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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.49 n.1 Concepción mar. 2004

http://dx.doi.org/10.4067/S0717-97072004000100001 

ANTIBACTERIAL ACTIVITY OF COMPOUNDS ISOLATED
OF THE RESINOUS EXUDATE FROM HELIOTROPIUM SINUATUM
ON PHYTOPATHOGENIC BACTERIA.

BRENDA MODAK1*, RENÉ TORRES1* , MARCELA WILKENS2 AND ALEJANDRO URZÚA1.

Facultad de Química y Biología, 1Departamento de Ciencias del Ambiente,
2
Departamento de Biología, Universidad de Santiago de Chile, Santiago, Chile;
E-mail: rtorres@lauca.usach.cl.

(Received: January: 29, 2003 - Accepted : June 2, 2003)

ABSTRACT

From the resinous exudates of Heliotropium sinuatum have been isolated and characterized two compounds, pentaeicosanol 1 and 1-hydroxy-3-doeicosanone 2. Those structures and previously isolated pure compounds have been tested in their antibacterial activity on phytopathogenic bacteria. Ketone 2 and the flavonoid hesperetine 10 showed antibacterial activity on Erwinia carotovora subsp. carotovora, while the flavonoid 3-O-methylgalangine 4 showed activity on Clavibacter michiganensis subsp. michiganensis .

Keywords: Heliotropium sinuatum, resinous exudate, antibacterial activity, Erwinia carotovora carotovora, Clavibacter michiganensis michiganensis.

INTRODUCTION

Heliotropium sinuatum Miers. (Boraginaceae) grows in the north of Chile. This specie is characterized by its production of a resinous exudate that covers leaves and stems 1-2).

Previous chemical studies on the resins of this species reported flavonoids 3) and an arylphenol 4). A new study of this resinous exudate allowed the isolation and characterization of the alcohol pentaeicosanol 1 and the ketone 1-hydroxy-3-doeicosanone 2.

The production of resinous exudates on vegetals have been asociated to antimicrobial, antioxidant and citotoxic properties due to the presence of secondary metabolites on that oily sticky surface 5-8).

Previosusly we reported) antibacterial activity of the resin and their pure compounds on E.coli., bacteria not related with vegetal microbial flora9, therefore it´s not a good model for searching the type of protection of the resinous exudate. A good approach will be to use phytopathogenic bacteria, such as Erwinia carotovora subsp. carotovora and Clavibacter michiganensis subsp. michiganensis for testing antibacterial activities.

Erwinia carotovora subsp. carotovora is a Gram negative bacterium and is responsible for the disease named bland rotting on the beefy tissue from species such as Solanum tuberosum (potato)10-11). On the other hand, Clavibacter michiganensis subsp. michiganensis is a Gram positive bacterium and produce ulcers and chancres in vegetables species, mainly in Licopersicum sp (L.esculentum)12).


EXPERIMENTAL

Tested compounds: the compounds were obtained of the resinous exudate from Heliotropium sinuatum Miers. through column cromatography in silica gel and were identified by spectroscopy 1H-NMR and 13C-NMR, IR, UV and mass spectrometry 3-5).Carbon kind were determined by DEPT experiments. The 13C-NMR spectrum were confirmed by ACD-Labs Demo Version 1.0 for MS Windows.

Pentaeicosanol: mp: 71-73ºC. IR (KBr) (cm-1): 3309, 2916, 2848, 1473, 1463, 1060. 1H-NMR ( 200 MHz, CDCl3 ), d (ppm): 0.88 (t,3H), 1.25 (m,22H), 1.54 (m,2H), 3.64 (t,2H). 13C-NMR ( 200 MHz, CDCl3 ), d (ppm): 14.1 (CH3); 22.7(CH2); 25.7(CH2); 29.4(CH2); 29.7(CH2); 31.9(CH2); 32.8(CH2); 63.0(CH2).

EIMS (m/z, %) : 365 (1), 322 (1), 126 (12), 99 (5), 98 (42), 71 (31), 70 (21), 57 (74), 43 (100), 42 (24). Analysis combustion calcd for C25H52O, C = 81.50 %, H = 14.19 %, O = 4.31 %.

