Antimicrobial Principle from Aframomum longifolius

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolates
• Antimicrobial assay
• Results and Discussion
• Acknowledgements
• References

Abstract

Antimicrobial activity-directed fractionation of the seeds of Aframomum longifolius (Zingiberaceae) afforded two new labdane-type diterpenoids, 15-hydroxy-15-methoxylabda-8(17), 12(E)-dien-16-al (aframolin A) (1) and 8β(17)-epoxy-15,15-dimethoxylabd-12(E)-en-16-al (aframolin B) (2), together with the known diterpenes labda-8(17),12(E)-diene-15,16-dial (3) and aframodial (4). Their structures were determined by spectroscopic methods. Compound 4 showed significant antimicrobial activity against Cryptococcus neoformans, Staphylococcus aureus and methicillin-resistant S. aureus (MRS) while 1, 2 and 3 were found to be inactive.

Introduction

Many species of Aframomum (Zingiberaceae) are used in Cameroon for medicinal, ethnodietary and spiritual purposes [1]. The genus Aframomum was found to contain flavonoids and diterpenoids [1], [2], [3]. Aframodial, a diterpene dialdehyde isolated from Aframomum daniellii (Hook. F.) K. Shum [2] was reported to possess antifungal, cytotoxic and hypercholesterolemic properties [4], [5]. Although plants of this genus find an extensive use in folklore medicine, no phytochemical study has so far been reported on A. longifolius. We herein report an antimicrobial activity-guided fractionation of the seeds of this plant.

Materials and Methods

General experimental procedures

NMR spectra, including COSY, HMQC, DEPT and HMQC experiments, were recorded on a Varian Mercury 400 or a Bruker DRX 500 spectrometer at 400 or 500 MHz (¹H) and 100 or 125 MHz (¹³C), with chemical shifts given in ppm (δ) using TMS as an internal standard. IR spectra were recorded on an ATI Mattson Genesis Series FTIR^TM. MS were recorded either on a Hewlett Packard 5890 Series II, GC/MS, Finnigan AQA or Brucker Biopex FTMS. Optical rotations were measured on a Rudolph Research Analytical (Autopol IV) automatic polarimeter. Silica gel, 40 μm (flash chromatography packing; 6 Å pore diameter) was used for CC, silica gel 60 F₂₅₄ (Merck) was used for TLC, and silica gel GF was used for preparative TLC.

Plant material

The seeds of A. longifolius were collected in September 2002 at Koladom, a village situated 12 km from Ebolowa, South Cameroon and authenticated by Dr. Zapfack of the Botany Department, University of Yaounde I, Cameroon. A voucher specimen (UD 516) is deposited at the herbarium of the Botany Department of the University of Dschang, Cameroon.

Extraction and isolation

The seeds of A. longifolius (284 g) were macerated three times with a methylene chloride/methanol mixture (1 : 1, 600 mL × 3). Filtration and removal of solvent under vacuum afforded 62 g of crude extract. This extract was loaded on a silica gel column (7 × 50 cm, 600 g) and eluted with hexane-EtOAc (19 : 1, 7 L; 9 : 1, 3 L; 17 : 3, 2.5 L; 4 : 1, 1.75 L; 7 : 3, 1.75 L; 3 : 2, 3.25 L; 2 : 3, 2.25 L) to obtain 170 fractions (125 mL each). The fractions were combined into nine major fractions (F₁ - F₉, F₁ : 1 - 20, F₂ : 21 - 56, F₃ : 56 - 68, F₄ : 69 - 92, F₅ : 93 - 100, F₆ : 101 - 114, F₇ : 115 - 139, F₈ : 140 - 153, F₉ : 154 - 170) based on TLC behavior. IC₅₀ values of active fractions are summarized in Table [1]. F₁, F₇, F₈ and F₉ were found to be inactive. Fraction F₂ (4.6 g) was chromatographed on a silica gel column (4 × 40 cm, 60 g) eluting with hexane-EtOAc (39 : 1, 2.7 L) to yield four subfractions: B₁ (1.5 L), B₂ (0.5 L), B₃ (0.3 L), B₄ (0.4 L). Subfraction B₁ was further chromatographed on a silica gel column (2 × 30 cm, 15 g) using hexane-Et₂O (9 : 1, 1 L) as eluent to yield compound 1 (10.6 mg). Subfraction B₄ was purified on a chromatotron with hexane-EtOAc (10 : 0, 39 : 1, 19 : 1, 33 : 3, 0.5 L each) to give compound 3 (9.1 mg). Fraction F₃ (3.2 g) was loaded on a silica gel column (3 × 45 cm, 50 g) using hexane-EtOAc (9 : 1, 17 : 3, 4 : 1, 3 : 1, 7 : 3, 0.5 L each) to yield 4 (80.1 mg). Part of fraction F₄ (3.5 g) was chromatographed on silica gel column (3 × 45 cm, 50g) using hexane-EtOAc (9 : 1, 17 : 3, 4 : 1, 3 : 1, 7 : 3, 0.5 L each) to yield 4 (70.5 mg). Subfractions from F₄ were further purified by preparative TLC with hexane-EtOAc (2 : 3) to obtain 2 (38 mg). Fraction F₅ (1.5 g) was loaded on a silica gel column (4 × 45 cm, 60 g) using hexane-EtOAc (9 : 1, 17 : 3, 4 : 1, 3 : 1, 7 : 3, 1.5 L each) as eluent to yield 134.5 mg of 4.

