Phytochemical analysis of Phyllanthus niruri L. (Phyllanthaceae) extracts collected in four geographical areas in the Democratic Republic of the Congo

1 Department of Biotechnology and Molecular Biology, Division of Life Sciences, General Atomic Energy Commission, Regional Center of Nuclear Studies of Kinshasa (GAEC/RCNS-K.), BP 868 Kinshasa XI, P.O.BOX 868 KIN.XI, Democratic Republic of the Congo (DRC). 2 Plant Physiology and Molecular, PHD Student, Department of Biology, Faculty of Sciences, University of Kinshasa, P.O.BOX 190 KIN.XI, DRC. 3 Biochimie et Physiologie Moléculaire des plantes, UMR 5004 (Agro-M/CNRS/INRA/UM2), Montpellier SupAgro; Montpellier/France. 4 Department of Microbiology, General Atomic Energy Commission. Regional Center of Nuclear Studies of Kinshasa .P.O.BOX 868 KIN.XI, DRC. 5 Faculty of Pharmacy, University of Kinshasa, P.O.BOX 834, Kinshasa XI, DRC. 6 Laboratory of Plant Physiology and Biochemistry at the University of Montpellier II (UM2), Montpellier/France.


INTRODUCTION
Plants have been used in traditional medicine since a long time.About 13,000 plant species have been used as drugs throughout the world, and approximately 25% of the current materia medica are derived from plants in form of teas, extracts, or pure substances (Adjanohoum, 1982;Oksman-Caldentey and Barz, 2002).Traditional medicine using plant extracts continues to provide health coverage for over 80% of the world's population, especially in the developing world (Igbinosa et al., 2009).In the Democratic Republic of Congo (DRC), among the species used in the treatment against malaria, Phyllanthus niruri is well positioned for different previous studies on this plant (Pauwels, 1993;Tona et al., 1999;Cimanga et al., 2004).P. niruri is one of the most important medicinal plants used in different regions in the world for the treatment of various diseases such as jaundice, asthma, hepatitis, flu, dropsy, diabetes, fever causing by malaria (Kerharo and Adam, 1974;Ishimari et al., 1999;Paranjape, 2001) but its availability is drastically decreasing because of numerous harvests.
The antiplasmodial activity of various parts of P. niruri according to its geographical distribution showed that the biological activity of P. niruri depending on its environment of harvest (Soh et al., 2009).
The purpose of this study is to characterize the major chemical groups by (Thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) of the aerial parts, roots and calli of P. niruri from the four areas of DRC and, so as to give an explanation of the difference on the in vitro antiplasmodial activity of P. niruri obtained by Soh et al. (2009) according to the geographical distribution and to suggest a study chase on phenolic constituents and steroidal sapogenins respon-sibles for antiplasmodial activity in P. niruri plant.

Plant material
For this work, the aerial parts of P. niruri were harvested from August to October 2007 respectively in 4 different areas of the DRC whose Kimwenza, Kisantu, Unikin and UPN.The localities Unikin and UPN correspond to the neighborhood of University of Kinshasa and of the neighborhood of National Pedagogic University.The distances between Unikin and Kimwenza, Kisantu and Kimwenza, Unikin and Kisantu, Unikin and UPN are 4, 116, 120 and 7 Km respectively.The geographic situations for those localities were illustrated in Figure 1 and Table 1.Callus was obtained by in vitro cell culture (Luyindula et al., 2004).The botanical identification was assured by M. Nlandu of the INERA (Institut National pour l'Etude et la Recherche Agronomiques), Herbarium -University of Kinshasa.The herbarium samples were deposited at the herbarium under the numbers respectively 72 bis (Kisantu), 66 (Kimwenza), 83 (Unikin) and 66 bis (UPN).All plant materials were dried at 40°C in GCA Precision Mechanical Convection Oven (USA) and reduced to powder, crushed in the mill Thomas (USA).

