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Journal of Ethnopharmacology 104 (2006) 188–192 Antibacterial and antifungal activity of sulfur-containing compounds from Petiveria alliacea L.
Seokwon Kim , Roman Kubec , Rabi A. Musah a Department of Chemistry, State University of New York at Albany, 1400 Washington Avenue, Albany, NY 12222, USA b Department of Chemistry, University of South Bohemia, ˇ Cesk´e Budˇejovice, Czech Republic Received 23 February 2005; received in revised form 28 August 2005; accepted 29 August 2005 Abstract
A total of 18 organosulfur compounds originating from Petiveria alliacea L. roots have been tested for their antibacterial and antifungal activities.
These represent compounds occurring in fresh homogenates as well as those present in various macerates, extracts and other preparations madefrom Petiveria alliacea. Of the compounds assayed, the thiosulfinates, trisulfides and benzylsulfinic acid were observed to be the most active, withthe benzyl-containing thiosulfinates exhibiting the broadest spectrum of antimicrobial activity. The effect of plant sample preparation conditionson the antimicrobial activity of the extract is discussed.
2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Petiveria alliacea; Petiveriin; 6-Hydroxyethiin; Petivericin; Thiosulfinate; Antimicrobial activity 1. Introduction
homogenates of Petiveria alliacea, with (3) and (6) predomi-
nating. However, both thiosulfinates and sulfines are known to
Petiveria alliacea L. (Phytolaccaceae) is a perennial shrub be labile compounds that undergo many subsequent reactions, that grows primarily in South and Central America, some areas particularly when subjected to heat and/or storage in non-polar of Africa, and the southeastern United States. It is popularly solvents. Thus, compounds (3)–(7) can readily decompose,
used in folk medicine for treating a wide variety of disorders.
giving rise to a variety of secondary products including sulfides, Various preparations from this plant reportedly exhibit anti- benzylsulfinic and sulfonyl acids, (E/Z)-stilbenes, and benzalde- inflammatory, anticancer and stimulant effects, among many others. Recently, we reported the isolation and characterization of several novel sulfur-containing amino acids from Petiveria There are considerable discrepancies in the bioactivity assay (RSRC)/(SSRC)-S-benzylcysteine results reported for Petiveria alliacea-derived compounds and sulfoxides (petiveriins A and B) (1) and (RSRC)/(SSRC)-S-(2-
extracts. Whereas some authors have reported the plant to be hydroxyethyl)cysteine sulfoxides (6-hydroxyethiins A and B) (2). We also showed that these cysteine derivatives are enzymat-
ically cleaved by a C–S lyase enzyme upon disruption of the tis- observed significant antimicrobial properties in various Petive- sue, yielding four thiosulfinates (47) (
ria alliacea extracts ver, several poly- Additionally, we identified the lachrymatory sulfides, some of which were demonstrated to exhibit relatively principle of the plant as the unique sulfine, (Z)-thiobenzaldehyde strong antifungal activity, have been isolated from Petiveria alli- S-oxide (3), which is yet another likely derivative of the petiveri-
ins (). Compounds (3)–(7) are the main
From our previous studies, we are aware that the compounds sulfur-containing components present in freshly prepared present in Petiveria alliacea extracts vary as a function of dif-ferences in sample preparation. We propose that the aforemen-tioned discrepancies in Petiveria alliacea antimicrobial assay ∗ Corresponding author. Tel.: +1 518 437 3740; fax: +1 518 437 3741.
results are a consequence of differences in extraction and sam- E-mail address: (R.A. Musah).
ple preparation procedures that ultimately result in significant 0378-8741/$ – see front matter 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2005.08.072 S. Kim et al. / Journal of Ethnopharmacology 104 (2006) 188–192 changes in the relative concentrations of bioactive compounds from Becton Dickinson and Company (Sparks, MD, USA). Cells present, with freshly prepared plant macerates possessing a dif- were grown at 30 ◦C in Mueller–Hinton broth to an OD420 = 1.9 ferent organosulfur compound profile than those from dried (approximately 105 CFU/mL), and were passaged at least twice plant and/or heat-treated extracts. Thus, we embarked on the on proper agar (brain heart infusion agar for bacteria, Sabouraud present study in order to determine the possible antimicrobial dextrose agar for yeasts, and potato dextrose agar for fungi).