Isolates

Aframolin A (1): Colorless oil; [α]_D ²⁷: 8.33° (c 0.24; CHCl₃); IR (CHCl₃): ν_max = 3380, 1680, 1464, 1125, 1077, 978, 750 cm^-1; EI-MS: m/z (rel. int.) = 316 [M - 18]⁺ (5), 258 (9), 137 (11), 122 (14), 91 (8), 75 (100), 47 (13), 41 (15); HR-ESI-MS: m/z = 357.2389 [M + Na]⁺ (calcd.: 357.2405); ¹H-NMR (CDCl₃, 400 MHz): δ = 0.74 (3H, s, H-20), 0.82 (3H, s, H-19), 0.88 (3H, s, H-18), 1.14 (1H, dd, J = 12.8, 2.4 Hz, H-5), 1.89 (1H, brd, J = 10.8 Hz, H-9), 2.60 (2H, dd, J = 6.0, 3.6 Hz, H-14), 2.65 (2H, dd, J = 6.4, 3.2 Hz, H-11), 3.34 (3H, s, OMe-15), 4.41 (1H, d, J = 1.6 Hz, H-17′), 4.43 (1H, t, J = 6.0 Hz, H-15), 4.83 (1H, d, J = 1.6 Hz, H-17), 6.54 (1H, t, J = 6.4 Hz, H-12), 9.33 (1H, s, H-16); ¹³C-NMR (CDCl₃, 100 MHz): δ = 14.6 (C-20), 19.5 (C-2), 21.9 (C-19), 24.3 (C-6), 24.7 (C-11), 29.2 (C-14), 33.7 (C-18), 33.8 (C-4), 38.1 (C-7), 39.4 (C-1), 39.8 (C-10), 42.2 (C-3), 54.5 (OMe), 55.6 (C-5), 56.7 (C-9), 104.1 (C-15), 108.1 (C-17), 138.3 (C-13), 148.4 (C-8), 160.2 (C-12), 195.2 (C-16).

Aframolin B (2): Colorless oil; [α]_D ²⁶: 22.2° (c 1.44; CHCl₃); IR (CHCl₃): ν_max = 2928, 2843, 1681, 1461, 1388, 1121, 1077, 973, 756 cm^-1; LR-ESI: m/z = 382 [M + NH₄]⁺, 365 [M + H]⁺; HR-ESI-MS: m/z = 387.2509 [M + Na]⁺ (calcd.: 387.2505); ¹H-NMR (CDCl₃, 400 MHz): δ = 0.88 (3H, s, H-19), 0.91 (3H, s, H-18), 0.95 (3H, s, H-20), 1.04 (1H, dd, J = 11.6, 2.8 Hz, H-5), 1.66 (1H, brd, J = 8.0 Hz, H-9), 2.30, 2.49 (each 1H, d, J = 4.0 Hz, H-17), 2.55 (2H, dd, J = 8.8, 5.2 Hz, H-14), 3.32 (6H, s, 2 × OMe-15), 4.28 (1H, t, J = 5.2 Hz, H-15), 6.47 (1H, t, J = 6.8 Hz, H-12), 9.35 (1H, s, H-16); ¹³C-NMR (CDCl₃, 100 MHz): δ = 14.9 (C-20), 18.8 (C-2), 20.2 (C-6), 21.9 (C-19), 22.2 (C-11), 29.0 (C-14), 33.7 (C-18), 33.7 (C-4), 36.0 (C-7), 39.4 (C-1), 40.0 (C-10), 42.1 (C-3), 49.0 (C-17), 53.1 (C-9), 54.1 (OMe), 54.3 (OMe), 55.2 (C-5), 57.8 (C-8), 103.6 (C-15), 138.5 (C-13), 160.6 (C-12), 194.8 (C-16).