Callus culture conditions
Fresh apical stems of P. niruri water were sterilized in 70% ethanol
for 1 min and then in mercuric chlorid 0.125% (w/v) solution for 3 min.They were finally washed four times with sterile distilled water and aseptically cut in 0.5 cm segments.These segments were cultured in sterile polystyrene flasks containing 30 ml of Murashige and Skoog (MS) medium (Murashige and Skoog, 1962;Hall, 1999) according to the nature of growth factors and the time of cultivation.The medium was supplemented with 1 ml of 1-naphtylacetic acid (NAA: 4 mg/ml) and 0.5 ml of 6-benzylaminopurine (BAP: 2 mg/ml) and albumen liquid of Coco nucifera L. (100 ml/l) (Ishimari et al., 1999).The initiated callus was routinely subcultured onto a fresh medium 2 to 3 weeks.Callus obtained were dried at 40°C in GCA Precision Mechanical Convection Oven (USA) and reduced to powder.Callus of 3 and 6 month-old were used for our analysis.

Preparation of crude extracts and sample for TLC
One of an equal mixture of 5-methoxyflavone 10 -4 M and α-gluconolactone 10 -4 M in methanol 80% at pH 2 is added to each sample (50 mg) followed by vortexing for 30 s. Treatment for 15 min twice at the ultrasonic agitation allows a better solubilization of the samples.At the 15 min interval, the samples were agitated manually to be returned to the ultrasonic agitation.The samples were then centrifuged at 13,400 rpm for 10 min using an Eppendorf centrifuge Minispin.
The TLC plates were dried under fume hood.Observations of phenolic substances (flavonoids) were made first under 254-366 nm UV without application of any reagent and after application of 4dimethylamino-cinnamaldehyde (DMACA) (2 g in 100 ml ethanol and 100 ml of concentrated HCl 6N).The liquid nitrogen was used for the eventual fluorescence stimulation of compounds.The revelation of spots for steroidal sapogenins was obtained after heating at 110°C under the Memmert Oven during 10 min after spraying triantimony chloride (SbCl 3 ) solution in HCl 12N (1/1; w/v) (Uematsu et al., 2004).Then, the plates were sprayed with solution of H 2 SO 4 10% in methanol.

High performance liquid chromatography (HPLC)
The HPLC analysis was carried out on a HPLC Waters 2695 Alliance (Germany) equipped with Alltech Altima C18 column (5 µm; 250×4.6 mm) and a diode array detector (Waters 2996).18 µl of    each samples (pure or hydrolyzed extracts) were injected in the loop sample.The flow rate was 1 ml/min and the peaks were detected at 275 nm.Gradient elution was performed using acetonitrile (solvent A) and water with 1%HCl (solvent B) as memorized in Table 2 (Von Hoist et al., 2001).

Calculation of relative area of peaks
The area of each peak is proportional to the concentration of compound which is then dosed at the wavelength selected for analysis by reference to the control compounds.The relative area of each peak was calculated by the relation (Macheix et al., 2005;Audigié et al., 1995): A x = area of peak × in the sample/area of peak × in the internal standard

RESULTS AND DISCUSSION
Results from the chemical analysis of hydrolyzed extracts from P. niruri parts or callus extracts are illustrated in Figures 2, 3 and 4.

Flavonoids compounds
The Figures 2, 3 and 4 shown chromatograms of the hydrolyzed extracts of P. niruri (stems, leaves, roots and callus respectively).Figures 2 and 3 showed that all aerials part of P. niruri from Unikin (Uk), UPN (Up), Kimwenza (Km) and Kisantu (Ks) contain flavonoids revealed in bleu by fluorescence or by UV in liquid nitrogen.P. niruri from Km presented again the yellow spot corresponding to control (quercetin) (Mabry et al., 1970).In nitrogen phosphorescence (366 nm) control and P. niruri from Kimwenza location no showed coloration or fluorescence.
Figure 4 revealed the presence of flavonoids regarding the hydrolyzed extracts of P. niruri from Unikin and UPN.Several of those compounds correspond at same level than control compounds.Figure 5  stem explants no coloration spots difference was observed.

