SHORT COMMUNICATION

Inhibition of the Passive Diffusion of Cholic Acidby the Ilex paraguariensis St. Hil. Saponins

Fernando Ferreiraw, Alvaro Vázquez, Carlos Güntner and Patrick MoynaCátedra de Farmacognosia y Productos Naturales, Facultad de Química, General Flores 2124, CP 1157, Montevideo, Uruguay

The Ilex paraguariensis St. Hil. (yerba mate) saponins effectively inhibit the passive diffusion of cholic acidthrough dialysis membranes. The formation of large macromolecular mixed micelles between cholic acid andyerba mate saponins could explain this effect. The observed inhibition of the passive diffusion of cholic acid usedas a model of bile acids, could have an antihypercholesterolaemic effect in vivo due to an increased sterolsexcretion. © 1997 by John Wiley & Sons, Ltd.

Keywords: Ilex paraguariensis; matesaponins; saponin–bile acid complexes; saponins; cholesterol; antihypercholester-olaemic effect.

INTRODUCTION

The habit of mate drinking extends over Uruguay, Paraguay,Argentina and Southern Brazil (Samaniego, 1927; Villa-nueva, 1968). At present the main producing countries areBrazil (States of Paraná and Santa Catarina), Paraguay andArgentine (Province of Misiones) (Samaniego, 1927; Villa-nueva, 1968).

The chemical composition of yerba mate and I. para-guariensis leaves has been studied extensively, especiallyfor purine alkaloids. Apart from the xanthines; tannins,chlorogenic and neo chlorogenic acids, essential oils,vitamins, triterpenoids and saponins have been reported(Vazquez and Moyna, 1968; Bertoni et al., 1992; Gosman etal., 1989, 1995). Saponins are known to form large mixedmicelles with steroids and bile acids (Oakenfull, 1986;Oakenfull et al., 1984). Ingested saponins remain in thegastrointestinal tract, and interact with cholesterol and bileacids producing complexes which prevent their absorption(Malinow et al., 1977). Complexed bile acids are thenexcreted in the faeces and are replenished by the metabo-lism of cholesterol in the liver. Thus the increase of faecalelimination of bile acids in the form of bile acid–saponincomplexes results in a decrease in the serum cholesterollevel (Eastwood and Hamilton, 1968; Balmer and Zilver-smit, 1974).

In Uruguay the prevalence of coronary and other heartdiseases and the age distribution are similar to Europeancountries (Coniglio et al., 1994) whilst the consumption ofmeat per capita is the highest in the world (Peterson, 1978).As the habit of mate drinking is so widespread and the dietis abundant in cholesterol-rich food, it appeared interestingto test the feasiblity of antihypercholesterolaemic activity ofthe saponins of this traditional drink.

MATERIALS AND METHODS

Plant material. Aerial parts (stem and leaves) of Ilexparaguariensis St. Hil. were collected in the Department of

Trienta y Tres, Uruguay. A voucher sample was kept at theFacultad de Química Herbarium under catalogue numberMVFQ 3394, leg. D. Miniccelli. The samples were air driedin the dark.

Saponin extract. Dried leaves and small twigs (1.5 kg) wereextracted with ethanol (4 L) for 48 h at room temperature.The solvent was evaporated under vacuum to yield a darkgreen–brown extract (159 g). The extract was suspended indistilled water (800 mL), defatted with chloroform(33400 mL) and the saponins extracted with n-butanol(33400 mL). The solvent was evaporated, the residuedissolved in boiling methanol (200 mL), and the saponinsprecipitated by the addition of ethyl ether (400 mL). Theprecipitate was redissolved in water and the solution freeze-dried yielding a saponin fraction (13.4 g).

Commercial yerba mate (Las Canarias, 30.7 g) wasmoistened with warm distilled water (40°C, 90 mL) andextracted with hot distilled water (80°C, 300 mL) simulat-ing the common infusion preparation technique (Vázquezand Moyna, 1986). The water extract was filtered throughglass-wool and freeze-dried to give the water extract(6.9 g).

