New Aromatic Compounds from the Marine Mangrove Bruguiera gymnorrhiza

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

Abstract

The phytochemical investigation of the stem of Bruguiera gymnorrhiza yielded five new aromatic compounds (1 - 5), of which the bruguierols A - C (1 - 3) represent a new structural skeleton in natural product chemistry. All structures have been determined by NMR spectroscopic studies. Among them, 3 showed moderate activity against Gram-positive and Gram-negative bacteria including mycobacteria and resistant strains (MICs 12.5 μg/mL).

Introduction

Among marine plants, mangroves represent a unique ecological system which can be found on tropical and sub-tropical coast lines. Because of tidal changes, mangroves have to deal with regular and drastic changes of conditions (salt exposure, nutrient supply). Due to the necessary physiological adaptations, they are considered to also harbor a variable secondary metabolism, thus being a rich source for natural products [1], [2]. As part of our investigations on mangroves for the search of new natural products, we have systematically investigated the chemical constituents of the stem of Bruguiera gymnorrhiza. Material of this large evergreen tree that belongs to the Rhizophoraceae family, was collected from the coast of Xiamen in the south of China. Recent studies on B. gymnorrhiza from India [3], [4], [5], [6] showed the presence of triterpenes, flavonoids and diterpenes in its leaves and the outer layer of the root bark. In this paper, we describe the isolation and structural elucidation of five new aromatic compounds, bruguierol A (1), bruguierol B (2), bruguierol C (3), 1-(3-hydroxyphenyl)-hexane-2,5-diol (4) and 3-(3-hydroxybutyl)-1,1-dimethylisochroman-6,8-diol (5). Results of biological testing are presented and the biosynthetic origins of the compounds are discussed.[]

Materials and Methods

General experimental procedures

Column chromatography, silica gel 60M (Macherey-Nagel, 230 - 400 mesh), Sephadex LH-20 (Pharmacia); TLC, silica gel plates (Macherey-Nagel, Sil G/UV₂₅₄, 0.20 mm), spots were detected under a UV lamp and after staining with anisaldehyde/H₂SO₄; Optical rotation: Propol Digital Automatic Polarimeter; IR spectra: Bruker IFS55 Spectrometer; UV spectra: Varian UV-Visible Cary spectrophotometer, 2 drops of 2 M HCl or 2 M NaOH, respectively, were added to 2 mL of compound solution; ¹H- and ¹³C-NMR spectra: Bruker DPX-300, chemical shift values in ppm and coupling constants in Hz, 2D-NMR: Bruker DPX-500; ESI-MS: Thermo Electron LCQ; HR-ESI-MS: Thermo Electron MAT95XL, EI-MS, 70eV, direct inlet, high resolution with perfluorokerosine as a standard: Thermo Electron MAT95XL.

Plant material

Samples of the stem of B. gymnorrhiza were collected in Xiamen, China, in June 2002 and authenticated by Prof. Peng Lin, Xia Men University, China. A voucher sample of the plant is deposited at the National Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China (Mangrove XM004). The stem was air-dried and milled.