Sapogenins steroids
Figure 6 illustrated saponins and steroidal sapogenins obtained in P. niruri plant using hydrolyzed extracts from stems with their leaves after TLC investigation.All samples of aerials parts of P. niruri showed the presence of steroidal sapogenins.These steroidal sapogenins revealed by yellow-colored spots (all samples) and violet, bleu or yellow-green spots depending on that degree of unsaturation of the molecules (Figure 6).In Unikin and UPN samples, yellow spots are more accentuated than of Kimwenza and Kisantu samples.Kimwenza samples presented others colored spots (violet, blue, green and yellow-green).These colored spots are absents in Unikin, UPN and Kisantu samples.The violet spot in Kimwenza sample may be due to lipids and the blue spot corresponded to that obtained by Uematsu et al. (2004).These authors have identified the blue spot compounds as hydroxymethylfurfral and its ethyl adducts.The same authors reported that yellowgreen colored spot obtained with standard sarsasapogenin was characteristic of steroidal sapogenin after application of anisaldehyd reagent.The violet colored spot from Kimwenza's sample was obtained also by Yung et al. (2005) with the butanol extract from aqueous layer of aerials portions of Pleurospermum kamtschaticum after several extraction procedures.
In roots and callus obtained from stems, the results revealed the absence of steroidal sapogenins.The TLC plates were not revealed specific coloration by the application of 10% sulfuric acid (H 2 SO 4 ) solution on the plate after splaying the tri-antimony chloride (SbCl 3 ).These sapogenins compounds from P. niruri as diosgenin, ruscogenin and other triterpenic genins would explain several effects of P. niruri on diabetes, anti-inflammatory activity, flu, arthritis, hepatitis treatment (Uematsu et al., 2004;Huang et al., 2008;Kang et al., 2011).It is noted that in general, sapogenins are bio-active principles rather than the saponins themselves (Yung et al., 2005).

High performance liquid chromatography
The results obtained by high performance liquid chromatography analysis of hydrolyzed extracts of P. niruri from 4 agglomerations were presented in Figures 7a to 7h.For comparison of chromatograms of hydrolyzed extracts from P. niruri obtained by HPLC, it was revealed that the number of major peaks differs depending on geographical location.If consider as the major peak with a relative area equal to or greater than 2.5: Unikin samples have got more than seven major peaks with their respective relative areas 5. 21, 4.82, 4.11, 2.55, 5.65, 2.71 and 3.81 which eluted respectively at 3.83, 4.28, 4.7, 5.59, 7.81, 26.5 and 27.13 min (Figure 7a).Five major peaks for UPN samples with the following relative areas: 7. 58, 4.78, 3.24, 3.32 and 4.98 eluted respectively at 3.84, 4.28, 4.7, 5.59 and 7.8 min (Figure 7b).Chromatograms of Kimwenza (Figure 6c) and Kisantu (Figure 7d) samples  revealed respectively 2 and 4 major peaks.The peaks from Kimwenza have the retention times as 4.28 and 7.8 min with their relative areas as 13.73 and 7.27.The Kisantu samples presented 4 major peaks at the retention times as 6.37, 4.04, 2.5 and 3.89 who have eluted to the following retention times 3.83, 4.28, 5.58  The major peak of which products settled at 4.28 min is present in all samples of P. niruri (stems with their leaves, roots and callus) (Figure 7).However, the relative area of those peaks is different.For example, callus of 3 monthold (Figure 7g) showed a major peak at 4.28 min which exceeds all peaks of all samples with the relative area of 10.41.The callus samples showed difference in number of major peaks while the major peaks are the same.The root samples from Unikin (Figure 7e) and Kisantu (Figure 7f) showed difference neither in number of peaks nor in number of major peaks.
This finding is also observed on TLC chromatograms.However, if the spots obtained (Figures 2, 3, 4 and 5) concord, Kimwenza shows yellow spot corresponding to those of quercetin control (Figure 1).That is also believed to differ from the compounds in different samples.This difference for the same species of P. niruri can be  explained according to the variety and the evolution stage physiology (Macheix et al., 2005;Hess, 1975;Wilkins, 1984).In the first, Macheix et al. (2005) reported that the absorption spectrum of chlorogenic acid (λmax = 328 nm, marker shoulder around 300 nm) reflects number of natu-ral esters of hydroxycinnamic acid, that of (+)-catechin (maximum at 280 nm) will be found in condensed tannins, that of quercetin (λmax = 375 nm, with a second maximum around 265 nm) reflects number of flavonols and their glycosylates derivatives.On the other hand, Ludwig-Müller et al. ( 2008) reported that four wavelengths (280, 325, 375 and 450 nm) correspond to the aromatic substances, derivatives of cinnamic acid, flavonoids and many carotenoids and other pigments.
The presence of (+)-catechin or the condensed tannins could be confirmed in the sample content peaks with maximum of wavelength at 280.6 nm (peak n°16 for Unikin sample (Figure 7a) and in the sample containing peak n°3 with λmax = 281.8nm from Kimwenza (Figure 7c) for further analysis by nuclear magnetic resonance (NMR).Previously, Soh (2008) reported that a polyphenol as ellagic acid was identified in the active fractions of three medicinal plants whose Chrozophora senegalensis, Sebastiania chamaelae and P. niruri.The study of antimalarial properties of ellagic acid has shown high activity in vitro against all P. falciparum strains used.
The antiplasmodial activity of different P. niruri according to their geographical distribution obtained by Soh et al. (2009) showed that crude extracts of P. niruri aerial parts from Kisantu presented a higher antiplasmodial activity than to those of Unikin and Kimwenza.In the case of root extract no difference was observed and all samples presented a low antiplasmodial activity.The antiplasmodial activity could be explained by the presence of a particular compound with retention time around 22 min (Figures 7a to 7h).The peak n°12 of Kisantu sample (Figure 7d) has two maxima at 255.3 nm and at 366.3 nm with a relative area of 2.32 and peak exceed all relative area of compounds those settle around 22 min for other samples (UPN and Kimwenza).For Unikin sample (Figure 7a), there is no compound that eluted at 22 min but at 19.43 min (peak 11); compound would be comparing to quercetin with an absorption spectrum containing two maxima (255.7 and 366.3 nm).For UPN sample, there are two compounds those elute around 22 min such as 22.34 min (peak 13) and 22.58 min (peak 14).The peak 13 with an absorption spectrum carrying two maxima (266.3 and 365.1 nm) is similar to the peak 12 of sample from Kisantu but has a lower relative area of 1.45 than this one.Some effort were made to identified same compounds: Peak 11 Unikin was identified to quercetin, peak 13 UPN was identified to chlorogenic acid and peak 12 Kisantu was identified to catechin by comparing the UV spectra to those of respective reference products.This may also explain the lower antiplasmodial activity of crude extracts from samples of UPN than the crude extracts from samples of Kisantu or Kimwenza obtained by Soh et al. (2009).