1-hydroxy-3-doeicosanone: mp: 65-67 ºC. IR (KBr) (cm-1): 3303, 2916, 2848, 1697. 1H-NMR (200 MHz, CDCl3), d (ppm): 0.88 (t,3H), 1.25 (s,30 H), 1.58 (s,4H), 2.44 (m,3H), 2.67 (t,2H), 3.85 (m,2H). 13C-NMR (200 MHz, CDCl3), d (ppm): 14.1(CH3); 22.7(CH2); 23.7(CH2); 29.2(CH2); 29.4(CH2); 29.5(CH2); 31.9(CH2); 43.4(CH2); 44.3(CH2); 57.9(CH2); 212.1. EIMS ( m/z, %) : 340 (4), 88 (73), 73 (48), 43 (100). Analysis combustion calcd for C22H44O2 ,C = 77.58 %, H = 13.02 %, O = 9.40 % .

Bacterial strains: Clavibacter michiganensis subsp. michiganensis Cmm 623, was isolated from L. esculatum and donated by Dr. Jorge Delgado ( Microbiology Laboratory of Pontificia Universidad Católica of Chile). Erwinia carotovora subsp. carotovora IC 2610 was isolated from potato and donated by Dra. Inés Calderón (Pontificia Universidad Católica of Chile).

Antibacterial activity determination: The antimicrobial activity was determinated by the agar overlay method 13). Bacteria growing overnight in broth suitable according to the bacterial use, were diluted to Mc Farland 0,5 ( 1x10 8 cells/mL) and 100 mL of this dilution were mixed with 3 mL of melted soft agar (0.7%) at 50ºC. The soft agar was poured over Petri dishes containing 20 mL of 1.5% agar. Drops of 5 mL of the test compounds (1 mg/mL) in methanol and the solvent were deposited over solidified agar. After 24 h of incubation at environmental temperature, the diameter of the inhibition zone was determinated. Control measurements were carried out with methanol.

Broth for Clavibacter michiganensis subsp. michiganensis Cmm 623: sacarose 2.0 g, hydrated casein 1.6 g, ferment extract 0.8 g, K2HPO4 0.4 g, MgSO4 x 7H2O 0.06 g, water 200 mL, agar at 1.5 % for hard agar and 0.75 % for softagar.

Broth for Erwinia carotovora subsp. carotovora IC 2610: K2HPO4 2.625 g, KH2PO4 1.125 g, (NH4)2SO4 0.250 g, sodium citrate x 2 H2O 0.125 g, water 250 mL, agar at 1.5 % for hard agar and 0.75 % for soft agar.

After of sterilize were additioned MgSO4 x 7 H2O 1M 0.25 mL and glucose 20 % 2.5 mL solutions.

Statistical analysis: was carried out by the Mann-Whitney test.

RESULTS AND DISCUSSION

From resinous exudate of Heliotropium sinuatum was obtained pentaeicosanol with white solid. The presence of the hydroxyl group was evident from the IR absorption band at 3309 cm-1. This spectrum showed absorption at 1060 cm-1 corresponding to stretching C-O of satured alcohol. The 1H-NMR spectrum showed only aliphatic region, indicating that was a large chain alcohol. This structure was supported by 13C-NMR spectrum and by 13C-NMR simulator ACD-Labs Demo Version 1.0 for MS Windows. No parent pick was observed, what is typical in large chain alcohol. This chain suffered very fast a -hydrogen elimination to give an ion M-3 at m/z = 365 (1%) consistent with the formula C24H49O. This compound was isolated previously from oil olive 14).

From the same extract, was isolated 1-hydroxy-3-doeicosanone as a white solid. The presence of the hydroxyl group was evident from the IR absorption band at 3303 cm-1. This spectrum showed besides absorption band at 1697 cm-1 corresponding to stretching of the carbonyl group. The 1H-NMR spectrum showed a wide singlet at 1.58 ppm which integrated for 4 protons and was assigned to the 2 methylene hydrogen a and b at a carbonyl group. At 2.44 ppm was observed a multiplet which integrated for 3 protons and was assigned to CH2 group adyacent to carbonyl and the proton of hydroxyl group, which disappeared for exchange with deuterium. At 3.85 ppm was observed a multiplet which integrated for 2 protons and was assigned to methylene group a to hydroxyl group and b to carbonyl group. Carbonyl was confirmed by the signal at 212.1 ppm of the 13C-NMR spectrum. In the mass spectrum was observed a parent ion at m/z = 340 (4%) consistent with the formula C22H44O2. This formula was confirmed by combustion analysis. This compound was isolated previously from Elaeugnus angustifolia 15).