activity of the compounds present in freshly prepared macer- Broth cultures were swabbed onto agar to achieve a lawn of ates, and to also determine the antimicrobial activity of their confluent bacterial growth. Paper disks impregnated with a test compound (50 and 200 ␮g) or antibiotic (10 ␮g for gentamycinand ampicillin, 30 ␮g for chloramphenicol and tetracycline) 2. Materials and methods
were placed on each plate. The plates were incubated at 30 ◦Cfor 24 h. Active compounds were those for which a zone of inhi- (RCRS)/(RCSS)-S-Benzylcysteine sulfoxide (1), (RSRC)/
2.4. Determination of minimum inhibitory concentration (SSRC)-S-(2-hydroxyethyl)cysteine sulfoxide (2) and (Z)-
thiobenzaldehyde S-oxide (3) were prepared as previously
described (
The microdilution broth methods (M7-A5 for bacteria, M27- All four thiosulfinates [S-(2-hydroxyethyl) phenylmethanethio- A for yeasts, and M38-P for fungi) of the NCCLS were employed. Briefly, 100-␮L aliquots of inoculum dilution that (5), S-benzyl phenylmethanethiosulfinate (6), and S-(2-
were twice the test concentration were dispensed into sterile 96- well plates (Corning Incorporated, Corning, NY, USA) to final inoculum concentrations of approximately 5 × 105 CFU/mL for zyl sulfide (8), bis(2-hydroxyethyl) sulfide (10), benzyl disulfide
bacteria, 2.5 × 103 CFU/mL for yeasts, and 5 × 104 CFU/mL (11) and benzyl trisulfide (14) were purchased from Aldrich.
for fungi. Determination of MIC was performed with a dual- Benzyl 2-hydroxyethyl sulfide (9), benzyl 2-hydroxyethyl
wavelength microplate reader (model EL311; Bio-Tek Instru- disulfide (12), bis(2-hydroxyethyl) disulfide (13), benzyl 2-
ments, Winooski, VT, USA), measuring the optical density at hydroxyethyl trisulfide (15) and bis(2-hydroxyethyl) trisulfide
570 nm, with reference reading at 690 nm. MIC is defined as the (16) were obtained as described in the literature (
lowest concentration that completely inhibits organism growth.
Sodium benzylsulfonate (17) was synthesized
Thus, the growth in each well was compared with that of the con- trol (drug-free) well. Tetracycline and amphotericin B (Sigma) and benzylsulfinic acid (18) was prepared by hydrolysis of
were used as controls for the antibacterial and antifungal tests, benzylsulfinyl chloride. The identities of the compounds were confirmed by 1H, 13C NMR and IR spectroscopy. Their puritywas >99%.
3. Results
In the present study, we investigated the antibacterial and anti- fungal properties of 18 sulfur-containing compounds originating The microorganisms tested were obtained from the Ameri- from Petiveria alliacea L. roots. The compounds include those can Type Culture Collection (Manassas, VA, USA). They were present in intact tissue (1 and 2), as well as their primary decom-
Aspergillus flavus (ATCC 9643s), Mucor racemosus (ATCC position products, namely (Z)-thiobenzaldehyde S-oxide (3) and
7924), Pseudallescheria boydii (ATCC 760), Issatchenkia thiosulfinates (47), which occur in freshly disrupted plant tis-
orientalis (ATCC 6258), Candida tropicalis (ATCC 750), sue. Additionally, the degradation products of (37), which are
Candida albicans (ATCC 10231), Bacillus cereus (ATCC likely to be present in various Petiveria alliacea preparations 11778), Staphylococcus aureus (ATCC 25923), Micrococ- (818), were also tested. The structures of the tested compounds
cus luteus (ATCC 4698), Mycobacterium smegmatis (ATCC 19420), Streptococcus agalactiae (ATCC 13813), Escherichia The antibacterial activity of these compounds was initially coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), evaluated by the disk diffusion method, using five strains of Stenotrophomonas maltophila (ATCC 13637), and Klebsiella Gram-positive bacteria (Bacillus cereus, Mycobacterium smeg- matis, Micrococcus luteus, Streptococcus agalactiae, Staphy-lococcus aureus) and four strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Stenotrophomonasmaltophila, Klebsiella pneumoniae). (Z)-Thiobenzaldehyde S- Assays were performed according to the standard guidelines oxide (3), the three benzyl-containing thiosulfinates (46), two
(M2-A7) of the National Committee for Clinical Laboratory trisulfides (15, 16), and benzylsulfinic acid (18) exhibited rela-
Standards (NCCLS), using a modified Kirby–Bauer disk dif- tively strong antibacterial activity. Unlike its benzyl containing fusion method. All the organisms were stored at −80 ◦C until counterparts, the fourth thiosulfinate tested, S-(2-hydroxyethyl) use. BBL blank paper discs (6 mm diameter) were purchased 2-(hydroxyethane)thiosulfinate (7), showed only weak activity.
