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Laboratoire de Pharmacognosie, Faculte des Sciences Pharmaceutiques et Biologiques, B.P. 83, 59006 Lille Cedex, France b Laboratoire de Bacteriologie, Faculte des Sciences Pharmaceutiques et Biologiques, B.P. 83, 59006 Lille Cedex, France c Laboratoire de Pharmacognosie, Faculte des Sciences Pharmaceutiques et Biologiques, U.R.A. au C.N.R.S. no. 1310, 4, A6enue de lObser6atoire, 75006 Paris, France Received 20 November 1998; received in revised form 1 February 1999; accepted 8 February 1999
Abstract In addition to the previously isolated phenylpropanoid glycosides verbascoside 1, forsythoside B 2, arenarioside 3 and ballotetroside 4, another four compounds were isolated from generative aerial parts of Ballota nigra: three phenylpropanoid glycosides, alyssonoside 5, lavandulifolioside 6 and angoroside A 7 and a non-glycosidic derivative ( +)-(E)-caffeoyl-L-malic acid 8.The antibacterial activity of the ve major compounds (1 4 and 8) was tested against Gram-positive and Gram-negative bacteria. Three of them (1 3) exhibited a moderate antimicrobial activity against Proteus mirabilis and Staphylococcus aureus including one methicillin-resistant strain. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Ballota nigra; Phenylpropanoid non-glycosidic ester; Phenylpropanoid glycosidic esters; Antimicrobial activity
1. Introduction The genus Ballota L. consists of about 33 species of Lamiaceae belonging to the tribe Stachydae, subtribe Ballotae. Most of them grow in the Mediterranean region and adjoining Europe and Asia Minor. Only four species originate from Eastern and Southern Africa (Patzak, 1958, 1959, 1961). Ballota nigra L. is commonly distributed in Western Europe where the generative aerial parts
* Corresponding author.
and their aqueous and hydroalcoholic extracts are used in medicine for treating inuenza, cough, and more especially to evoke neurosedative activities. Previously chemical investigation of aerial parts resulted in isolation of the avonoids apigenin-7glucoside, vicenin-2 (Darbour et al., 1986) and tangeretin (Kisiel and Piozzi, 1995) and the labdane diterpenoids ballotinone (Savona et al., 1976a), ballonigrine (Savona et al., 1976b), 7aacetoxymarrubiin (Savona et al., 1977a), ballotenol (Savona et al., 1977b), preleosibirin (Bruno et al., 1986) and 13-hydroxyballonigrino-
0378-8741/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 1 9 - 7
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lide (Seidel et al., 1996a). Recent studies of a hydroalcoholic extract of aerial parts led to the isolation of four phenylpropanoid glycosides: verbascoside 1, forsythoside B 2, arenarioside 3, and ballotetroside 4 (Seidel et al., 1996b, 1997). The present work deals with the isolation of four other known phenylpropanoid derivatives from a polar extract of aerial parts of Ballota nigra, including three glycosides: alyssonoside 5, lavandulifolioside 6, angoroside A 7, and a nonglycosidic derivative: (+ )-(E)-caffeoyl-L-malic acid 8 (see Fig. 1). The well-known antibacterial properties of numerous phenolics and especially of several natural phenylpropanoids (Jimenez and Riguera, 1994) prompted us in a second time to investigate the antimicrobial activity of the ve major compounds 14 and 8. They were evaluated against several strains of both Gram-positive and Gramnegative bacteria.
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ment of 5 7 (10 mg each) with acetic anhydride (1 ml) and pyridine (1 ml). The remaining aqueous solution (1/5) was adjusted to pH 2 (HCl) and extracted twice with ether. The ether solutions were concentrated in vacuo and the residue (3.4 g) was submitted to column chromatography (CC) on Sephadex LH 20 using EtOHH2O 1:1 as eluent. Fractions containing 8 were further puried by CC on silica gel H using EtOAc MeOH H2O, 70:40:20 to give pure 8 (0.9 g).
2.5. Microorganisms
Bacteria (24 strains) were isolated from human clinical samples in the Dron Hospital (135, rue President Coty, F-59200 Tourcoing). The following tested microorganisms were Staphylococcus aureus (ve strains including one methicillin-resistant strain [MRSA]) and Enterococcus faecalis (ve strains) for Gram-positive bacteria, Pseudomonas aeruginosa (three strains), Escherichia coli (ve strains), Proteus mirabilis (three strains) and one of each strain of Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca for Gram-negative bacteria. Staphylococcus aureus were isolated from cutaneous pus, other strains from urinary tract infections.
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An agar plate without phenylpropanoids was inoculated and incubated to determine the viability of the organisms and to serve as a control for the comparison of growth. Previous control with DMSO indicated that the growth of the bacterial strains was not affected by the DMSO.