Antimicrobial assay

All organisms were obtained from the American Type Culture Collection (Manassas, VA) and included Candida albicans ATCC 90 028, Cryptococcus neoformans ATCC 90 113, Staphylococcus aureus ATCC 29 213, and methicillin-resistant S. aureus ATCC 43 300 (MRS). Susceptibility testing was performed using a modified version of the National Committee on Clinical Laboratory Standards (NCCLS) methods [6], [7], [8]. Samples (dissolved in DMSO) were serially-diluted using 0.9 %/20 % saline/DMSO and transferred in duplicate to 96-well microplates. Microbial inocula were prepared after comparison of the OD₆₃₀ of cell suspensions to the 0.5 McFarland standard and diluting the suspensions in broth Sabouraud Dextrose and cation-adjusted Mueller-Hinton (Difco) for the fungi and bacteria, respectively, to afford recommended inocula. Microbial inocula were added to the samples to achieve a final volume of 200 μL and final sample concentrations starting with 20 - 50 μg/mL. Growth, solvent and media controls were included on each test plate. All organisms were read at 630 nm prior to and after incubation. Percent growth was calculated and plotted versus test concentration to afford the IC₅₀.

Results and Discussion

The dichloromethane-methanol (1 : 1) extract of seeds of A. longifolius exhibited significant activities against Candida albicans, C. neoformans, S. aureus and MRS, with IC₅₀ values of 20, 9, 40 and 25 μg/mL, respectively. Bioassay-guided fractionation of the crude extract and purification of the active fractions led to the isolation of compounds 1 - 4.[*]

Compound 1 was obtained as a colorless oil, molecular formula C₂₁H₃₄O₃, based on HR-ESI-MS, m/z = 357.2389 [M + Na]⁺ (calcd.: 357.2405). The ¹³C-NMR spectrum exhibited 21 carbon signals, including four methyls (among which is one methoxy group), eight methylenes, five methines and four quaternary carbons. The ¹H-NMR and IR spectra indicated the presence of an α,β-unsaturated aldehyde (δ_H = 9.33; ν = 1680 cm^-1) [9]. This was evident from the ¹³C-NMR spectrum wherein one olefinic carbon appeared at δ_C = 160.2 and the aldehyde carbon at δ_C = 195.2. Further examination of the ¹H-NMR spectrum revealed a signal for an olefinic proton at δ_H = 6.54 and an exocyclic methylene group at δ_H = 4.41 and 4.83. The remaining signals of the ¹H- and ¹³C-NMR suggested a labdane skeleton for 1 [5], [9], [10], [11]. The structure of the side-chain C-11 to C-16 was assigned by a combination of HMQC and HMBC experiments. The HMQC spectrum enabled to assign each proton to the corresponding carbon. In the HMBC spectrum (Fig. [1]), pertinent correlations were observed between H-12 at δ_H = 6.74 (1H, t, J = 6.4 Hz) and C-9 (δ_C = 56.7), C-14 (δ_C = 29.2), and C-16 (δ_C = 195.2). In addition, protons at δ_H = 2.60 (2H, dd, J = 6.0, 3.6 Hz, H-14) and δ_H = 3.34 (3H, s, OMe) were correlated with the methine carbon at δ_C = 104.1 (C-15). Accordingly, the structure of the side-chain showed great resemblance with that of labda-8(17),12(E)-dien-15,16-dial (3) [5], [9], [10] also isolated. They differed at position 16 where the aldehyde group is replaced by a hemiacetal group in 1. Thus, compound 1 was determined as 15-hydroxy-15-methoxylabda-8(17),12(E)-dien-16-al. The new compound was named aframolin A. Aframolin A is a demethylated derivative of calcaratarin A, isolated from Alpinia calcarata [11].

