COMPARISON OF SAPONINS AND FLAVONOIDS IN VARIOUS AFRICAN SPECIES OF Desmodium adscendens FROM DIFFERENT COUNTRIES : Analysis and OPLC Purification N. GALAND1, J. RAGOT1, J. DOLLET1 and J. POTHIER1* W. AMOYAL2, N. BRYSON2, H. KORB3, and M. MANACH2

INTRODUCTION

OPLC Purification

Results

Genus Desmodium is a forest shrub found in Africa and South America. In traditional medicine, decoctions of Desmodium adscendens have several "virtues" and have been used to treat pain, hepatitis, muscle contraction or spasms, asthma and several other indications. The active ingredients in Desmodium adscendens are thought to be Soyasaponins and Flavonoids which are proposed to have anti-inflammatory and hepato-protecting properties. As part of an ongoing study, we are particularly interested in the hepato-protection that can be offered by the active ingredients from this species and we have sought to characterize the decoctions, and when possible isolate and characterize the species present. Optimum Performance Laminar Chromatography was the preferred method for purification of this species from the Togo variety of Desmodium. In this study, we have characterized the content of 3 different Flavonoids and 2 Soyasaponins in Desmodium adscendens from four geographical locations in Africa : Ghana, Nigeria, Sierra Leone and Togo. First, we obtained TLC qualitative fingerprints of the extracts, and then, we purified and isolated Soyasaponins for use in a more detailed evaluation (to be published). Traditional reproduce extraction, performed analysis.

1) Pharmacognosy Laboratory, Faculty of Pharmacy, 31 av. Monge - 37200 Tours, France. Contact: [email protected] 2) BIONISIS SA Parc Technologique Le Carnot 9, 18-22 av Ed Herriot - 92350 Le Plessis Robinson, France. Contact: [email protected] 3) BIONISIS Inc. 135 Sullivan Road, Salem, CT 06420, USA - www.bionisis.com.

extraction methods were performed, ie in boiling water so as to decoctions for phytotherapy. In addition, percolated methanolic more classically employed in research for active species was also with additional steps to further clean-up the samples prior to

The results of the TLC fingerprint analysis of extracts are shown below with standard reference materials for Rutin, Vitexine and Isovitexine. In Figures 1 and 2, we observe that the relative composition of flavonoids differs depending on the origin of the plant material. For example, samples from Sierra Leone and Togo are devoid of the quercetin flavonoid, Rutin, while the Togo variety is devoid of Vitexine and Isovitexine. More precise quantitative measurements are needed to determine the exact proportions of each in the samples studied here.

Figure 1. TLC Flavonoids : Rutin (1), Ghana decoction (2), Vitexine (3), Ghana MeOH extract (4), Isovitexine (5), Rutin (6), Nigeria decoction (7), Vitexine (8), Nigeria MeOH extract (9), Isovitexine (10).

Figure 2. TLC Flavonoids : Rutin (1), Sierra Leone decoction (2), Vitexine (3), Sierra Leone MeOH extract (4), Isovitexine (5), Rutin (6), Togo decoction (7), Vitexine (8), Togo MeOH extract (9), Isovitexine (10).

FLAVONOIDS

Soyasaponin I OH

Standards. Vitexine, Isovitexine and Rutin were obtained from Sigma-Aldrich (St Quentin-Fallavier, France) Rha-Gal-Glc- O

HO

O OH OH

O

Quercetin Rutin : Quercetin-3O-glucoside

50 bar pressure applied Solvent pump

Fraction Collector

Soyasaponin VI is an O-22 maltol conjugate of soyasaponin I

metal support

Figure 4. Description of OPLC Technique. Solvent is pumped through an HTSorbTM flat column, sandwiched between teflon and aluminum sheets compressed to 50bar to ensure solvent tightness. Compounds can be applied directly on the stationary phase, separated detected on the column and/or eluted to a fraction collector. Figure 5. TLC Verification of Soyasaponins after purification using an OPLC. CHCl3/MeOH/H2O 65/35/2 Lanes 34-40 are analyses performed on different fractions obtained during the OPLC purification, Lane 1 is a reference sample of Soyasaponin I. Lanes 34-39 were combined.