The compounds before described 1 and 2, the flavonoids 5,7,4'-trihydroxy-flavanone (naringenine) 3, 5,7-dihydroxy-3-methoxyflavone (3-O-methylgalangin) 4, 5,3',4'-trihydroxy-7-methoxyflavanone (7-O-methyleriodictyol) 5, 5,7,4'-trihydroxy-3,3'-dimethoxyflavone (3-O-methylisorhamnetin) 6, 5,4'-dihydroxy-3,7,3'-trimethoxyflavone (pachypodol) 7, 3-acetoxy-5,7-dihydroxyflavanone (pinobanksin-3-acetate) 8, 5,7-dihydroxyflavanone (pinocembrin) 9, 5,7,3'-trihydroxy-4'-methoxyflavanone (hesperetin) 10 and 4-(3',5'-dihydroxynonadecyl)phenol 11, were tested against Erwinia carotovora subsp. carotovora y Clavibacter michiganensis subsp. michiganensis. The results and the statistical analysis are in tables 1 and 2. With the compounds that showed prevented bacterial growth (2,4,10) were realized MIC (minimum inhibitory concentration) determinations (see table 3).

Table 1.- Inhibition zone for C.michiganensis subsp. michiganensis.


Test compound

Diameter of the
inhibition zone (mm) ± sd


Metanol

0 ± 0

1

zd

2

zd

3

zd

4

11.33 ± 0.47

5

zd

6

zd

7

zd

8

0 ± 0

9

zd

10

zd

11

zd


 

zd:zone diffuse.

Table 2: Inhibition zone for E.carotovora subsp. carotovora.


Test compound

Diameter of the
inhibition zone (mm) ± sd


Metanol

0 ± 0

1

zd

2

9 ± 0.82

3

0 ± 0

4

0 ± 0

5

0 ± 0

6

0 ± 0

7

0 ± 0

8

0 ± 0

9

zd

10

5.33 ± 0.58

11

0 ± 0


zd:zone diffuse.

Table 3: Minimum inhibitory concentration for compounds 2, 4 y 10.


 

COMPOUND

MIC ( mg / mL)

4

(with C. michiganensis michiganensis)

250

2

(with E.carotovora carotovora)

500

10

(with E.carotovora carotovora)

500

When the group of compounds was tested with Gram negative bacterium Erwinia carotovora subsp. carotovora, only the flavonoid hesperetin 10 and the ketone 1-hydroxy-e-doeicosanone 2 were active. On the other hand, when were tested with Clavibacter michiganensis subsp. michiganensis, only the flavonoid 3-O-methylgalangin 4 showed activity and structures just formed a zone diffuse, in other words , the bacteria present a very small growth inhibition, and only pinobanksin-3-acetate 8 was inactive. The determination of MIC value gave for metylgalangine a good activity. Though there is not general conclusion about structure-activity relationship for the flavonoids, is very important the cathecol system on the A or B ring, so that the compounds are active. Also is important the total number of hydroxyl groups16-19). However, in our results, those characteristics are not important in the activity of 3-O-methylgalangin against C.michiganensis subsp. michiganensis, which does not present cathecol system and have a low hydroxylation level compared with other tested flavonoids.

Recent studies show that hydroxylated ketones have antibacterial activity, but test on not phythophatogenic bacteria such as B. cereus, E. coli and S. epidermidis 20).

The MIC values and the different effects against the bacteria show that the resin is a complex system protection against bacteria, with compounds highly specific on their action.

ACKNOWLEDGEMENTS

This work was supported by FONDECYT Grant Nº 2950053 and DICYT (Usach).

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