Conclusion
Through the present work, we found that effectively the metabolites profiles of P. niruri change with its geographical distribution certainly because of the change in soil composition or the environmental conditions.These factors can alter yield of the secondary metabolism pathways.All extracts from P. niruri aerial parts (stems with leaves) were exhibited in the HPLC profiles of hydrolyzed extracts indicated that P. niruri from UPN was able to synthesize more phenolic constituents than P. niruri from Unikin, Kisantu or Kimwenza.
Results obtained in the present study confirm the difference of in vitro antiplasmodial activity of P. niruri according to its geographical location.Roots revealed the same concentration of compounds and the same antiplasmodial activity.Callus showed difference in the secondary metabolites composition and the difference in the in vitro antiplasmodial activity.Those results are confirmed by TLC and the extracts showed presence of flavonoids compounds and steroidal sapogenins.These compounds, flavonoids, saponins and steroidal sapogenins, would be explained the many effects which are played by P. niruri in the treatments of several diseases.Thus, studies could be continued for the characterization of flavonoids or others compounds responsible of antiplasmodial activity and steroidal sapogenins who would be presents in P. niruri.pellier through M. Didier Bogusz (Supervisor) for providing us with a framework for the successful completion of a course on modern techniques of molecular biology.

Figure 7b .
Figure 7b.HPLC of hydrolyzed extract from aerial parts of P. niruri L. from UPN.

Figure 7c .
Figure 7c.HPLC of hydrolyzed extract from aerial parts of P. niruri L. from Kimwenza.

Table 1 .
The geographical data of P. niruri L. harvested sites samples.

Table 2 .
Gradient elution HPLC of phenolic compounds in extracts of P. niruri.