CH2OH

OH

OH HO

Results Preparative isolation of the Soyasaponin I in Togo Desmodium adscendens was demonstrated using Optimum Performance Laminar Chromatography (OPLC). OPLC is a forced-flow, planar chromatography technique with an open column format that facilitates the identification and separation of substances with weak chromophores relative to classical LC methods. Furthermore, as the column is semi-disposable, it is convenient for crude or semi-prepared extracts which may leave considerable amounts of non-migrating species on the column. Figure 4 shows the TLC analysis of the fractions recovered after an OPLC separation, a standard reference material in found in Lane 1. As can be seen, the OPLC provided an efficient separation with several fractions of pure Soyasaponin I.

SOYASAPONINS Experimental Standards. Soyasaponin I was obtained from Promochem (Molsheim, France) and Soyasaponin VI was provided by Prof. Lavaud (Univ Reims, France).

Experimental

OH

Experimental 80g of dried leaves were extracted by percolation with MeOH. After evaporation, and dissolution in water, the sample was defatted with hexane and finally extracted into BuOH. Evaporation left a residue (0.7g) which was dissolved in MeOH and applied on a silica stationary phase HTSorbTM BSLA001 (Bionisis, Le Plessis Robinson, France) using a Camag Linomat (Muttenz, Switzerland). The purification was performed on an OPLC50 Chromatography System using the following parameters: Pressure 50bar, Flowrate 400µl/min; Flash Vol 400µl, Eluent EtOAc/MeOH/H2O 70:20:10, volume 39ml, Runtime 1h40min.

O

OH

O

Apigenin Vitexine : Apigenin-8C-glycoside Isovitexine : Apigenin-6C-glucoside

Decoction : Extraction of the active ingredients was performed by grinding leaves and stems and boiling in water for a period of 1.5h. The suspension was filtered and the filtrate lyophilized. The product was dissolved in methanol for analysis. Methanolic Extraction A methanol extract was obtained by refluxing of leaves and stems for 1.5h, followed by filtration and evaporation. TLC Analysis of Flavonoids : Separation was performed on Merck Silica 60 using the mobile phase EtOAc/Formic acid/AcOH/H2O 100/11/11/26. Detection was made after pulverization with NP/PEG Reagent.

Extracts. Dried leaves were extracted by percolation with hot methanol. After evaporation, the residue was dissolved in water and defatted by extraction with hexane. In a final step, the soyasaponins were extracted into butanol and evaporated to dryness. TLC Analysis Soyasaponins : Separation was performed on Merck Silica 60 using the mobile phase CHCl3/MeOH/H2O 60/40/10. Detection was made after pulverization with 1% Anisaldehyde in Sulfuric Acid and heating to 115°C. Results The TLC analysis of Soyasaponins from Desmodium adscendens of Ghana, Sierra Leone, Nigeria and Togo are shown in Figure 3. Soyasaponin was present in nearly identical amounts in each, except for the extract of the Sierra Leone variety, which was poorer in this component.

Figure 3. TLC Soyasaponins : BuOH extracts from Desmodium adscendens Ghana (1), Ghana (2), Soyasaponin I (3), Nigeria (4), Soyasaponin VI (5), Sierra Leone (6), Togo (7)

CONCLUSIONS TLC is a good method for obtaining qualitative fingerprints of natural product extracts. The fingerprint analysis of Desmodium adscendens originating from different regions in Africa show that these species can be easily distinguished and that this method may provide an efficient means of measuring the phytotherapeutic value of their respective decoctions containing the active compounds, flavonoids and soyasaponins. Optimum Performance Laminar Chromatography (OPLC) provided an efficient means to isolate the soyasaponin fraction from the Togo variety of Desmodium adscendens, which will allow to continue our search for new hepato-protecting agents from natural sources.