Chemical composition fluctuations in roots of Plumbago scandens L . in relation to floral development

Plumbago scandens L. is a Brazilian tropical/subtropical species that occurs along the coast. Chemically it is mainly represented by naphthoquinones, flavonoids, terpenoids and steroids. The aim of the present work is to study quantitative changes in the root metabolic production of Plumbago scandens during different physiologic developmental stages relative to floration. The results indicated the presence of four substances in the extracts: plumbagin, epi-isoshinanolone, palmitic acid and sitosterol, independent on developmental stage. The naphthoquinone plumbagin has always showed to be the major component of all extracts. Naphthoquinones exhibited their highest content during floration, while the content of the two others components decreased during this stage, revealing an inverse profile. The chemical composition changed depending on the plant requirements.


INTRODUCTION
Plumbago scandens L. belongs to the family Plumbaginaceae, order Plumbaginales, superorder Plumbaginanae, according to Dahlgren 1989.In this system of classification, Plumbaginales is positioned in the vicinity of Caryophyllales and Polygonales, suggesting possible affinities.According to the system proposed by the Angiosperm Phylogeny Group, the family Plumbaginaceae is placed with Polygonaceae, in the order Caryophyllales (APGIII 2009).Although they are systems that use different tools, in both cases the relationship with Caryophyllaceae and Polygonaceae was pointed.
Plumbago scandens is a subshrub with white flowers quite widespread in Brazil, being found along the coast.It is a native species found in a typical vegetation of "restinga", which is characterized by high luminous intensity, sandy soil and water restriction.This plant has already disappeared in many places due to human's activity.It can also be found in Southerm Florida, Texas and Arizona, Mexico, Central America, the west Indies and South America (Verhoek-Williams 1970).Popularly this species is known as "louco" since their leaves are used as nape compresses in people with mental disorders in order to calm them down (Braga 1960 apud Lorenzi andMatos 2002).P. scandens is also known as "caataia", "caapomonga", "folha-de-louro", "erva-dodiabo", among others (Lorenzi and Matos 2002).According to Mors et al. 2000 apud Lorenzi andMatos 2002, several uses are attributed to preparations obtained from roots, such as purgative and local anesthetic.It is also used as decoction or infusion to soothe toothaches and earaches, as well as to reduce joints inflammation.There are few studies in the literature about the chemistry of the species.However, there is a record production of naphthoquinones, steroids and flavonoids (Bhattacharyya and Carvalho 1986, Paiva et al. 2004, 2002).Among the isolated compounds, the naphthoquinone plumbagin deserves attention by the description of numerous pharmacological activities, such as antimicrobial (Paiva et al. 2003), anti-tumoral (Devi et al. 1999, Lin et al. 2003), trypanocidal (Sepúlveda-Bozza and Cassels 1996) and antimalarial (Suraveratum et al. 2000), among others.This naphthoquinone is mostly found in roots of several species of the genus Plumbago (Van der Vijver 1972).
Organic compounds make up the food for the cell and the structural components of the wall and the protoplasm, besides other special compounds, such as hormones, pigments and enzymes (Weier et al. 1982).Depending on the plant physiological stage, these compounds show changes in their contents.
The production of secondary metabolites is the result of complex interactions among biosynthesis, transport, reservoir and degradation.Each process is controlled by genes, hence it will be influenced by three main factors: hereditarity, ontogenesis and surroundings (Robbers et al. 1996).
This work aimed to verify the micromolecular composition changes in roots of Plumbago scandens L. during the floral development in order to analyze the influence of physiological stages in chemical production.

SOLVENTS
The solvents used (chloroform and ethyl acetate) were PA grade.

PLANT MATERIAL
Plumbago scandens L. was collected at Fundação Oswaldo Cruz campus, Rio de Janeiro, Brazil.A voucher of this plant was deposited at the Instituto de Pesquisas Jardim Botânico do Rio de Janeiro Herbarium (RB) under the number 340.340.

EXTRACTS PREPARATION
The roots of P. scandens were collected in 2001 in different periods (before, during and after floral development, which corresponded to the months of March, April and May, respectively).The dried powdered roots of each plant material (9,5g) were submitted to a dynamic extraction with 300 ml of chloroform (three extractions of 100ml).All extracts were evaporated to dryness under reduced pressure.

