MATERIALS AND METHODS

The plant materials viz., leaf, stem, seed and root of the Jatropha species were collected from different localities in the Niger Delta between September 2009 and March 2010. The research was conducted from March to October 2010. The plants were identified with the help of The Flora of West Tropical Africa (Hutchinson and Dalziel, 1958) and are deposited in the Herbarium, Department of Plant Science and Biotechnology, University of Port Harcourt.

Qualitative and quantitative determination of secondary metabolites in Jatropha species: Extracts of samples of leaf, stem, seed and root from each species were subjected to preliminary screening for the presence of secondary metabolites (alkaloids, tannins, flavonoids, phenols and saponins) using standard procedures (Trease and Evans, 1989) with some modifications. The test procedure for each compound is presented below.

Alkaloids: Five milliliter of 2% HCl was added to 2 mL of each plant extract in a test tube placed on a steam bath and warmed. It was filtered and divided into two parts for the following tests:

In both cases, the formation of precipitation indicated the presence of alkaloids.

Tannins: Five milliliter of the extract was treated with 2 mL of HCl and boiled for 5 min. The presence of red precipitate confirmed the presence of tannins.

Flavonoids: One milliliter of the extract was treated with 1 mL of dilute NaOH. The presence of a cloudy precipitate confirmed the presence of flavonoids.

Saponins: One milliliter of the extract was added to 4 mL of distilled water and shaken. A stable frothing or foaming indicated the presence of saponins.

Phenols: One milliliter of the extract was added to 1 mL of 10% FeCl₂ and mixed together. The presence of blue precipitate confirmed the presence of phenols.

After preliminary analysis to determine presence of these phytochemicals, the samples were further subjected to quantitative analysis to determine the percentage of each of these secondary metabolites in each plant part of each species. The following procedures were adopted:

Percentage alkaloids were computed as follows:

Where:

Tannins were computed thus:

Where:

Percentage Flavonoid was calculated by:

Where:

Where:

Saponin concentration was computed thus:

Where:

The experiments were performed in triplicates (or three times) for each parameter and the resultant percentages were summed up to deduce the average percentage for comparison of means for each parameter.

RESULTS

Alkaloids Qualitative assessment (Table 1) revealed that alkaloids were observed to be deeply present (++) in the leaves and present (+) in the root and stem of all four species. However, the quantitative analysis results (Table 2) show alkaloid distribution in the different plants to be in the following decreasing order: leaf (J. curcas 4.54%; J. multifida 2.85%; J. gossypifolia 2.81%; J. podagrica 0.33%), stem (J. gossypifolia 2.16%; J. curcas 1.94%; J. multifida 1.72%; J. podagrica 0.15%), seed (J. curcas 2.85%; J. multifida 2.63%; J. gossypifolia 2.36%; J. podagrica 0.18%) and root (J. curcas 1.83%; J. multifida 1.68%; J. gossypifolia 1.60%; J. podagrica 0.26%). A similar order was observed in the leaf and root.

Tannins: Tannin was present (+) in the stem of the four species investigated. It was very deeply present (+++) in the leaf of J. curcas and J. multifida and deeply present (++) in the seed of J. curcas and J. gossypifolia and root of J. multifida (Table 1). A decreasing order of the observed results of quantitative analysis of tannin concentration in the leaves and seeds of the species follows thus: leaf (J. curcas 7.43%; J. podagrica 6.79%; J. multifida 5.16%; J. gossypifolia 5.14%), seed (J. curcas 4.23%; J. multifida and J. podagrica 3.89% each; J. gossypifolia 3.52%). Also, the same tannin concentration of 2.73% (J. gossypifolia and J. podagrica) and 1.21% (J. multifida and J. podagrica) were recorded in the root and stem of the species respectively (Table 2).

Flavonoids: Flavonoids were observed to be very deeply present (+++) in the leaves of J. curcas and J. multifida and deeply present (++) in that of J. gossypifolia. They were observed to be present (+) in all other plant parts of the species (Table 1). The highest flavonoid concentration of 3.25% was observed in the leaf of J. podagrica (Table 2). Flavonoid was 2.76% in J. curcas leaf, 2.41% in J. gossypifolia and 2.18% in J. multifida. The same concentration of 2.14% (seed) and 1.28% (stem) were observed in J. multifida and J. podagrica. The least flavonoid concentration of 1.20% was observed stem of J. gossypifolia.

Table 1:	Preliminary (qualitative) screening of secondary metabolites in Jatropha species
+: Present, ++: Deeply present, +++: Very deeply present

Table 2:	Concentration of secondary metabolites present in Jatropha species

Saponins: The saponins were deeply present (++) in the leaf of the four species and seed of J. podagrica. They were observed to be present (+) in all other plant parts of the species. A descending order of the concentration of saponins in the leaf and stem of the species as revealed from the quantitative analysis follows thus: leaf (J. curcas 4.89%; J. gossypifolia 4.15%; J. multifida and J. podagrica 3.15% each), stem (J. podagrica 2.77%; J. multifida 2.73%; J. curcas 2.27%; J. gossypifolia 2.18%). The least saponin concentration of 2.16% was observed in the root of J. multifida while the highest concentration of 4.89% was in the leaf of J. curcas.