S. Kim et al. / Journal of Ethnopharmacology 104 (2006) 188–192 Fig. 1. Structures of the Petiveria alliacea-derived organosulfur compounds tested.
The other compounds were not found to be active at the lev- mum inhibitory concentration (MIC) of each. The results of this els tested. With the exception of Pseudomonas aeruginosa, the assay are outlined in MIC is defined as the minimum growth of Gram-negative bacteria was significantly inhibited by concentration of a compound required for complete inhibition unsymmetrical thiosulfinates (4 and 5).
of microorganism growth. Since none of the compounds were Based on the results of the disk diffusion assay, the antibac- observed to be active against Pseudomonas aeruginosa, this bac- terial activity of 8 compounds (37, 15, 16, and 18) out of
terium was not included in the MIC assay. Compounds (4)–(6)
the total of 18 were further evaluated to determine the mini- were found to be the most active. No significant difference Table 1Minimum inhibitory concentration (in ␮g/mL) of Petiveria alliacea-derived compounds against Gram-negative and Gram-positive bacteria S. Kim et al. / Journal of Ethnopharmacology 104 (2006) 188–192 Table 2Minimum inhibitory concentration (in ␮g/mL) of Petiveria alliacea-derived compounds against fungi between the activities of the two regiomeric thiosulfinates (4 and
5) was observed. Sulfine (3) showed activity only against some
of the Gram-positive bacteria. In general, the compounds tested our study focused on compounds that have been observed in exhibited only modest antibacterial activity, compared with that Petiveria alliacea fresh root macerates, whereas the previous of the reference compound tetracycline.
studies focused on leaf extracts. Because the cysteine sulfoxides Antifungal activities of the compounds tested are summa- (1 and 2) that serve as precursors of the organosulfur compounds
rized in Those compounds showing no antifungal are present in the leaves in only very low concentrations, the activity by the disk diffusion assay method (2, 10, 17) are
primary products of their C–S lyase-mediated decomposition, omitted from the table. As can be seen, the sulfine (3), the
such as thiosulfinates (47), are present at much lower levels
benzyl-containing thiosulfinates (46), all three trisulfides
compared to the root. Indeed, we have found that the concen- (1416) and benzylsulfinic acid (18) possess varying degrees
tration of S-benzylcysteine sulfoxides in the root is ∼3 mg/g of activity, with (18) being the most active. Interestingly,
fresh weight whereas in the leaves it is on the order of 0.08 mg/g one of the cysteine sulfoxide precursors, S-benzylcysteine sulfoxide (petiveriin, 1), also exhibited modest antifungal
that the thiosulfinates possess the highest antibacterial activity, the reported absence of antibacterial activity in Petiveria alli-acea leaf extracts may be a consequence of significantly lower 4. Discussion
levels of cysteine sulfoxide precursors in the leaves of the ana-lyzed plants. The second important difference between our study, The purpose of this study was to assess the antimicrobial and those in which Petiveria alliacea extracts were reported not potential of various sulfur-containing compounds derived from to have antibacterial activity, is related to sample preparation.
Petiveria alliacea L. We believe the compounds tested in the Whereas the compounds used in our study are those found both present study to be the most important of these. They may serve, in extracts of fresh macerates and also in heat-treated samples, in part, as a component of the defense system of the plant, pro- other studies were based on an analysis of dried samples, or tecting it from fungi and predators.
fresh samples that were extracted after heating in water or alco- The primary products of enzymatically mediated decomposi- tion of petiveriins A and B [i.e. the sulfine (3) and thiosulfinates
(46)], were found to be the most active, whereas the prod-
results in the formation of the organosulfur compounds that ucts of further thiosulfinate decomposition (i.e. sulfides and we have observed in fresh macerates is mediated by C–S lyase disulfides) exhibited much lower or no activity in the antimi- enzymes. Inactivation of these enzymes through sample drying crobial assays. Our study has confirmed the antifungal activity and/or application of heat would effectively curtail the forma- of benzyl 2-hydroxyethyl trisulfide (15) that was first reported
tion of the sulfine (3), thiosulfinates (47), and consequently,
other antimicrobially active compounds (e.g. 15 and 16). Thus,
by All three trisulfides tested (1416)
the reported absence of antimicrobial activity in previous stud- were significantly more active than the corresponding mono- ies may have been the result of the absence of antimicrobial and disulfides (813).
compounds, since these compounds would not be present in There are two important distinctions between our studies, the extracts if the enzymes that mediate their formation were and those in which Petiveria alliacea extracts were reported to destroyed through treatment with heat.
