Antimicrobial Triterpenoids from Licania heteromorpha

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Six triterpenoids having a lupane and oleane skeleton were isolated from the leaves and young branches of Licania heteromorpha Bentham var. heteromorpha and were identified as: betulinic acid 1, alphitolic acid 2, 3β-O-trans-p-coumaroyl alphitolic acid 3, 3β-O-cis-p-coumaroyl alphitolic acid 4, 3β-O-trans-p-coumaroyl maslinic acid 5, 3β-O-cis-p-coumaroyl maslinic acid 6. The antimicrobial activity of these compounds was evaluated in vitro on clinically isolated microorganisms employing a microdilution method. Compounds 2, 3, 5, and 6 showed antimicrobial activity on Gram-positive bacteria and yeasts, whereas none of the six triterpenoids were active against Gram-negative organisms.

Licania heteromorpha Bentham, var. heteromorpha (Chrysobalanaceae), is a tree up to 30 m high native of the Amazonian forest [1]; in Venezuelan traditional medicine the leaves are used as anti-inflammatory remedies [2]. The ethanolic extract of this plant showed cytotoxic activity in vitro in colon carcinoma 38 and B16 melanoma models [3]. In this paper we report the isolation of six triterpenoids and their antimicrobial activity; triterpenoids are widely spread in plant extracts [4] and some of them, e.g., ursolic acid, are reported to have antimicrobial activity [5].

Six triterpenoids (1 - 6) were isolated from the chloroform extract of L. heteromorpha by silica gel column chromatography followed by reversed-phase HPLC. Structural identification of compounds 1 - 6 was performed by spectral methods, such as 1D and 2D NMR (¹H, ¹³C, HSQC); the data obtained were compared with those reported in the literature [6], [7], [8], [9] and were confirmed by chromatography in comparison with standards. These triterpenoids were identified as: betulinic acid 1 ([α]²⁵ _D: + 6.8°, c = 2.0, pyridine) [6], alphitolic acid 2 ([α]²⁵ _D: - 4.0°, c = 1.0 pyridine), 3β-O-trans-p-coumaroyl alphitolic acid 3 ([α]²⁵ _D: - 30°, c = 2.5 pyridine), 3β-O-cis-p-coumaroyl alphitolic acid 4 ([α]²⁵ _D: + 40°, c = 1.0 pyridine), [7], [8], 3β-O-trans-p-coumaroyl maslinic acid 5 ([α]²⁵ _D: + 1°, c = 1.0 pyridine), 3β,O-cis-p-coumaroyl maslinic acid 6 ([α]²⁵ _D: + 9°, c = 1.0 pyridine) [9], (copies of the original spectra are obtainable from the corresponding author). Their optical rotation values agreed with those reported in the literature [8], [9]. Although triterpenoids 4 - 6 are typical of other families, to our knowledge it is the first time they have been detected in the genus Licania. []

Compounds 2, 3, 5, and 6 showed antimicrobial activity, with a different spectrum of action, on Gram-positive bacteria and yeasts (Table [1]). The broadest activity was observed for compounds 3 and 6 that inhibited the growth of S. capitis, S. agalacticae, C. albicans, and C. krusei; in particular 3 showed a MIC of 12.5 μg/ml on S. capitis and C. albicans. Alphitolic acid (2) inhibited S. aureus (100 μg/ml) and C. albicans (50 μg/ml); 4 was active at the maximum concentration tested (100 μg/ml) only against S. agalacticae. Compound 5 inhibited S. aureus, S. agalacticae and C. albicans with different activity. The antimicrobial activity of the triterpenoids tested was generally bactericidal. Betulinic acid (compound 1) was inactive against all microorganisms tested. None of these triterpenoids inhibited the Gram-negative bacteria Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa.

The stereoisomery of the compounds appeared to affect their activity particularly in the case of 3β-O-trans-p-coumaroyl alphitolic acid 3 and 3β-O-cis-p-coumaroyl alphitolic acid 4; the latter inhibited only one bacterium at the maximal concentration tested while the former inhibited almost all tested microorganisms at low concentration.