3. Results and discussion Seven phenylpropanoid glycosidic esters 1 7 and one phenylpropanoid non-glycosidic ester 8 were isolated from aerial parts of Ballota nigra. The structures of 1 4 which were previously described in this species (Seidel et al., 1996b; Seidel et al., 1997) were determined by direct comparison ([h]D, ms, 1H and 13C NMR) with authentic samples. The structures of compounds 5 8 which are described for the rst time in the genus Ballota were deduced from their physical and spectral data ([h]D, ms, 1H and 13C NMR) and from those of their peracetylated derivatives in the case of glycosides 5 7. The complete spectroscopic assignment of the different moieties of these three phenylpropanoid glycosides as well as the determination of interglycosidic linkages have been conrmed by extensive experiments using 1 H 1H COSY, 13C 1H HETCOR and COLOC techniques and in comparison with previously reported data (Calis et al., 1987, 1992; Basaran et al., 1988; Hahn and Nahrstedt, 1993). The total phenylpropanoid derivatives in aerial parts of Ballota nigra determined colorimetrically accounted for 5.5% of dry weight. Quantitatively, compounds 14 and 8 which are present in almost equal amounts are the major phenylpropanoids, compounds 5 7 being present in very small amounts. Therefore each of compounds 14 and 8 accounts for ca 1% of the dried aerial parts. Compounds 14 and 8 were evaluated with the agar dilution method at concentrations up to 128 mg/ml against a panel of gram-positive and gram-negative bacteria. Amongst this last type of microorganisms, we chose three strains of each Pseudomonas aeruginosa and Proteus mirabilis,
ve strains of Escherichia coli and one strain of Enterobacter aerogenes, Klebsiella pneumoniae and Klebsiella oxytoca. Regarding gram-positive bacteria, ve strains of each Enterococcus faecalis and Staphylococcus aureus (one of them methicillin-resistant) were used for testing the compounds named above. It is interesting to take in account that some infections produced by Staphylococcus aureus are commonly very difcult to eradicate. Therapy with several type of antibiotics (Hiramatsu et al., 1997) is frequently accompanied by the appearance of unexpected side effects and acquisition of resistance. Methicillin-resistant Staphylococcus aureus (MRSA) are one of the major nosocomial pathogens in hospitals (Voss and Doebbeling, 1995) and although some studies devoted to looking for new anti-MRSA agents have been reported (Tsuchiya et al., 1996; Chambers, 1997), other compounds alternative to existing antibiotics are still needed. Results obtained with the ve major phenylpropanoids tested 14 and 8 showed that 4 and 8 do not possess antimicrobial activity up to 128 mg/ml. Compounds 13 possess a moderate inhibitory effect against Staphylococcus aureus and Proteus mirabilis (see Table 1). Compounds 1 and 2 inhibited one out of three strains of Proteus mirabilis and two out of ve strains of Staphylococcus aureus including the methicillinresistant strain, at 128 mg/ml. The more signicant results were observed with compound 3: three out of ve strains of Staphylococcus aureus were inhibited at a concentration of 128 mg/ml and one out of three strains of Proteus mirabilis at 64 mg/ml. Although the moderate antimicrobial activity of verbascoside, forsythoside B and arenarioside could not allow their use as single agents we hope that results reported here open the possibility of further investigations on their antimicrobial properties and studies of their combinations with other antibiotics. In addition, the fact that compound 3 shows inhibition of the methicillin-resistant Staphylococcus aureus strain, could aid in the development of new agents against one of the major nosocomial pathogens in hospitals.
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Table 1 Antimicrobial activity of compounds 1, 2, 3 at concentrations of 64 mg/ml and 128 mg/ml against Staphylococcus aureus and Proteus mirabilis a Bacterial strains Compounds 1 64 mg/ml Staphylococcus aureus 1 2 3 4 5 (MRSA) 1 2 3 + + + + + + + + + + + + + + + + + + + + + + + 2 3 1 128 mg/ml + + + + + + + + + + + + + + 2 3
Proteus mirabilis
MRSA, methicillin-resistant Staphylococcus aureus; +, growth; , no visible growth. feuilles de Ballota foetida Lamk. (Labiees). Pharmazie 41, 605 606. Hahn, R., Nahrstedt, A., 1993. Hydroxycinnamic acid derivatives, caffeoylmalic and new caffeoylaldonic acid esters from Chelidonium majus. Planta Medica 59, 71 75. Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T., Tenover, F.C., 1997. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy 40, 135 146. Jimenez, C., Riguera, R., 1994. Phenylethanoid glycosides in plants: structure and biological activity. Natural Products Reports 11 (6), 591 606. Kisiel, W., Piozzi, F., 1995. Tangeretin from Ballota nigra. Polish Journal of Chemistry 69, 476 477. National Committee for Clinical Laboratory Standards, 1997. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Fourth Edition, Approved Standard. M7-A4. NCCLS, Villanova, PA. Patzak, A., 1958. Revision der Gattung Ballota section Ballota. Annales der Naturhistorischen Museums in Wien 62, 57 86. Patzak, A., 1959. Revision der Gattung Ballota section Acanthoprasium und section Beringeria. Annales der Naturhistorischen Museums in Wien 63, 33 81. Patzak, A., 1961. Zwei neue Ballota Arten aus der Turckei nebst einem nachtrag zur Gattung Ballota. Annales der Naturhistorischen Museums in Wien 64, 42 56. Savona, G., Piozzi, F., Hanson, J.R., Siverns, M., 1976a. Structure of ballotinone, a diterpenoid from Ballota nigra. Journal of the Chemical Society Perkin Trans 1, 1607 1609. Savona, G., Piozzi, F., Hanson, J.R., Siverns, M., 1976b. New diterpenoids from species of genus Ballota. La Chimica e lIndustria, Milan 58, 378. Savona, G., Piozzi, F., Hanson, J.R., Siverns, M., 1977a. Structure of three new diterpenoids from Ballota species. Journal of the Chemical Society Perkin Trans 1, 322 324.
Acknowledgements The authors would like to thank N. Derensy and E. Singer, Department of Bacteriology, University of Lille, for their technical assistance in the course of this work and Professor I. Calis, Hacettepe University, Faculty of Pharmacy of Ankara, for supplying authentic samples.
References
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