454 P. AVATO ET AL.

Copyright © 2006 John Wiley & Sons, Ltd. Phytother. Res. 20, 454–457 (2006)

Copyright © 2006 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 20, 454–457 (2006)Published online 18 April 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ptr.1876

Antimicrobial Activity of Saponins fromMedicago sp.: Structure-Activity Relationship

Pinarosa Avato1*, Rossella Bucci1, Aldo Tava2, Cesare Vitali1, Antonio Rosato1,Zbigniew Bialy3 and Marian Jurzysta3

1Dipartimento Farmaco-Chimico, Via Orabona 4, Università, I-70125 Bari, Italy2Istituto Sperimentale Colture Foraggere,V.le Piacenza 29, I-26900 Lodi, Italy3Institute of Soil Science and Plant Cultivation, PL-24 100 Pulawy, Poland

The antimicrobial activity of saponins from Medicago sativa, M. arborea and M. arabica against a selectionof medically important yeasts, Gram-positive and -negative bacteria was investigated. Structure-activity growthinhibitory effects of related prosapogenins and sapogenins are also described. Increasing antibiotic activity wasobserved going from the saponin extracts to the sapogenin samples, suggesting that the sugar moiety is notimportant for the antimicrobial efficacy. Activity was especially high against Gram-positive bacteria (Bacilluscereus, B. subtilis, Staphylococcus aureus and Enterococcus faecalis) with M. arabica being the speciesshowing a broader spectrum of action. Discrete antifungal activity was also observed, mainly againstSaccharomyces cerevisiae. The observed antimicrobial properties of M. sativa and M. arborea were related tothe content of medicagenic acid, while hederagenin seems to contribute to the bioactivity of M. arabica totalsapogenins. Copyright © 2006 John Wiley & Sons, Ltd.

Keywords: Medicago; saponins; antimicrobial activity; medicagenic acid; hederagenin.

Received 29 April 2005Accepted 13 January 2006

* Correspondence to: Professor P. Avato, Dipartimento Farmaco-Chimico,Via Orabona 4, I-70125 –Bari, Italy.E-mail: avato@farmchim.uniba.it

The present study reports on the in vitro antibioticactivity of saponins from M. sativa L., M. arboreaL. and M. arabica (L.) Huds. against a selection ofmedically important yeasts, and Gram-positive andGram-negative bacteria. The structure-activity growthinhibitory effects of related prosapogenins andsapogenins are also described.

METHODS

Plant material. Roots and tops from M. sativa L. (syn.M. media Pers.) and M. arabica (L.) Huds., andtops from M. arborea L. were used for saponinextraction. M. sativa and M. arborea were grown atthe Istituto Sperimentale per le Colture Foraggere, Italy;M. arabica was grown at the Institute of Soil Scienceand Plant Cultivation, Poland. Ground desiccated(40 °C) plant material was used in the successiveextraction procedures.

Saponin extraction and purification. Saponins of rootsand aerial parts of Medicago species were extracted aspreviously reported by Tava et al. (1993) and purifiedaccording to Timbekova et al. (1989). Pure medicagenicacid (Jurzysta, 1982a) and hederagenin (Jurzysta, 1982b)were obtained from M. sativa and bayogenin (Bialyet al., 2004) from M. arabica.

Saponin derivatization. Saponins from M. sativa andarborea tops were subjected to basic hydrolysis (2 N

KOH in MeOH:H2O, 30:70) to give relatedprosapogenins (Nowacka and Oleszek, 1992). Acidhydrolysis of saponins from M. sativa and M. arabica(tops and roots) and M. arborea (tops) was carried outfollowing the procedure described by Tava et al. (1993).

INTRODUCTION

The genus Medicago (Leguminosae, Papilionaceae)includes 83 different species, the most known repre-sented by M. sativa L., alfalfa, highly valued as a foragecrop (Lesins and Lesins, 1979; Heyn, 1963; Small andJomphe, 1989). The genus contains characteristicsecondary metabolites such as coumarins, isoflavones,naphthoquinones, alkaloids and saponins (Barnes et al.,2002). The chemical structure of saponins from differ-ent species of Medicago especially has been studied(Morris et al., 1961; Morris and Hussey, 1965; Gestetner,1971; Timbekova and Abubakirov, 1984, 1985; Massiotet al., 1988; Oleszek et al., 1990; Timbekova et al., 1989;Bialy et al., 1999; Oleszek, 1996); they generally consistof a mixture of triterpene glycosides with medicagenicacid, hederagenin, zahnic acid and soyasapogenols asthe main aglycones (Oleszek, 1996).