SAMPLE PREPARATION
A portion of each crude chloroform extract (2,0 mg) was dissolved in ethyl acetate (1 ml) and injected into a gas chromatograph coupled with a mass spectrometer.The injections were performed in triplicates.

CHROMATOGRAPHIC CONDITIONS
The following conditions were used: helium as the carrier gas, mass detector, detector temperature = 280 • C, injector temperature = 270 • C, flow rate 1,0 ml/min, split of 1:20, injection volume = 1,0 μl, initial temperature = 120 • C and oven programme from 5 • C/min to 290 • C followed by an isotherm period of 20 min.

RESULTS AND DISCUSSION
The three extracts revealed interesting aspects.The results indicated a trend towards an increased production of extracts on the basis of flowering.The major extract yield was obtained after the floral development.However, the highest increase of extract production to achieve the blooming was up to 23% (Fig. 1).
The quantitative determination of chemical constituents in the extracts was evaluated by a gas chromatography coupled to a mass spectrometry.It was verified the presence of four compounds in all extracts (Fig. 2).The analysis of the spectral data allowed to identify the compounds as plumbagin (1), epi-isoshinanolone (2), palmitic acid (3) and sitosterol (4) (Table I).The naphthoquinone plumbagin has always been the major compound present in the root extracts.The lowest level of this substance in roots was observed before the floral development.During floration, the content of the naphthoquinone plumbagin had a great increase, reaching its highest level.Previous studies (Paiva unplublished data) showed that, in P. scandens, this naphthoquinone is found predominantly on the roots, with low concentrations in aerial parts.In other species of the genus Plumbago, such as P. rosea, the literature data also point to the accumulation of plumbagin in roots (Panichayupakaranant and Tewtrakul 2002).A study carried out by Verma et al. 2002, revealed the potentialities of the hairy root cultures of P. zeylanica for the production of plumbagin.However, it is important to point out the work of Kitanov and Pashankov (1994), which showed that the flowers of the inflorescences of P. europaea have high concentrations of this substance.
It may function in the plant chemical defense against pathogens.During floral development, the plant may be more susceptible to microorganisms attack, once its metabolism can be involved in the transition of the vegetative caulinar apex into a reproductive apex.Moreover, the roots are always exposed to different soil microorganisms, which may explain the accumulation of defense compounds such as the naphthoquinone plumbagin.
The other naphthoquinone, epi-isoshinanolone,  functioned in the same way as plumbagin, showing the major yield during the floral development.
The analysis of other components showed a great variation on their contents.Sitosterol and palmitic acid levels demonstrated the same profile, decreasing during the floral development.Root tips are a metabolically active tissue with a high energy demand, and the role of fatty acid β-oxidation in this tissue is likely to be in membrane lipid turnover (Graham and Eastmond 2002).P. scandens roots do not realize photosynthesis, and the energy demand is higher than the arrival of products (carbohydrates) from the aerial parts, especially during the floral development.It could be said that the roots represent a physiological drain since they demand energy and food for their growth and maintenance.
According to Graham and Eastmond (2002), under carbohydrate starvation conditions, the respiratory demand for C-skeletons derived from fatty acid β-oxidation is expected to increase the triggering induction of the breakdown pathway.The decrease in palmitic acid and sitosterol level suggests their consumption for energy supply and for the construction of plasma membranes in the new cells generated by the activity of the root apical meristem.
Moreover, cellular sterol content and composition may also play a regulatory role in important events in the life cycle of higher plants.The analysis of the sterol content and the composition of developing floral apices from Lolium temulentum confirmed that the sterol content decreased during the inductive period, and a much An Acad Bras Cienc (2011) 83 (4) greater amount of cholesterol was present in the apex, compared to other parts of the plant.It was suggested that such changes in sterol composition mediate membrane permeability, and this might affect the transport of metabolites during evocation (Hobbs et al. 1996).

CONCLUSION
Root chemical composition of Plumbago scandens is affected by the plant physiological stage.Plant requirements, depending on the stage, can vary, and this fact influences the chemical production.The increase in the content of defense compounds such as naphthoquinones during the floral development may protect the plant during a susceptible period.The content of steroids and fatty acids in roots decreased in the same stage, suggesting their consumption and a declined supply of organic material to the roots.

ACKNOWLEDGMENTS
The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for fellowships.