Cholic acid quantification. Standard cholic acid was preparedfrom a commercial sample (32 g) by recrystallization frommethanol. The purity of the product was checked by TLC.Yield 89%, m.p. 196°–202°C (not corrected), [a]25

D =32° (c0.97, CH3OH). Samples and different volumes of standardsolutions of cholic acid in methanol (0.249 mg/mL: 3, 6 and9 mL; 1.245 mg/mL: 3 and 5 mL) were applied in duplicateon precoated silica-gel TLC plates. The upper phase of anacetic acid–toluene–water mixture (5.5:5.0:1.0) was used asthe mobile phase. The spots were developed by sprayingwith an 8% CuSO4 solution in 10% H3PO4 and heating theplate at 120°C for ca. 15 min. The spots wer measured at500 nm using zig-zag scanning on a Shimadzu CS-9000TLC scanner.

Effect of the saponin extract on cholic acid passive diffusionassay. A KHCO3 buffer solution (pH 8.4, 30 mM) was usedto prepare all the solutions and to perform the dialysisw Author to whom correspondence should be addressed.

CCC 0951–418X/97/010079–03© 1997 by John Wiley & Sons, Ltd. Accepted (revised) 18 July 1996

PHYTOTHERAPY RESEARCH, VOL. 11, 79–81 (1997)

assays. A saponin working solution (SWS) was prepared bydissolving the saponin extract (6.31 g) in buffer solution(100 mL). A cholic acid working solution (CAWS) wasprepared by dissolving cholic acid (499.4 mg) in buffersolution (500.0 mL). Different volumes of SWS (1, 5, 10and 15 mL) were mixed with CAWS (15 mL) and thevolume adjusted to 30 mL with buffer solution, to givedifferent final concentrations of saponins (2.1, 10.5, 21.0and 31.6 g/L respectively). Control blanks were preparedusing buffer solution (15 mL) and CAWS (15 mL). Thesolutions were kept at room temperature for 15 min, put intodialysis bags and dialysed against buffer solution (30 mL) atroom temperature in a shaker. After 24 h an aliquot from theexternal chamber (2.00 mL) was taken. To each aliquot 2 M

HCL (2 mL) was added and the aqueous solution wasextracted with chloroform (332 mL). The chloroformextracts were combined, washed with distilled water(2 mL), dried with Na2SO4, and the solvent evaporated byflushing with N2. The residue was dissolved in methanol(200 or 300 mL) and aliquots of the solution (5 or 8 mL,respectively) were applied by duplicate onto a TLC plate,together with different volumes of the standard cholic acidsolutions, and the cholic acid quantified as described above.Data are expressed as percentage of cholic acid dialysedrelative to the blank.

Effect of extract on cholic acid passive diffusion assay. Yerbamate water extract (2.87 g) was dissolved in buffer solution(50 mL). A cholic acid solution was prepared by dissolvingcholic acid (20.8 mg) in buffer solution (50.0 mL). Aliquotsof the yerba mate water extract solution (10 mL) induplicate were mixed with the cholic acid solution (15 mL)and the volume adjusted to 30 mL with buffer solution. Thediffusion assay and the diffused cholic acid quantificationwas performed as before. The results are expressed aspercentage of cholic acid dialysed relative to a blankprepared as described but using buffer solution instead ofyerba mate water extract solution.

Mass spectrometry. The mass spectrometry matrix assistedlaser desorption ionization-time of flight (MALDI-TOF)spectra were recorded on a Linear LDI 1700XP instrument,using 2,5-dihydroxybenzoic acid as matrix. For betterprecision tomatidine and solanine were used as internalstandards.

RESULTS AND DISCUSSION

For the present study we developed a quantitative TLCmethod, which is fast, demands little sample preparationand fulfils our present analytical requirements. The detec-

tion limit, defined as the concentration at which thesignal-to-noise ratio is greater than two (S/N>2) was lessthan 0.3 mg of applied cholic acid. The method showed agood linear response (r=0.993, Sn-1 =5.1023 for n=10) inthe 0.75 mg–8.75 mg range.