Saponins are widely distributed in the plant kingdomand display several biological properties (Oleszek, 1996;Timbekova et al., 1996; Hostettmann and Marston, 1995;Tava and Odoardi, 1996). Purified saponins from M.sativa have also been tested in vitro as antitumor agentsand for their ability to inhibit the growth of humanleukemic cell lines (Tava and Odoardi, 1996). Previousinvestigations (Oleszek, 1996; Timbekova et al., 1996)on the saponins isolated mainly from M. sativa haveshown that, depending on their structure, they possessantimicrobial activity, principally against plant patho-gens and some yeasts pathogenic to humans (Zehaviand Polacheck, 1996; Jurzysta and Waller, 1996; Gruiz,1996; Polacheck et al., 1986).

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Copyright © 2006 John Wiley & Sons, Ltd. Phytother. Res. 20, 454–457 (2006)

The recovered sapogenins were derivatized in the formof Na salts to improve solubilization in the test broth.

Test organisms. The following microorganisms wereused: Gram-positive bacteria: Bacillus subtilis (ATCC6633), B. cereus (ATCC 11778), Staphylococcus aureus(ATCC 6538P, 25923, 29213), Enterococcus faecalis(ATCC 29212, 19433); Gram-negative bacteria:Pseudomonas aeruginosa (ATCC 27853), Acinetobacterbaumanii (ATCC 19606), Escherichia coli (ATCC25922, 8739); yeasts: Candida albicans (ATCC 14053,10231), C. tropicalis (ATCC 10750), Saccharomycescerevisiae (ATCC Y139), Cryptococcus laurentii (ATCC18803) and Blastomyces capitatus (ATCC 10663). Bac-terial strains were cultivated on triptone soya agar,Oxoid (15 g/L) while Sabouraud dextrose agar, Oxoid(20 g/L) was used instead to grow yeast strains. Cellcultures were incubated at 37 °C for 24 h. Bacterialsuspensions were adjusted to 108 CFU/mL and fungalsuspension tests to 106 CFU/mL by comparison witha McFarland 0.5 turbidity standard according to theNCCLS (2003). Logarithmic phase fresh cultures wereprepared from the above by diluting the standardizedinoculum into the suitable medium (Mueller Hintonbroth, 21 g/L, for bacteria and yeast malt broth, 20 g/L,for yeasts) to obtain a final inoculum of 2.5 × 106 CFU/mL for bacteria and 103 CFU/mL for yeasts. Incubationwas performed at 36 °C. Conventional sterile conditionswere adopted.

Samples preparation. Test samples (crude mixtures ofsaponins, prosapogenins and sapogenins; medicagenicacid; hederagenin and bayogenin) were solubilized inMueller Hinton broth (0.5 mg/mL). Two-fold serialdilutions were prepared from the above starting stand-ardized solutions to give a range of concentrations from500 to 15.625 µg/mL for each of the test samples.Sonication of the samples was generally adopted tofacilitate solubilization of the metabolites; sterilization(121 °C for 15 min) of the test solutions was performedbefore the assay.

Minimal inhibitory concentration (MIC). MICs for eachof the samples were determined by the broth micro-dilution method according to the NCCLS (2003).Aliquots (200 µL) from each of the test drug dilutions(see above) were dispensed in each well; microtiterplates were then inoculated with the microorganismsuspensions (20 µL and 100 µL for bacteria and yeasts,respectively). A positive control consisting in a drug-free inoculum was also included. Parallel controlswith reference antibiotics (miconazole, against yeastsand piperacillin, against bacteria) were also run. Plateswere incubated at 36 °C for 24 h (bacteria) and 48 h(yeasts). MICs were interpreted as the lowest concen-tration of drug totally inhibiting the growth of the testedmicroorganisms compared with the blank experiment,after the preset incubation time. Assays were alwaysrun in duplicate.

RESULTS AND DISCUSSION

Saponins are a major family of plant secondarymetabolites characterized by numerous biological

properties; among them, their antimicrobial activity hasbeen particularly studied (Oleszek, 1996; Hostettmannand Marston, 1995; Oleszek, 2000). The presence ofsaponins in the plant has been related with a defenserole against pathogenic microbes (Osbourn, 1996, 2003).It has been shown, in fact, that wounding of planttissues by pathogenic fungi involves the production ofsaponin-detoxifying enzymes. The fungicidal activityof M. sativa saponins against some plant fungi, such asTrichoderma viride and Sclerotium rolfsii, has beenproved also in in vitro assays (Zehavi and Polacheck,1996; Jurzysta and Waller, 1996; Gruiz, 1996; Oleszek,2000). The previous work on the antimicrobial efficacyof saponins from Medicago against human pathogenshas mainly concerned a limited number of yeasts anddermatophytes; moreover, mainly M. sativa has beeninvestigated (Zehavi and Polacheck, 1996; Jurzysta andWaller, 1996; Gruiz, 1996).