Phenols: Phenols were generally low qualitatively and quantitatively. They were observed to be deeply present (++) only in leaves of J. curcas and J. gossypifolia and present in all other plant parts investigated in the four species. The highest and least phenol concentrations of 0.59 and 0.13% were recorded in the stems of J. curcas and J. gossypifolia, respectively. While the seed of J. podagrica, J. multifida and J. gossypifolia were observed to have the same phenol concentration of 0.18%, the leaves of J. multifida and J. gossypifolia had 0.16%.

DISCUSSION

The results of the comparative qualitative and quantitative phytochemical analysis of secondary metabolites in the leaf, root, seed and stem of Jatropha species in the Niger Delta are presented in Tables 1 and 2. Burkill (1994) reported the presence of tannins and saponins in the leaf of J. gossypifolia and J. multifida respectively which is confirmed by this study. The qualitative screening from the presence study revealed the presence of alkaloids, tannins, saponins, flavonoids and phenols in all plant parts investigated but to varying intensities/qualities as shown in Table 1. Preliminary qualitative test according to Mallikharjunah et al. (2007) is useful in the detection of bioactive principles and subsequently may lead to drug discovery and development. Qualitative phytochemical screening of two species of Avicennia (Ganesh and Vennila, 2011) and three cucurbits (Irshad et al., 2010) revealed the presence of alkaloids, flavonoids, tannins, saponins and phenols in a manner suggesting close relationship of the species studied. Similar results (Table 1) were obtained in this research work which confirm the relatedness of the investigated Jatropha species as well as reveal their potentials in the drug industry. Specifically, the qualitative screening revealed that tannins were very deeply present in the leaves and stems of J. curcas and J. multifida and deeply present in the roots of J. curcas, J. gossypifolia and J. multifida. Likewise, alkaloids were deeply present in the leaf of J. multifida, the stem of all species except in J. multifida and the roots of J. curcas, J. gossypifolia and J. multifida. Olawale-Abulude (2007) undertook phytochemical screening of leaves of twenty-eight woody species from different plants families in Nigeria and discovered the presence of tannins, alkaloids and flavonoid in all samples. Thus these secondary metabolites seem cosmopolitan in plants but to varying degrees and types. Their different degrees of occurrence in plant parts confer taxonomic usefulness on them. This is similar to the findings of Nyananyo et al. (2010) on some Niger Delta plants.

Among the five groups of phytochemicals investigated from the leaf, root, seed and stem of the species, tannins were found to be the most abundant followed by saponins and alkaloids. Phenols were the lowest in concentration and together with flavonoids varied across the different plant parts. Specifically, tannins were concentrated to 7.43% in the leaves of J. curcas with corresponding concentration of 0.38% by phenols. Similarly, saponin (the most abundant phytochemical in the root of the species) with concentration of 3.62% (the highest in all seed samples) in the root of J. curcas had corresponding phenol concentration of 0.20%. A similar sequence with tannins being the most abundant phytochemical in plant parts, followed by saponins, alkaloids, flavonoids and then phenols as seen in this study has been reported by Mallikharjunah et al. (2007). Other recent investigation showcasing presence, distribution in plant parts and importance of these phytochemicals similar with the results of this investigation include Ogunkunle and Ladejobi (2006), Sunita and Abhishek (2008), Ferreira et al. (2009), Kumar et al. (2009) and Ganesh and Vennila (2011). It is noteworthy that while all these phytochemicals are present in the different Jatropha species connoting taxonomic affinity, the differences in their concentrations uniquely confers individualism on each species and thus support their being treated as taxonomic species. This agrees with Irshad et al. (2010) where phytochemical results established closer relationship between Lagenaria siceraria and Luffa cylindrical than Cucumis maxima.

This study reveals the presence of the subject secondary metabolites in the different Jatropha species to varying concentrations which is taxonomically useful; it also brings to bare the fact that the species are potential sources of these important phytochemicals. For instance, flavonoids are one of the most popular secondary metabolites possessing a variety of biological activities at nontoxic concentrations (Irshad et al., 2010). Dietary flavonoids are noted to play effective roles in cancer prevention (Ren et al., 2003; Aggarwal and Shishodia, 2006). Flavonoids together with the other secondary metabolites identified in the Jatropha species have been severally reported in other plants to show curative activity against diverse pathogens, used traditionally as analgesic, antimicrobial and soothing herbs (Hassan et al., 2004; Faruq et al., 2004; Olafimihan, 2004; Singh et al., 2009; Thirunavukkarasu et al., 2010; Ganesh and Vennila, 2011).