Extraction and isolation

The pulverized plant material (6.1 kg) was macerated with methanol (25 L) at room temperature three times for 2 weeks. The combined methanolic extracts were concentrated and yielded 282.6 g of a crude extract. A suspension of this crude extract in distilled water (1.5 L) was extracted with EtOAc (4 times, 4.5 L ) and n-BuOH (4 times, 4.5 L), respectively, to yield 23.6 g of a dried EtOAc extract, 39.6 g of a n-BuOH extract and an aqueous residue. The EtOAc extract was subjected to silica gel CC (6 × 50 cm) and eluted with CHCl₃/MeOH (50 : 1 2.5 L; 20 : 1 2 L; 9 : 1 1.5 L; 4 : 1 1.5 L: 1 : 1 1 L). The eluents were combined to 25 fractions (1 - 25) on the basis of TLC analysis. Fr. 15 (488 mg, 4.820 - 5.120 L) was subjected to Sephadex LH-20 CC (3.5 × 120 cm, CHCl₃ 600 mL) and yielded 8 fractions (A₁ - A₈) on the basis of TLC analysis. Fr. A₃ (38 mg, 248 - 296 mL) was further purified by silica gel CC (1.5 × 30 cm, cyclohexane/EtOAc, 3 : 1, 250 mL) to give 1 (4.5 mg, 102 - 120 mL). Fr. 18 (560 mg, 5.840 - 6.160 L) was separated by repeated silica gel chromatography (2.5 × 50 cm, cyclohexane/EtOAc, 2 : 1, 500 mL) to get 2 (4.0 mg, 96 - 114 mL). Fr. 19 (278 mg, 6.160 - 6.380 L) was subjected to Sephadex LH-20 CC (3.5 × 120 cm, MeOH, 560 mL). The eluents were combined to 4 fractions (B₁ - B₄) on the basis of TLC analysis. Fr. B₄ (85 mg, 318 - 378 mL) was re-subjected to silica gel CC (2.0 × 30 cm, cyclohexane/EtOAc, 2 : 1, 300 mL) to yield 3 (25.0 mg, 120 - 168 mL). Fr. 21 (336 mg, 6.780 - 7.120 L) was subjected to Sephadex LH-20 CC (3.5 × 120 cm, MeOH, 580 mL) and yield 7 fractions (C₁ - C₇). Fr. C₂ (56 mg, 144 - 192 mL) was further purified by silica gel CC (1.5 × 30 cm, cyclohexane/EtOAc, 2 : 3, 220 mL) to yield 4 (12.0 mg, 117 - 135 mL) and 5 (7.5 mg, 78 - 102 mL).

Bruguierol A (1): White solid; [α]_D ²⁰: + 14.4° (c 0.30, MeOH); ESI-MS: m/z = 191 [M + H]+, 213 [M + Na]+,189 [M - H]^-; HR-ESI-MS: m/z = 213.0902 (calcd. for C₁₂H₁₄NaO₂ : 213.0891); UV (MeOH): λ_max (log ε) = 278 (3.29) nm; (MeOH/HCl): 278 (3.07) nm; (MeOH/NaOH): 298 (3.46) nm; IR (film): ν_max = 3275(br), 2928, 2360, 2341, 1609, 1499, 1457, 1295, 1234, 1090, 994, 951, 816 cm^-1; Rf (TLC silica gel) = 0.67 (cyclohexane/EtOAc, 1 : 1), 0.49 (CHCl₃/MeOH 20 : 1); pink color after staining; NMR data see Table [1].

Bruguierol B (2): White solid; [α]_D ²⁰: + 8.9° (c 0.27, MeOH); ESI-MS: m/z = 229 [M + Na]+, 435 [2M + Na]+, 641 [3M + Na]+, 205 [M - H]^-, 411 [2M - H]^-; HR-ESI-MS: m/z = 229.0829 (calcd. for C₁₂H₁₄NaO₃ : 229.0841); UV(MeOH): λ_max (log ε) = 289 (3.86) nm; (MeOH/HCl): 289 (3.81) nm; (MeOH/NaOH): 302 (3.35) nm; IR (film): ν_max = 3257(br), 2973, 2360, 2341, 1605, 1517, 1446, 1293, 1227, 1098, 992, 865 cm^-1; Rf (TLC silica gel) = 0.48 (cyclohexane/EtOAc, 1 : 1), 0.30 (CHCl₃/MeOH, 20 : 1); purple color after staining; NMR data see Table [1].

Bruguierol C (3): White solid; [α]_D ²⁰: + 4.0° (c 0.50, MeOH); ESI-MS: m/z = 435 [2M + Na]+, 641 [3M + Na]+, 207 [M + H]+, 224 [M + NH4]+, 430 [2M + NH4]+, 205 [M - H]^-; HR-ESI-MS: m/z = 229.0848 (calcd. for C₁₂H₁₄NaO₃ : 229.0841); UV(MeOH): λ_max (log ε) = 285 (3.09) nm; (MeOH/HCl): 284 (2.95) nm; (MeOH/NaOH): 293 (3.29) nm; IR (film): ν_max = 3220(br), 2360, 2341, 1615, 1507, 1457, 1296, 1209, 1126, 1031, 997, 836, 790 cm^-1; Rf (TLC silica gel) = 0.50 (cyclohexane/EtOAc, 1 : 1), 0.25 (CHCl₃/MeOH, 20 : 1); orange color after staining; NMR data see Table [1].