The antimicrobial activity of saponin extracts andrelated prosapogenins and sapogenins from M. sativa,M. arborea and M. arabica against a selection of yeastsand Gram-positive bacteria pathogenic to humans isreported in Tables 1 and 2. Gram-negative bacteria havealso been assayed (see above), but no activity (MICs> 500) was detected against this class of microorgan-isms, in the concentration range used. Of the selectedmycetes, S. cerevisiae appeared the most susceptible,showing high growth inhibition when treated with thesapogenin mixtures from the different species ofMedicago (Table 1): MICs of 125, 62.5 and 175 µg/mLwere in fact recorded with M. sativa, M. arabica and M.arborea tops, respectively. A lower MIC (42.5 µg/mL)was observed when the same strain was treated withmedicagenic acid, which was also active against C.tropicalis and B. capitatus with an MIC of 125 µg/mL.A broader spectrum of efficacy was found against Gram-positive bacteria (Table 2), with the largest inhibitoryeffects observed for the sapogenin mixtures obtainedfrom the acid hydrolysis of M. arabica (tops and roots)saponins. Those samples resulted in fact active towardsalmost all the assayed Gram-positive bacteria. The twostrains of E. faecalis were affected particularly by thesapogenins from M. arabica tops (MIC = 95 and31.25 µg/mL). A common MIC value of 250 µg/mL wasrecorded for sapogenins of M. arabica roots against B.subtilis, B. cereus and the two strains of S. aureus 6538pand 25923. Lower MICs were instead detected againstS. aureus 29213 (95 µg/mL) and E. faecalis 29212(150 µg/mL) and 19433 (125 µg/mL). Good inhibitoryactivities were also observed (Table 2) for thesapogenins from M. arborea tops against B. subtilis(MIC = 125 µg/mL), B. cereus (MIC = 42.5 µg/mL) andE. faecalis (MIC = 95 and 225 µg/mL, respectively).Moreover, medicagenic acid (Table 2) showed consider-able antibacterial activity against S. aureus 29213 (MIC= 52.5 µg/mL) and the two strains of E. faecalis (MIC =50 and 32.5 µg/mL). The growth of some Gram-positive bacteria was also inhibited by hederagenin (B.subtilis, MIC = 62.5 µg/mL).

Previous investigations on the structure-activityrelationships of saponins have led to contrasting results(Oleszek, 1996, 2000): some suggested that sugarmoieties are important for their antifungal effects, whileaccording to others, sapogenins are more active. Ourdata clearly demonstrate that the presence of sugarsin the saponin molecule is not a determinant for

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Copyright © 2006 John Wiley & Sons, Ltd. Phytother. Res. 20, 454–457 (2006)

Table 1. MICs (µµµµµg/mL) of saponin constituents of Medicago sp. against selected mycetes

Mycetes

C. albicans C. albicans C. tropicalis C. laurentii B. capitatus S. cerevisiaeSample 10231 14053 10750 18803 10663 Y139

Total saponins M. sativa tops >500 >500 >500 >500 >500 500M. sativa roots >500 >500 >500 >500 >500 >500M. arabica tops >500 >500 62.5 >500 >500 200M. arabica roots >500 >500 125 >500 >500 500M. arborea tops >500 >500 >500 >500 >500 500

Prosapogenins M. sativa tops >500 >500 >500 >500 >500 250M. arborea tops >500 >500 >500 >500 >500 250

Sapogenin M. sativa tops >500 >500 >500 >500 >500 125mixtures M. sativa roots >500 >500 >500 >500 >500 400

M. arabica tops >500 125 500 >500 >500 62.5M. arabica roots 500 500 500 250 >500 250M. arborea tops 250 >500 200 250 500 175

