Identification of Compounds and Insecticidal Activity of the Root of Pride of Barbados ( Caesalpinia Pulcherrima L )

: Caesalpinia pulcherrima (Caesalpiniaceae) is an ornamental plant with several ethnomedicinal uses. The present study was designed to investigate the brine shrimp cytotoxicity and insecticidal activity of oil obtained from C. pulcherrima root. The powdered root was extracted with methanol and then defatted with petroleum ether (40-60 o C) to obtain a viscous oil. The oil was investigated for its brine shrimp cytotoxicity and insecticidal activity in vitro . The chemical constituents were identified by Gas chromatography-Mass spectrometry. The oil showed significant lethal effect against Artemia salina (Brine shrimp) with LC 50 of 23.85 µg/mL and mild insecticidal activity against Tribolium castaneum and Callosbruchus analis with percentage mortality of 20% and 40% respectively at 1 mg/cm 2 . GC-MS analysis identified 37 compounds mainly steroids, terpenoids and fatty acids.

The genus Caesalpinia consists of more than 500 species belonging to the family Caesalpiniaceae. The species are mostly woody occurring in tropical and subtropical regions of the world. The genus has been found to contain numerous phytochemicals such as alkaloids, tannins, saponins, glycosides, phenolics, steroids and terpenoids. Diterpenoids of the cassanetype have been isolated from various species of the genus. Literature reports have shown the species to possess interesting pharmacological activities, such as, antidiabetic, antiulcer, anti-inflammatory, analgesic, adaptogenic, antimicrobial, antirheumatic, antipyretic, antioxidant and anticancer activities (Carvalho et al, 1996;Sudhakar et al., 2006;Kannur et al., 2006;Srinivasan et al., 2007;Saravanan et al., 2008;Shukla et al., 2010;Sgariglia et al., 2011).
Caesalpinia pulcherrima L. Swartz is a well-known ornamental plant belonging to the family Caesalpiniaceae. The plant is commonly known as Pride of Barbados. The plant is native to Central America but well distributed in tropical and subtropical regions of Africa, Asia and Australia (Zanin et al., 2012). In traditional medicine, extracts from various parts of the plant have been used as stimulant, emenagogue and arbotificient (Srinivas et al., 2003;Chiang et al., 2003). Pharmacologically, the plant has been reported to possess antimicrobial, analgesic, anti-inflammatory, anthelmintic, antimalarial, antiulcer, cytotoxic, and antioxidant activities (Roach et al., 2003;Promsawan et al., 2003;Sudhakar et al., 2006;Pawar et al., 2009;Patel et al., 2010;Sharma and Rajani, 2011;Venkatesalu et al., 2012;Kumbhare et al., 2012). Phytochemicals including diterpenes, flavonoids, peltogynoids, steroids and glycosides have been isolated from various parts of the plant (McPherson et al., 1986;Cheet al., 1986;Ragasa et al., 2003;Maheswara et al., 2006;Pranithanchai et al., 2009;Das et al., 2009;Das et al., 2010;Yodsaoue et al., 2011). In the present study, we report the brine shrimp cytotoxicity and insecticidal activity of the oil obtained from the root of C. pulcherrima and the identification of the constituents by GC-MS analysis. Extraction: The powdered plant material (2.9 kg) was macerated in methanol (7.5 L) at room temperature for 7 days. The extract was concentrated to dryness using a rotary evaporator at reduced pressure. The crude oily extract (240 g) was suspended in 250 mL of methanol-water (4:1) and extracted with petroleum ether (3 X 500 mL) to obtained a viscous oil.

MATERIALS AND METHOD
Brine shrimp Lethality Assay: Hatchine of brine shrimp: Artemia salina (Brine shrimp) eggs were hatched in the hatching tray half-filled with brine solution incubated at 37 o C.
Sample Preparation: Test sample (20 mg) was dissolved in 2 mL of DMSO to make a stock solution of 10 mg/mL. Concentrations of 10, 100 and 1000 µg/mL were prepared by transferring 5, 50 and 500 µL to vials and the solvent was allowed to evaporate overnight.
Treatment with test sample: After 2 days of hatching and maturation as nauplii, 5 mL of artificial seawater containing 10 larvae was transferred into each well containing different concentration of the test sample (final concentration 2, 20 and 200 µg/mL). The vials were incubated at 25-27 o C for 24 h under illumination. Seawater and etoposide were used as negative and positive control respectively. Each concentration was done in triplicate and the LC 50 determined.
Insecticidal Activity Screening (Impregnated filter paper test): Test insects: Tribolium castaneum, Sitophilus oryzea, Rhyzopertha dominica, Trogoderma granarium and Callosobruchus analis were reared in the laboratory at 25-27 o C in 50% relative humidity in plastic bottles containing sterile breeding media. Insects of uniform age and size were used for the experiment.
Test procedure: Day-1: Filter papers (90 mm each) were put in petri plates and loaded with the test sample at 200 mg in 3 mL ethanol. The plates were left at room temperature for 24 h to evaporate the solvent completely. Day-2: After the evaporation of solvent, 10 insects of each specie were placed in each plate using a clean brush. The plates were Incubated at 27 o C for 24 h at 50% relative humidity in the growth chamber. Permethrin was used as the standard insecticide while ethanol served as control.
Day-3: The number of surviving insects belonging to each specie were counted and the Percentage mortality was calculated using the formula: Percentage mortality

RESULTS AND DISCUSSION
Prior to the discovery of the organochlorine and organophosphate insecticides in the late 1930s and early 1940s, botanical insecticides have remained an important weapon in managing insect pests disease of farm produce (Forim et al., 2012). Botanical source insecticides may serve as alternatives to popularly used synthetic chemical insecticides due to its biodegradability, and non-toxicity to none target organisms. Defatted methanol extract of Caesalpinia pulcherrima root bark yielded 9.23% w/w of the fixed oil. The oil was evaluated for its in vitro cytotoxicity using the brine shrimp (Artemia salina) lethality assay and its potential insecticidal activity was evaluated by the impregnated filter paper test.
The insecticidal activity was investigated against four common stored grain pests including red flour beetle (Tribolium castaneum), rice weevil (Sitophilus oryzea), lesser grain borer (Rhyzopertha dominica) and pulse beetle (Callosobruchus analis). The oil showed 20% mortality against Tribolium castaneum and 40% mortality against Callosobruchus analis at 1019.10 µg/cm 2 concentration. Compared to the standard insecticide (permethrin), the oil was significantly less active against the tested grain pests ( Table 2).
The LD 50 values recorded in this present study is lower than those reported previously, indicating higher cytotoxic activities of the oil extract. Compared to the standard insecticide (permethrin), the LD 50 values for the oil were higher. The lower LD 50 for permethrin was as a result of their highly purified state as against complex composition of the oil extract. Considering the effect of synthetic insecticides especially on environment and food, LD 50 values for botanical insecticides do not need to be low before it is accepted.

Conclusion:
The present investigation has shown the cytotoxic effect of C. pulcherrima root oil. The identified compounds may serve as potential cytotoxic agents and may find usefulness in the control of insect pests if properly harnessed.