The I. paraguariensis saponins extract was preparedusing classical methods for saponin extraction and purifica-tion (Dominguez, 1973). The saponins extracts werepositive in the foam and haemolytic tests (Dominguez,1973). To more fully characterize the saponins extract theMALDI-TOF spectra of the saponins extract with andwithout the addition of internal standards were measured.The mass spectra showed peaks attributable to two differentsaponin series. The first ion series ([M+H]+ ) (m/z: 1075,913, 751, 589) appears as a set of saponins with an aglyconeor aglycones having a molecular weight of 456. Some of thesaponins already known to be present in the plant couldbelong to this series, based on ursolic acid (MW 456), themolecular weights 912 (matesaponin 1) and 1074 (matesa-ponin 3) (Gosmann et al., 1989, 1995). This series seems toconsist of saponins differing in the number of hexoseresidues. The second ion series (m/z: 1426, 1264, 1102 and940) consist of [M+Na]+ ions, as was confirmed by thepresence of the corresponding [M+K]+ ions, could corre-spond to not yet described saponins present in this plant.The members of this group could also correspond to ananalogous series of saponins, differing in the number ofhexose residues. Interestingly, no ions corresponding tomatesaponins 2 and 4 (Gosmann et al., 1995) wereobserved.

Saponins and steroids, due to their amphilphilic naturecould interact to form mixed micelles. Their hydrophobicgroups interleave and the steric and electrostatic constraintsare relieved. The micelles are too large to pass through thegut wall and remain in the gastrointestinal tract (Oakenfull,1986).

The passive diffusion through cellulose membranes isknown to be a good model for the passive absorption of bileacids in the ileum and jejunum. After equilibration thenormal concentration of cholate was measured in theexternal chamber of the dialysis system (Table 1) giving theconcentration of free cholate which is in equilibrium withthe mixed micelles, since the micelles are too large to passthrough the dialysis membrane. This external concentrationis the effective concentration available for absorption. Ascan be seen in Table 1, the I. paraguariensis saponinsextract reduced the amount of diffusible cholic acid, and aninverse relation with the amount of saponins extract isclearly observed.

To confirm that the saponins responsible for the cholicacid passive diffusion inhibition are actually present in theconsumed beverage, the same diffusion assay was per-formed using the water extract. An amount of 576 mg of

Table 1. Dialysed cholic acid in relation to the amount of addedIlex paragueriensis saponins

Saponinsa Diffused cholic acid (%)b

(g/L) Assay 1 Assay 2 Assay 3 Average ±SEM

0.0 100 100 100 100 100 100 100 –

2.1 76 78 80 85 99 94 86 4

10.5 60 63 74 71 60 59 64 3

21.0 32 37 36 43 49 46 41 2

31.6 3 11 13 11 NA 19 12 3

NA, data not available; SEM, standard error of the mean.a Concentration of the saponin extract in the dialysis bag.b Expressed as percentage relative to the blank.

F. FERREIRA ET AL.80

water extract, corresponding to 2.56 g of yerba mate,produced a decrease of 28.5%±3.2% of diffused cholic acidcompared with the blank. That decrease corresponds to thecomplexing of 1.35±0.04 mg of cholic acid. In a typicalmate drinking session 50 g to 200 g of yerba mate is usedand completely extracted with hot water (Vazquez andMoyna, 1986). According to the above data, this consump-tion corresponds to an amount of saponins capable ofcomplexing between 26 mg to 105 mg of cholic acid. Thiswould mean that the saponins of I. paraguariensis arecapable in vivo of complexing bile acids. A similarconclusion was proposed by Jenkins and Atwal (1994),suggesting that dietary saponins would be effective inreducing elevated blood cholesterol concentrations. Ashabitual mate drinkers take the infusion once or twice a day,the associated increased bile acids excretion and decreased

sterol absorption would produce a significant increase in thefaecal excretion of total sterols, and thus have an anti-hypercholesterolaemic effect. This could be a possiblemechanism influencing the European distribution of heartdiseases in Uruguay, in spite of higher consumption of redmeat.

Acknowledgements

This work was supported by grants from the International Program in theChemical Sciences (IPICS, Project URU 03), Uppsala, Sweden, thePrograma de Desarrollo de las Ciencias Básicas (PEDECIBA), and theComisión Sectorial de Investigacióin Cientfica (CSIC, Universidad Mayorde la República), Uruguay. The authors gratefuly acknowledge Mr DiegoMiniccelli for some of the plant material, Professor Eduardo Alonso for thebotanical classification and Professor L. Kenne for access to the MALDI-TOF instrument.

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