1-(3-Hydroxyphenyl)-hexane-2,5-diol (4): White solid; [α]_D ²⁰: -5.7° (c 0.40, MeOH); ESI-MS: m/z = 233 [M + Na]+, 443 [2M + Na]+, 211 [M + H]+, 209 [M - H]^-; HR-ESI-MS: m/z = 233.1170 (calcd. for C₁₂H₁₈NaO₃ : 233.1154); UV(MeOH): λ_max (log ε) = 275 (3.17); (MeOH/HCl): 275 (3.06); (MeOH/NaOH): 290 (3.34) nm; IR (film): ν_max = 3329 (br), 2360, 2341, 1588, 1488, 1456, 1367, 1233, 1158, 1037 cm^-1; Rf (TLC silica gel) = 0.10 (cyclohexane/EtOAc, 1 : 1), 0.12 (CHCl₃/MeOH, 20 : 1); yellow color after staining with anisaldehyde/H₂SO₄; ¹H-NMR (300 MHz, CD₃OD): δ = 7.05 (1H, t, J = 7.9 Hz, H-5′), 6.67 (1H, brd, J = 7.9 Hz, H-6′), 6.65 (1H, brs, H-2′), 6.60 (1H, brd, J = 7.9 Hz, H-4′), 3.74 (1H, m, H-2), 3.67 (1H, m, H-5), 2.65 (2H, m, H-1), 1.63 (1H, m, H-3a), 1.61 (1H, m, H-4a), 1.46 (1H, m, H-4b), 1.37 (1H, m, H-3b), 1.13 (3H, d, J = 6.2 Hz, H-6); ¹³C-NMR (75 MHz, CD₃OD): δ = 158.3 (s, C-3′), 141.9 (s, C-1′), 130.1 (d, C-5′), 121.7 (d, C-6′), 117.3 (d, C-2′), 114.0 (d, C-4′), 73.8 (d, C-2), 68.7 (d, C-5), 45.1 (t, C-1), 36.4 (t, C-4), 33.9 (t, C-3), 23.4 (q, C-6).

3-(3-Hydroxybutyl)-1,1-dimethylisochroman-6,8-diol (5): White solid; [α]_D ²⁰: -10.0° (c 0.05, MeOH); ESI-MS: m/z = 289 [M + Na]+, 555 [2M + Na]+, 267 [M + H]+, 533 [2M + H]+; HR-EI-MS: m/z = 266.1526 (calcd. for C₁₅H₂₂O₄ : 266.1518); UV (MeOH): λ_max (log ε) = 282 (3.93); (MeOH/HCl): 279 (4.04); (MeOH/NaOH): 293 (3.83) nm; IR (film): ν_max = 3275 (br), 2926, 2360, 2341, 1595, 1450, 1378, 1340, 1274, 1158, 1042, 835 cm^-1; Rf (TLC silica gel) = 0.33 (cyclohexane/EtOAc, 1 : 1), 0.17 (CHCl₃/MeOH, 20 : 1); pink color after staining; ¹H-NMR (300 MHz, CD₃OD): δ = 6.10 (1H, d, J = 2.4 Hz, H-7), 6.01 (1H, d, J = 2.4 Hz, H-5), 3.76 (1H, m, H-3′), 3.71 (1H, m, H-3), 2.48 (2H, m, H-4), 1.63 (2H, m, H-2′), 1.61 (2H, m, H-1′), 1.56 (3H, s, CH3 - 1a), 1.54 (3H, s, CH3 - 1b), 1.17 (3H, d, J = 6.2 Hz, H-4′); ¹³C-NMR (75 MHz, CD₃OD): δ = 156.9 (s, C-6), 155.7 (s, C-8), 137.3 (s, C-4a), 122.0 (s, C-8a), 107.4 (d, C-5), 102.7 (d, C-7), 76.6 (s, C-1), 69.8 (d, C-3), 68.6 (d, C-3′), 37.3 (t, C-4), 36.2 (t, C-2′), 33.2 (t, C-1′), 29.0 (q, 1-CH₃a), 25.5 (q, 1-CH₃b), 23.5 (q, C-4′).

