ 166.05 (C-1´) confirmed the location of the trans-cinnamoyloxy side chain at C-6.Thus compound 1 (CP 13) was assigned to be 6β-cinnamoyloxyvouacapen-5α-ol and was named Pulcherrin J. Compound 2 (CP93-123) was isolated as colourless prism and had a molecular formula of C29H36O5 by HREIMS.The 1 H and 13 C are comparable to that of compound 1 except that the cinnamoyloxy group attached to C-6 in compound 1 has been interchanged and attached to C-7.In much the same way, the beta hydrogen attached to position 7 in compound 1 has been replaced by hydroxyl group.Compound 2 was successfully characterized through comparison of its spectra data with those reported in literature and was assigned as 6β-cinnamoyl-7βhydroxy-vouacapen-5α-ol. 41mpounds 1 and 2 were evaluated using the in vitro pLDH assay against the causative parasite of human malaria; P. falciparum [D6; chloroquine sensitive and W2; chloroquine-resistant strains].The investigation of the isolated compounds revealed significant inhibition of parasites growth in D6 and W2 clones with IC50 values of 10.25-10.62µM and 10.25-10.62µM, for compounds 1 and 2, respectively.The median lethal dose as calculated from equation 1 above is LD50 = 5656.9mg/kg.Compound 2 (IC50 value; 10.25 µM) showed a relatively higher antiplasmodial activity than compound 1 for both clones.The IC50 values of the standard test drugs (artemisinin and chloroquine) (table 5) were significantly lower than those of the isolated compounds.
The Selectivity Indexes (SI) of the isolated compounds were observed to be relatively lower than those of the standard drugs, revealing that the standard drugs are relatively less toxic than compounds 1 and 2.

Table 1 :
Percentage yield of crude extract and fractions of C. pulcherrima stem bark.

Table 2 :
Phytochemical analysis of the crude extract of C. pulcherrima stem bark.

Table 3 :
Oral acute toxicity results of the crude extract of C. pulcherrima stem bark in mice.

Table 4 :
Activity of C. pulcherrima fractions against P. falciparum.

Table 5 :
Antimalarial screening of compounds 1 and 2 against P. falciparum D6 and W2 Clones and their selectivity indexes.

Table 6 :
13C and 1 H NMR data of Compounds 1 and 2.