Compound 2, colorless oil, was assigned the molecular formula C₂₂H₃₆O₄ based on HR-ESI-MS, m/z = 387.2509 [M + Na]⁺ (calcd.: 387.2505). Its ¹H-NMR and IR spectra suggested the presence of an α,β-unsaturated aldehyde (δ_H = 9.35, ν = 1681 cm^-1) [9]. The ¹H- and ¹³C-NMR spectra showed signals characteristic of an 8(17)-epoxylabdane-type diterpenoid [1], [10]. The 8(17)-epoxide was indicated by the carbon signals at δ_C = 49.0 (C-17) and 57.8 (C-8). The spectral data of 2 were almost similar to those of aframodial (4) [5], [9], [10], also isolated. The ¹H-NMR of 2 compared to that of aframodial (4), displayed only one aldehyde proton at δ_H = 9.35 (s), one methine at δ_H = 4.28 (t, J = 5.2 Hz) and two methoxys at δ_H = 3.32. Based on HMBC correlations, the proton at δ_H = 4.28 was assigned to position 15, since it showed correlation with the two methoxy carbons at δ_C = 54.1 and 54.3, and the methylene at δ_C = 29.0 (C-14). The HMBC correlations are depicted in Fig. [1]. Thus, compound 2 was established as 8β(17)-epoxy-15,15-dimethoxylabd-12(E)-en-16-al. The new compound was named aframolin B.

Two known diterpenes were identified as labda-8(17),12(E)-dien-15,16-dial (3) and aframodial (4) from their spectral data [5], [9], [10].

The antimicrobial activity of the isolates had been evaluated, and compounds 1 - 3 did not show any appreciable activity. However, compound 4 showed significant activity against C. neoformans, S. aureus and MRS with IC₅₀/MIC 6.0/20, 10/20 and 10/20 μg/mL, respectively.

Acknowledgements

We gratefully acknowledge financial support from the ICBG ”Drug Development and Conservation in West and Central Africa” Grant No TW03004 from the Fogarty Centre, NIH. Materials have been reviewed by the Walter Reed Army Institute of Research, Washington DC, USA. We thank Dr. P. V. Srinivas, National Center for Natural Products Research, The University of Mississippi, for assistance in the interpretation of some spectral data.

References

• 1 Tomla C, Kamnaing P, Ayimele G A, Tanifum E A, Tsopmo A, Tane P. et al . Three labdane diterpenoids from Aframomum sceptum (Zingiberaceae).  Phytochemistry. 2002;  60 197-200
  CrossrefPubMedSuche in Google Scholar
• 2 Kimbu S F, Njini T K, Sondengam B L, Akinniyi J A, Connolly J D. The structure of labdane dialdehyde from Aframomum daniellii (Zingiberaceae). J Chem Soc Perkin Trans I 1979: 1303-4
  Suche in Google Scholar
• 3 Tsopmo A, Ayimele G A, Tane P, Ayafor J F, Connolly J D, Sterner O. A norbislabdane and other labdanes from Aframomum sulcatum .  Tetrahedron. 2002;  58 2725-8
  CrossrefPubMedSuche in Google Scholar
• 4 Masahiro T, Yuh-Dan C, Ken-ichi S, Yoshihiro K. Cholesterol biosynthesis inhibitory component from Zingiber officinale Roscoe.  Chem Pharm Bull. 1993;  41 710-3
  PubMedSuche in Google Scholar
• 5 Morita H, Itokawa H. Cytotoxic and antifungal diterpenes from the seeds of Alpinia galanga .  Planta Med. 1988;  54 117-20
  PubMedSuche in Google Scholar
• 6 Pfaller M A, Chaturvedi V, Espinel-Ingroff A, Ghannoum M A, Gosey L L, Odds F C. et al . Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, M27-A2.  National Committee on Clinical Laboratory Standards. 2002;  22 1-51
  PubMedSuche in Google Scholar
• 7 Pfaller M A, Chaturvedi V, Espinel-Ingroff A, Ghannoum M A, Gosey L L, Odds F C. et al . Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Approved standard, M-38A.  National Committee on Clinical Laboratory Standards. 2002;  22 1-30
  PubMedSuche in Google Scholar
• 8 Ferraro M J, Craig W A, Dudley M N, Eliopoulos G M, Hecht D W, Hindler J. et al . Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, M7-A5.  National Committee on Clinical Laboratory Standards. 2000;  20 1-58
  PubMedSuche in Google Scholar
• 9 Morita H, Itokawa H. New diterpenes from Alpinia galanga wild.  Chem Lett. 1986;  7 1205-8
  PubMedSuche in Google Scholar
• 10 Ngo K, Brown G D. Stilbenes, monoterpenes, diarylheptanoids, labdanes and chalcones from Alpinia katsumadai .  Phytochemistry. 1998;  47 1117-23
  CrossrefPubMedSuche in Google Scholar
• 11 Kong L, Qin M, Niwa M. Diterpenoids from the rhizomes of Alpinia calcarata .  J Nat Prod. 2000;  63 939-42
  CrossrefPubMedSuche in Google Scholar