Antimicrobial assays

Compounds 1 - 5 were tested for their antimicrobial activity against a series of microorganisms (sensitive and resistant Gram-positive and Gram-negative bacteria, mycobacteria and yeast) by a microbroth dilution method according to the NCCLS guidelines [7]. The bacteria were grown overnight at 37 °C in Mueller-Hinton broth (MHB, Difco). The yeast cells were grown in YNPG medium (Difco) with 1 % glucose at 30 °C. 50 μL of a compound solution of 400 μg/mL were serially diluted by factor two with MHB. The wells were then inoculated with 50 μL of bacteria to give a final concentration of 5 × 10⁶ CFU/mL. After the microtiter plates were incubated at 37 °C (bacterial strains) or at 30 °C (Candida albicans) for 24 h, the MIC values were read with a Nepheloscan Ascent 1.4 automatic plate reader (Labsystems) as the lowest dilution of antibiotic allowing no visible growth. Test organisms were taken from the stock of the Hans-Knöll Institute.

Results

To search for new natural products, we investigated the chemical constituents of the stem of Bruguiera gymnorrhiza. The crude methanol extract was partitioned to yield an EtOAc extract which was separated by gradient chromatography on a silica gel column followed by additional silica gel chromatography and gel permeation chromatography on Sephadex LH-20 to yield the pure compounds.

Compound 1 was obtained as a white solid. ESI-MS indicated the quasi-molecular weight at m/z = 191.2 [M + H]+, 213.2 [M + Na]+ and 189.3 [M - H]^-. The molecular formula of 1 was assigned as C₁₂H₁₄O₂ by HR-ESI-MS at m/z = 213.0902 (calcd. for C₁₂H₁₄NaO₂: 213.0891). In the UV spectra, a bathochromic shift was observed under basic conditions which is a typical property of phenolic hydroxy groups. The IR spectrum also indicated the presence of hydroxy groups (3275 cm^-1) and a benzene ring (1609, 1499, 1457 cm^-1). These structural elements were also confirmed through ¹H-NMR data (Table [1]) by three signals at δ = 6.52 (1H, d, J = 2.4 Hz), 6.58 (1H, dd, J = 8.4 Hz, 2.4) and 6.99 (1H, d, J = 8.4 Hz) forming an AMX system. The ¹³C-NMR spectrum (Table [1]) of 1 showed 12 signals, of which six carbons form a trisubstituted benzene ring and the others represent an oxygenated quaternary carbon, an oxymethine carbon, three methylene groups and one methyl group. Accounting of the functional groups and the unsaturation degree of 1 indicated the presence of a bicyclic skeleton together with a benzene ring. COSY data showed one additional proton spin system of a CH₂CH₂CH(OR)CH₂ fragment (H-6 to H-9) (Fig. [1]). Long-range proton-carbon couplings, deduced from an HMBC experiment (Fig. [1]), indicated that two quaternary carbons of the benzene ring (δ = 136.2, 133.5) have to be connected with both ends of the CH₂CH₂CH(OR)CH₂ fragment with the oxygenated quaternary carbon (δ = 80.4) inserted next to the ethylene moiety. At the same time, the correlation between C-5 and H-8 in HMBC indicated a tetrahydrofuran in this position. The positions of a hydroxy and a methyl group were also confirmed by HMBC. A NOESY experiment allowed the assignment of the relative stereochemistry of 1. NOEs were observed between H-3, H-6β, H-10 and H-4, between H-6β, H-9β and H-7β, between H-6α, H-8 and H-7α and between H-4, H-6α, H-8 and H-10 (Fig. [2]). From the NOE results and the coupling constants, the relative stereochemistry of 1 was determined as 5R*,8S*. Thus, 1 was assigned 2-hydroxy-5-methyl-5,8-epoxy-6,7,8,9-tetrahydro-5H-benzo-[a]cycloheptene and because it represents a new structural skeleton was named bruguierol A.

The molecular formula of 2 was determined as C₁₂H₁₄O₃ by HR-ESI-MS at m/z = 229.0829 (calcd. for C₁₂H₁₄NaO₃: 229.0841). The IR and UV spectra as well as the ¹H- and ¹³C-NMR (Table [1]) spectra of 2 indicated a close similarity of the skeleton to that of 1. Two sharp singlets at 6.39 (1H, s) and 6.52 (1H, s) in the ¹H-NMR spectra which were related to two aromatic hydroxy groups in an ortho arrangement indicated a difference to 1. This was also confirmed by the absence of an aromatic spin system in the COSY spectra (Fig. [1]). As in 1, the relative stereochemistry of 2 was determined by NOE correlations as 5R*,8S*. Therefore, 2 was assigned 2,3-dihydroxy-5-methyl-5,8-epoxy-6,7,8,9 -tetrahydro-5H-benzo-[a]cycloheptene and named bruguierol B.