S. Kim et al. / Journal of Ethnopharmacology 104 (2006) 188–192 Based on our findings, we suggest that the discrepancies in Screening of activity to bacteria, fungi and American trypanosomes of the data that have so far been published on the antimicrobial 13 native plants. Journal of Ethnopharmacology 62, 195–202.
activity of Petiveria alliacea could be the result of differing pro- Chatgilialoglu, C., Gilbert, B.C., Gill, B., Sexton, M.D., 1980. Electron spin resonance studies of radicals formed during the thermolysis and photolysis cedures used during preparation of the tested samples. Whereas of sulphoxides and thiolsulphonates. Journal of the Chemical Society freshly prepared samples are likely to contain mostly the Perkin Transactions II, 1141–1150.
sulfine (3) and thiosulfinates (47), other preparations (obtained
Furukawa, M., Tsuiji, S., Kojima, Y., Hayashi, S., 1973. The reaction of following heat treatment, distillation, and/or drying of plant benzyl phenylmethanethiosulfinate with amines. Chemical and Pharma- material) will mainly consist of various sulfides, stilbenes, Johnson, T.B., Ambler, J.A., 1914. Researches on amines. IV. The alkylation benzaldehyde and other secondary decomposition products.
and hydrolysis of aliphatic sulfonamides. A new synthesis of sarcosine.
This implies that fresh samples prepared by mild procedures are likely to show a significantly higher degree of antimicrobial Kice, J.L., Parham, F.M., Simons, R.M., 1960. The thermal decomposition of activity than those prepared employing harsher conditions. The thiolsulfonates. Journal of the American Chemical Society 82, 834–842.
latter ones may, however, still possess significant antifungal Kubec, R., Kim, S., Musah, R.A., 2002. S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea. Part II. Phytochemistry properties. Furthermore, root-derived preparations are likely to exhibit much higher antimicrobial activity compared to leaf Kubec, R., Kim, S., Musah, R.A., 2003. The lachrymatory principle of Petive- ria alliacea. Phytochemistry 63, 37–40.
Kubec, R., Musah, R.A., 2001. Cysteine sulfoxide derivatives in Petiveria Acknowledgements
alliacea. Phytochemistry 58, 981–985.
Misas, C.A.J., Hernandez, N.M.R., Abraham, A.M.L., 1979. Contribuci´on a la evaluaci´on biol´ogica de plantas cubanas III. Revista Cubana de Medicina This work was supported by the National Science Founda- tion (Grant 0239755). The authors would like to thank Denise P´erez, C., Anesini, C., 1994a. Inhibition of Pseudomonas aeruginosa by McKeon for helpful discussions and critical reading of the Argentinean medicinal plants. Fitoterapia 65, 169–172.
P´erez, C., Anesini, C., 1994b. In vitro antibacterial activity of Argentine folk medicinal plants against Salmonella typhi. Journal of Ethnopharmacology44, 41–46.
Smythe, J.A., 1922. Decomposition of benzyl disulphoxide. Journal of the Ayodele, E.T., Hudson, H.R., Ojo, I.A.O., Pianka, M., 2000. Organosulfur Szczepanski, C., Zgorzelak, P., Hoyer, G-A., 1972. Isolierung, Struktu- compounds as potential fungicides: the preparation and properties of some raufkl¨arung und Synthese einer antimikrobiell wirksamen Substanz aus substituted benzyl 2-hydroxyethyl oligosulfides. Phosphorus Sulfur Sili- Petiveria alliacea L. Arzneimittel-Forschung 22, 1975–1976.
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Torre Melis, R.A., Guerra, M.J.M., Cabrera, M.R.F., Rodr´ıguez, F.M., Benevides, P.J.C., Young, M.C.M., Giesbrecht, A.M., Roque, N.F., Bolzani, 1994. Caracterizacion farmacologica y toxicologica de Petiveria alliacea V.S., 2001. Antifungal polysulphides from Petiveria alliacea L. Phyto- (anamu) I Evaluacion de la actividad antimicrobiana. Revista Cubana de C´aceres, A., L´opez, B., Gonz´alez, S., Berger, I., Tada, I., Maki, J., 1998.
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