Pure sapogenins Medicagenic ac. >500 500 125 400 125 42.5Hederagenin >500 >500 >500 >500 >500 225Bayogenin >500 >500 >500 >500 >500 >500

antimicrobial efficacy. In fact a higher antibiotic activ-ity was observed going from the saponin raw extractsto the different samples of sapogenins, the only excep-tion being represented by the saponin extracts fromM. arabica (tops and roots) which had strong inhibitorypotency against C. tropicalis and S. aureus 6538p(Tables 1 and 2). Compositional studies have shownthat the saponin profile of M. arabica (Bialy et al., 2004)is different to that of M. sativa and M. arborea (Oleszek,1996; Tava et al., 1993). Medicagenic and zahnic acidare the two main aglycones identified in M. sativa (50%and 15%, respectively) and M. arborea (30% and 15%,respectively) tops (Oleszek, 1996, Tava and Odoardi,1996). Medicagenic acid is still dominant (65%) in theirroots, while zahnic acid is present in very low amounts(less than 2%). Bayogenin in relatively high amounthas also been detected in M. arborea tops (Tava,unpublished). In contrast, hederagenin (35%) andbayogenin (30%) are the dominant sapogenins in both

tops and roots of M. arabica (Bialy et al., 2004).Combining the above findings with our data (Tables 1and 2), it can be concluded that medicagenic acidand hederagenin possibly contribute to the activitydetected with M. sativa and M. arborea and withM. arabica, respectively. In comparison to the othertwo pure sapogenins, bayogenin displayed instead veryhigh MICs (>500; Table 2) against our test strains tobe considered an active ingredient of M. arabicaextracts. However, the highest antimicrobial efficacy ofM. arabica suggests that other sapogenins present inthe extract may be the active constituents.

Antibacterial activity of saponins from different plantsources has been already reported (Hostettmann andMarston, 1995; Oleszek, 2000); among Medicago sp.,however, only few data on M. sativa are known(Oleszek, 2000). Antimicrobial data on M. arabica andM. arborea, to the best of our knowledge, are reportedhere for the first time. The spectrum of action of

Table 2. MICs (µµµµµg/mL) of saponin constituents of Medicago sp. against selected Gram-positive bacteria

Gram-positive bacteria

B. subtilis B. cereus S. aureus S. aureus S. aureus E. faecalis E. faecalisSample 6633 11778 6538p 25923 29213 29212 19433

Total saponin M. sativa tops 500 500 >500 >500 >500 >500 >500M. sativa roots >500 >500 >500 >500 >500 >500 >500M. arabica tops >500 >500 125 >500 >500 >500 >500M. arabica roots >500 >500 150 >500 >500 >500 >500M. arborea tops >500 >500 >500 >500 >500 >500 >500

Prosapogenins M. sativa tops >500 >500 >500 >500 >500 >500 >500M. arborea tops >500 >500 >500 >500 >500 >500 >500

Sapogenin M. sativa tops 500 200 >500 500 >500 400 42.5mixtures M. sativa roots >500 >500 >500 >500 >500 >500 >500

M. arabica tops >500 110 150 125 250 95 31.25M. arabica roots 250 250 250 250 95 150 125M. arborea tops 125 42.5 >500 >500 500 95 225

Pure sapogenins Medicagenic ac. 250 400 500 500 52.5 50 32.50Hederagenin 62.5 250 500 500 400 400 >500Bayogenin >500 >500 >500 >500 >500 >500 >500

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M. arabica sapogenins against a large selection of medi-cally important Gram-positive bacteria and their con-siderable minimal inhibitory concentrations, can betaken as good evidence for a possible application ofthose compounds as antibacterial agents.

REFERENCES

Acknowledgement

We are thankful to Dr P. Martiniello, Istituto per le ColtureForaggere, Foggia, Italy, for providing us with M. arborea plantmaterial.

Barnes J, Anderson LA, Phillipson JD. 2002. Herbal Medicines.Pharmaceutical Press: London, 38– 41.

Bialy Z, Jurzysta M, Mella M, Tava A. 2004. Triterpene saponinsfrom aerial parts of Medicago arabica L. J Agric Food Chem52: 1095–1099.

Bialy Z, Jurzysta M, Oleszek W, Piacente S, Pizza C. 1999.Saponins in alfalfa (Medicago sativa L.) root and their struc-tural elucidation. J Agric Food Chem 47: 3185–3192.

Gestetner B. 1971. Structure of a saponin from Lucerne(Medicago sativa). Phytochemistry 10: 2221–2223.

Gruiz K. 1996. Fungitoxic activity of saponins: practical use andfundamental principles. In Saponins Used in Food andAgriculture, Waller GR, Yamasaki K (eds). Plenum Press:New York, 565–574.