Dr. Ikhlas A. Khan

National Center for Natural Products Research

The University of Mississippi

University

MS 38677-1848

USA

Fax: +1-662-915-7989

eMail: ikhan@olemiss.edu

References

• 1 Tomla C, Kamnaing P, Ayimele G A, Tanifum E A, Tsopmo A, Tane P. et al . Three labdane diterpenoids from Aframomum sceptum (Zingiberaceae).  Phytochemistry. 2002;  60 197-200
  CrossrefPubMedSuche in Google Scholar
• 2 Kimbu S F, Njini T K, Sondengam B L, Akinniyi J A, Connolly J D. The structure of labdane dialdehyde from Aframomum daniellii (Zingiberaceae). J Chem Soc Perkin Trans I 1979: 1303-4
  Suche in Google Scholar
• 3 Tsopmo A, Ayimele G A, Tane P, Ayafor J F, Connolly J D, Sterner O. A norbislabdane and other labdanes from Aframomum sulcatum .  Tetrahedron. 2002;  58 2725-8
  CrossrefPubMedSuche in Google Scholar
• 4 Masahiro T, Yuh-Dan C, Ken-ichi S, Yoshihiro K. Cholesterol biosynthesis inhibitory component from Zingiber officinale Roscoe.  Chem Pharm Bull. 1993;  41 710-3
  PubMedSuche in Google Scholar
• 5 Morita H, Itokawa H. Cytotoxic and antifungal diterpenes from the seeds of Alpinia galanga .  Planta Med. 1988;  54 117-20
  PubMedSuche in Google Scholar
• 6 Pfaller M A, Chaturvedi V, Espinel-Ingroff A, Ghannoum M A, Gosey L L, Odds F C. et al . Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, M27-A2.  National Committee on Clinical Laboratory Standards. 2002;  22 1-51
  PubMedSuche in Google Scholar
• 7 Pfaller M A, Chaturvedi V, Espinel-Ingroff A, Ghannoum M A, Gosey L L, Odds F C. et al . Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Approved standard, M-38A.  National Committee on Clinical Laboratory Standards. 2002;  22 1-30
  PubMedSuche in Google Scholar
• 8 Ferraro M J, Craig W A, Dudley M N, Eliopoulos G M, Hecht D W, Hindler J. et al . Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, M7-A5.  National Committee on Clinical Laboratory Standards. 2000;  20 1-58
  PubMedSuche in Google Scholar
• 9 Morita H, Itokawa H. New diterpenes from Alpinia galanga wild.  Chem Lett. 1986;  7 1205-8
  PubMedSuche in Google Scholar
• 10 Ngo K, Brown G D. Stilbenes, monoterpenes, diarylheptanoids, labdanes and chalcones from Alpinia katsumadai .  Phytochemistry. 1998;  47 1117-23
  CrossrefPubMedSuche in Google Scholar
• 11 Kong L, Qin M, Niwa M. Diterpenoids from the rhizomes of Alpinia calcarata .  J Nat Prod. 2000;  63 939-42
  CrossrefPubMedSuche in Google Scholar

Dr. Ikhlas A. Khan

National Center for Natural Products Research

The University of Mississippi

University

MS 38677-1848

USA

Fax: +1-662-915-7989

eMail: ikhan@olemiss.edu