Materials and Methods

Leaves and young branches of L. heteromorpha var. heteromorpha were collected in Puerto Ayacucho, Venezuela, in July 1996 and identified by Dr. A. Castillo of Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela, where a voucher specimen of leaves and young branches has been deposited at herbarium (n ·VEN296964).

¹H-NMR and ¹³C-NMR spectra (CD₃OD) were obtained with a Bruker DRX-600 spectrometer operating at 599.19 MHz for ¹H and 150.858 MHz for ¹³C; HSQC experiments were performed using the UXNMR software package. HPLC separations were performed on a Waters 590 series pumping system equipped with a Waters R401 refractive index dectector and with a Waters μ-Bondapak C₁₈ column and U6K injector. Silica gel column chromatography was carried out with Merck Kieselgel 60 (70 - 230 mesh). TLC was conducted on silica 60 F₂₅₄ gel-coated glass sheets.

The powdered dried leaves and young branches of L. heteromorpha (480 g) were defatted with n-hexane and then extracted in a Soxhlet apparatus with CHCl₃ and CHCl₃-MeOH (9 : 1) each for 24 h to yield 10.0 g and 13.0 g of residue, respectively. Part of the chloroform extract (6 g) was chromatographed on a silica gel column (5 × 50 cm, 50 g of silica gel for 1 g of crude extract), eluting with CHCl₃ followed by increasing concentrations of MeOH (between 1 % and 50 %) in CHCl₃; the following volumes of solvents were used: 17.58 L of CHCl₃, 5.85 L of CHCl₃ : MeOH 99 : 1, 1.28 L of CHCl₃ : MeOH 98 : 2, 0.68 L of CHCl₃ : MeOH 97 : 3, 1.48 L of CHCl₃ : MeOH 95 : 5, 2.1 L of CHCl₃ : MeOH 9 : 1, and 0.6 L of CHCl₃ : MeOH 1 : 1. Fractions of 25 mL were collected and analysed by TLC (silica gel plates, in CHCl₃ or mixtures CHCl₃-MeOH 99 : 1, 98 : 2, 97 : 3, 95 : 5, 9 : 1, 8 : 2; spray reagent was ceric sulphate in 65 % of sulphuric acid). The dried fractions 261 - 281, 844 - 859, and 885 - 898 were subjected to reversed-phase HPLC on a C₁₈μ-Bondapak column (300 × 7.8 mm, 125 Å, 10 μm, flow rate 2.0 mL min^-1) using respectively MeOH-H₂O (9 : 1), (15 : 5), and (17 : 3) as eluent to yield the pure triterpene 1 (10 mg, t_R = 13 min) from the first fraction, triterpene 3 (12 mg, t_R = 22 min), 4 (15 mg, t_R = 15 min), 5 (9 mg, t_R = 27 min), and 6 (10 mg, t_R = 20 min) from the second, and pure triterpene 2 (13 mg, t_R = 16 min) from the third.

The minimal inhibitory concentration (MIC) was determined on 96 well culture plates by a microdilution method using Muller-Hinton Broth (Becton-Dickinson, Milan, Italy). Eleven two-fold dilutions of test substances were carried out starting from the concentration of 100 μg/mL (2.5 % of ethanol). All preparations were sterilized with a 0.22 μm filter. The wells were inoculated with a microorganism suspension at a density of 10⁵ cells/mL. The plates were incubated for 24 h (or 48 h for the yeasts) at 37 °C. The cultures that did not present growth were used to inoculate plates of solid medium in order to determine the minimal bactericidal concentration (MBC). Proper blanks were assayed simultaneously. Samples were tested in triplicate.

Acknowledgements

Dr. Lucia Battinelli was supported by the “Noopolis Foundation” (Italy).