Heyn CC. 1963. The Annual Species of Medicago. Magnes Press:Hebrew University, Jerusalem.

Hostettmann K, Marston A. 1995. Saponins. Cambridge Univer-sity Press: Cambridge.

Jurzysta M. 1982a. Habilitation’s Thesis, Ser. R (170), pp. 1–64,IUNG Pulawy.

Jurzysta M. 1982b. Polski Urzad Patentowy, Patent No. 114171.Jurzysta M, Waller GR. 1996. Antifungal and haemolytic activity

of aerial parts of alfalfa (Medicago) species in relation tosaponin composition. In Saponins Used in Food and Agri-culture, Waller GR, Yamasaki K (eds). Plenum Press: NewYork, 565–574.

Lesins KA, Lesins I. 1979. Genus Medicago (Leguminosae). ATaxogenetic Study, Junk W (ed.). Kluwer, Dozdzecht: London.

Massiot G, Lavaud C, Le Men-Olivier L, Binst G, Miller SF, FalesHM. 1988. Structural elucidation of alfalfa root saponins byMS and NMR analysis. J Chem Soc Perkin Trans 12: 3071–3079.

Morris RJ, Dye WB, Gisler PS. 1961. Isolation, purification, andstructural identity of an alfalfa root saponin. J Org Chem26: 1241–1243.

Morris RJ, Hussey EW. 1965. A natural glycoside of medicagenicacid. An alfalfa blossom saponin. J Org Chem 30: 166 –168.

NCCLS. 2003. National Committee for Clinical Laboratory Stand-ards, USA. http://www.mccls.org

Nowacka J, Oleszek W. 1992. High performance liquid chroma-tography of zanhic acid glycoside in alfalfa (Medicagosativa). Phytochem Anal 3: 227–230.

Oleszek W. 1996. Alfalfa saponins: structure, biological activity,and chemotaxonomy. In Saponins Used in Food and Agri-culture, Waller GR, Yamasaki K (eds). Plenum Press: NewYork, 155–170.

Oleszek W. 2000. Saponins. In Natural Food AntimicrobialSystems, Naidu AS (ed.). CRC Press: London, 1–30.

Oleszek W, Price KR, Colquhoun IJ, Jurzysta M, Ploszynski M,Fenwick GR. 1990. Isolation and identification of alfalfa(Medicago sativa L.) root saponins: their activity in relationto fungal bioassay. J Agric Food Chem 38: 1810 –1817.

Osbourn A. 1996. Saponins and plant defence – a soap story.Trends Plant Sci 1: 4–9.

Osbourn A. 2003. Saponins in cereals. Phytochemistry 62: 1– 4.Polacheck I, Zehavi U, Naim M, Levy M, Evron R. 1986. Activity

of compound G2 isolated from alfalfa roots against medi-cally important yeasts. Antimicrob Agents Chemother 30:290–294.

Small E, Jomphe M. 1989. A synopsis of the genus Medicago(Leguminosae). Can J Bot 67: 3260–3292.

Tava A, Odoardi M. 1996. Saponins from Medicago ssp.: chemi-cal characterization and biological activity against insects.In Saponins Used in Food and Agriculture, Waller GR,Yamasaki K (eds). Plenum Press: New York, 97–109.

Tava A, Oleszek W, Jurzysta M, Berardo N, Odoardi M. 1993.Alfalfa saponins and sapogenins: isolation and quantifica-tion in two different cultivars. Phytochem Anal 4: 269–274.

Timbekova AE, Abubakirov NK. 1984. Triterpene glycosidesof alfalfa. II. Medicoside G, a novel bisdesmoside fromMedicago sativa. Khim Prir Soedin 4: 451–458.

Timbekova AE, Abubakirov NK. 1985. Triterpene glycosides ofalfalfa. III. Medicoside C. Khim Prir Soedin 6: 805–808.

Timbekova AE, Isaev MI, Abubakirov NK. 1996. Chemistry andbiological activity of triterpenoids glycosides from Medicagosativa. In Saponins Used in Food and Agriculture, WallerGR, Yamasaki K (eds). Plenum Press: New York, 171–182.

Timbekova AE, Larin MF, Yagudaev MR, Abubakirov NK. 1989.Triterpene glycosides of alfalfa. V. Medicoside H. Khim PrirSoedin 5: 673–677.

Zehavi U, Polacheck I. 1996. Saponins as antimycotic agents:glycosides of medicagenic acid. In Saponins Used in Foodand Agriculture, Waller GR, Yamasaki K (eds). Plenum Press:New York, 535–546.