Variation of naphthoquinones in different stages of Euclea natalensis seedling ( P b 0.01). Each value of a bar is a mean of four replicates. Values of the bars within each compound not followed by the same letter are significantly different. 

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... 32.5: 0.5) and was used in isocratic mode at a flow rate of 0.8 mL/min at 25 °C. The run time for each injection was 22 min. Each crude extract was dissolved in 2 ml acetonitrile, the sample injection volume was 10 μ l and three injections per replicate were conducted. Individual naphthoquinones were identified based on the retention time and UV spectrum of purified standards. For quantitative analysis, pure compounds were dissolved in acetonitrile and a range of dilutions from 22.5 μ g/ml to 2.25 μ g/ ml was prepared. The dilutions were injected into the HPLC at a volume of 10 l, in triplicates. The absorbance wavelengths of pure 7-methyljuglone and diospyrin were 430 nm, whereas shinanolone was detected at 325 nm. Each sample extract was injected four times and their particular quantities were determined by standard curves generated for each compound. Mass of ground material (g), the standard curves slopes, volume of injection and areas of individual peaks were used to calculate the concentration of each naphthoquinone in mg/kg of plant material. The mean values of four concentrations of each secondary compound from each sample were considered for analysis. The results were statistically analysed using one-way analysis of variance (ANOVA) and least significant differences ( P = 0.05) were determined according to the MSTATC computer program. Preliminary examination of the chloroform extracts from shoots and roots of E. natalensis showed little variation in the chemical profiles of the respective samples analyzed (not shown). All three naphthoquinones (Fig. 1) were detected in all extracts except the seeds from which shinanolone ( 1 ) was the only compound visualized. There were very few compounds present in the fingerprints of shoot extracts and the number of compounds decreased with growth. More compounds were depicted by TLC chromatograms of root extracts. The qualitative analysis was only significant in prior assessment on the presence of the specified naphthoquinones in the crude extracts and therefore could not be used for quantification of the individual metabolites. HPLC analysis of naphthoquinones in seed extracts revealed the presence of diospyrin ( 3 ), which was not detectable from the TLC plates. Levels of the three naphthoquinones were very low or less detectible at dormancy, with shinanolone ( 2 ) displaying relatively higher concentrations (87.5 mg/kg) than diospyrin ( 3 ) and 7-methyljuglone ( 2 ) (6.2 and 0 mg/kg respectively). This could be attributed to the fact that at dormancy, seeds are mainly protected from attack by microorganisms by dehydration and the impermeability of the seed coat (Baskin and Baskin, 1998; Ceballos et al., 1998). In general, secondary metabolites in seeds accumulate in relatively low concentrations when compared to primary products such as starch and lipids (Mayer and Poljakoff-Mayber, 1982). The quantitative analysis of chloroform extracts from shoots clearly showed a decrease in naphthoquinone accumulation during growth (Fig. 2). The three naphthoquinones increased rapidly during the third growth stage, with an obviously elevated content of 7-methyljuglone ( 2 ) (1310 mg/kg), which was approximately three times higher than shinanolone ( 1 ) and diospyrin ( 3 ) (260.4 and 369.5 mg/kg respectively). The observed increase correlated with the shoot development initiation of the hypocotyls, which were partly underground and therefore required the same protection as the subterranean parts. This was followed by a decline in the content of the three naphthoquinones in the fourth and fifth growth stages. There was a significant correlation ( P b 0.01) in the interaction between growth and accumulation of naphthoquinones in shoot development of E. natalensis seedlings. Comparative examination of chloroform extracts from roots showed marked quantitative differences in the mean concentrations of naphthoquinones under consideration (Fig. 2). A significant correlation was established ( P b 0.01) between root growth and the accumulation of naphthoquinones. The concentration of shinanolone ( 1 ) fluctuated marginally during root growth and its levels ranged between 208.2 and 311.5 mg/ kg. The accumulation of 7-methyljuglone ( 2 ) peaked during the third growth stage (3693 mg/kg) and decreased significantly during the fifth growth stage (319.8 mg/kg). The mean concentrations of diospyrin ( 3 ) showed a different pattern to those depicted by 7-methyljuglone and shinanolone, and its accumulation was directly proportional to the relative growth of the seedlings. Diospyrin ( 3 ) was also detected at high levels during the fifth growth stage (6048 mg/kg), when the concentrations of the other naphthoquinones were very low. This was the highest mean value quantified from the separate shoot and root samples studied. Considering the plant as a whole, the mean concentrations of the respective naphthoquinones studied varied significantly ( P b 0.01) during the five growth stages (Fig. 3). All the chemical constituents considered were very low at dormancy and increased progressively up to the third stage. From this stage onward, they fluctuated independently, with the exception of diospyrin ( 3 ), which accrued incrementally with growth. The content of shinanolone ( 1 ) ranged from 87.5 mg/kg to 1047 mg/ kg and its high concentration was detected during the fourth seedling stage. 7-Methyljuglone ( 2 ) was quantified at a high level of 5003 mg/kg during the third seedling stage and was not detected from the seed samples. The minimum content of diospyrin ( 3 ) was detected initially at the first growth stage (6.2 mg/kg), whereas its highest mean levels were apparent during the fifth growth stage (6182 mg/kg). The level of consistency shown by the production of diospyrin ( 3 ) could also suggest its involvement in maximizing the fitness of seedlings. Seedlings are vulnerable to pathogen attacks and for efficient protection chemical defenses must be deployed and accumulate very early in the seedling development (Ceballos et al., 1998). From these results it is evident that naphthoquinones are synthesized from early stages of development. The results from this study indicate that naphthoquinones accumulated mainly in the roots, with the concentrations in shoots intermediate and those in the seeds comparatively low. Repcak et al. (2000) indicated that the amount of naphthoquinones vary in different tissues of the same plant, and during the growing seasons in a given population. Based on our study, increased yields of naphthoquinones can be obtained from cultivated seedlings of E. natalensis. Elevated yields of shinanolone ( 1 ) can be obtained from the fourth seedling stage. 7-Methyljuglone ( 2 ) and diospyrin ( 3 ) could be harvested for optimum levels at the third and fifth seedling stages respectively. Large-scale production of seedlings will help in reducing the pressure that is exerted on natural population by plant gatherers. We thank the National Research Foundation for their financial support. Mahdi Ziaratnia is acknowledged for his assistance with statistical ...