References

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• 2 Pittier H. Manual de las Plantas usuales de Venezuela. Caracas; Editorial Fundacion Eugenio Mendoza 1978: 579
• 3 Suffness M, Abbott B, Statz D, Wonilowicz E, Spjut R. The utility of P388 leukemia compared to B16 melanoma and colon carcinoma 38 for in vivo screening of plant extract.  Phytotherapy Research. 1988;  2 89-97
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  CrossrefPubMedSearch in Google Scholar
• 6 Reher G, Budesinsky M. Triterpenoids from plants of the Sanguisorbeae.  Phytochemistry. 1992;  31 3909-14
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• 7 Siddiqui B S, Farhat Begum S, Siddiqui S. Isolation and structural elucidation of acylated pentacyclic triterpenoids from the leaves of Eucalyptus camaldulensis var. obtusa .  Planta Medica. 1997;  63 47-50
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• 8 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishiota I. Studies on the constituents of Zizyphi Fructus. I. Structure of the three new p-coumaroylates of alphitolic acid.  Chemical and Pharmaceutical Bulletin. 1978;  26 1798-802
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• 9 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishiota I. Studies on the constituents of Zizyphi Fructus. II. Structure of new p-coumaroylates of maslinic acid.  Chemical and Pharmaceutical Bulletin. 1978;  26 3075-9
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Prof. Gabriela Mazzanti

Dipartimento di Farmacologia delle Sostanze

Naturali e Fisiologia Generale

Università di Roma “La Sapienza”

Piazzale Aldo Moro 5

00185 Rome

Italy

Email: gabriela.mazzanti@uniroma1.it

Fax: +39 06 49912480

References

• 1 Toledo C L, Kubitzki K, Prance G H. Flora de Venezuela. Vol IV Ediciones Fundacion Educacion Ambiental Caracas; 1982: 367-8
  Search in Google Scholar
• 2 Pittier H. Manual de las Plantas usuales de Venezuela. Caracas; Editorial Fundacion Eugenio Mendoza 1978: 579
• 3 Suffness M, Abbott B, Statz D, Wonilowicz E, Spjut R. The utility of P388 leukemia compared to B16 melanoma and colon carcinoma 38 for in vivo screening of plant extract.  Phytotherapy Research. 1988;  2 89-97
  PubMedSearch in Google Scholar
• 4 Hernàndez-Pèrez M, López-García R E, Rabanal R M, Darias V, Arias A. Antimicrobial activity of Visna mocanera leaf extracts.  Journal of Ethnopharmacology. 1994;  41 115-9
  CrossrefPubMedSearch in Google Scholar
• 5 Collins M A, Charles H P. Antimicrobial activity of carnosol and ursolic acid: two anti-oxidant constituents of Rosmarinus officinalis L.  Food Microbiology. 1987;  4 311-4
  CrossrefPubMedSearch in Google Scholar
• 6 Reher G, Budesinsky M. Triterpenoids from plants of the Sanguisorbeae.  Phytochemistry. 1992;  31 3909-14
  CrossrefPubMedSearch in Google Scholar
• 7 Siddiqui B S, Farhat Begum S, Siddiqui S. Isolation and structural elucidation of acylated pentacyclic triterpenoids from the leaves of Eucalyptus camaldulensis var. obtusa .  Planta Medica. 1997;  63 47-50
  PubMedSearch in Google Scholar
• 8 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishiota I. Studies on the constituents of Zizyphi Fructus. I. Structure of the three new p-coumaroylates of alphitolic acid.  Chemical and Pharmaceutical Bulletin. 1978;  26 1798-802
  PubMedSearch in Google Scholar
• 9 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishiota I. Studies on the constituents of Zizyphi Fructus. II. Structure of new p-coumaroylates of maslinic acid.  Chemical and Pharmaceutical Bulletin. 1978;  26 3075-9
  PubMedSearch in Google Scholar

Prof. Gabriela Mazzanti

Dipartimento di Farmacologia delle Sostanze

Naturali e Fisiologia Generale

Università di Roma “La Sapienza”

Piazzale Aldo Moro 5

00185 Rome

Italy

Email: gabriela.mazzanti@uniroma1.it

Fax: +39 06 49912480