According to HR-ESI-MS at m/z = 229.0848 (calcd. for C₁₂H₁₄NaO₃: 229.0841), the molecular formula of 3 was determined as C₁₂H₁₄O₃. The IR and UV spectra as well as the NMR data (Table [1]) of 3 were indicative of the same skeleton as 1 and 2. The major difference was concerning two additional signals at δ = 6.01 (1H, brs) and 6.07 (1H, brs) in the ¹H-NMR of 3 which were indicating a meta-dihydroxylation of the aromatic system. The positions of the hydroxy groups and all other protons were confirmed by an HMBC experiment (Fig. [1]). As in 1, the relative stereochemistry of 3 was determined by NOE correlations as 5R*,8S*. Thus, the structure of 3, named brugierol C, was determined to be 2,4-dihydroxy-5-methyl-5,8-epoxy-6,7,8,9-tetrahydro-5H-benzo[a]cycloheptene.

Compound 4 was obtained as white amorphous powder. The molecular formula was established as C₁₂H₁₈O₃ by HR-ESI-MS at m/z = 233.1154 (calcd. for C₁₂H₁₈NaO₃: 233.1170). A bathochromic UV shift under basic conditions indicated the presence of a phenolic hydroxy group which was confirmed by IR absorption peaks at 3329 cm^-1 (hydroxy groups) and 1588, 1488, 1456 cm^-1 (benzene ring). Correspondingly, the ¹H-NMR data showed four aromatic methine protons at δ = 6.60 (1H, brd, J = 7.9 Hz), 6.67 (1H, brd, J = 7.9 Hz), 7.05 (1H, t, J = 7.9 Hz) and 6.65 (1H, brs). The ¹³C-NMR and DEPT spectra of 4 revealed six carbons of a benzene ring, a methyl, two oxymethine, and three methylene groups. A COSY experiment indicated two spin systems (Fig. [1]): one aromatic AMX system and a CH₂CH(OR)CH₂CH₂CH(OR)CH₃ fragment. HMBC data (Fig. [1]) allowed the determination of the combined arrangement of these fragments. Therefore, the structure of 4 is 1-(3-hydroxyphenyl)-hexane-2,5-diol.

Compound 5 was obtained as a white amorphous powder and showed a molecular formula of C₁₅H₂₂O₄ as determined by HR-EI-MS of the molecular ion at m/z = 266.1526 (calcd. 266.1518). The UV and IR spectra indicated a benzene ring with hydroxy substituents. The ¹H-NMR data of 5 showed two signals at δ = 6.01 (1H, d, J = 2.4 Hz) and 6.10 (1H, d, J = 2.4 Hz) which were identical to the aromatic protons of 3. Next to two additional methyl groups, other ¹H-NMR data of 5 were similar to those of 4 and according to COSY data, part of the same CH₂CH(OR)CH₂CH₂CH(OR)CH₃ spin system. The ¹³C-NMR spectra showed 15 signals and except for the additional two methyl groups and one oxygated quaternary carbon, are identical with those of 4. HMBC (Fig. [1]) experiments indicated that the two methyl groups were connected to the oxygenated quaternary carbon (δ = 76.6) which forms an oxetane with C-3 in the spin system. Therefore, the structure of 5 is 3-(3-hydroxybutyl)-1,1-dimethylisochroman-6,8-diol.

Within our biological profiling program of new natural products, the antimicrobial activity of 1 - 5 was tested. Product 3 was found to have a moderate effect against Gram-positive and Gram-negative bacteria including one mycobacterial strain and one resistant Enterococcus, whereas 1 and 2 showed no significant activity (Table [2]) and 4 and 5 have no activity against all these microorganisms. These results indicate the importance of the spatial arrangement of the aromatic hydroxy groups of the brugierol skeleton for antibacterial activity.

Discussion

The bruguierols A - C (1 - 3) represent a structural skeleton that is otherwise not found in the literature. Only the icetexane diterpenoids, e. g., 5,6-dihydro-6α-hydroxysalviasperanol 6, show correspondence in some of the structural features [8]. However, we suggest that 1 - 3 are not derived from the terpene biosynthetic pathway. Due to the oxidation level they seem also unrelated to catechin-type metabolites like theaflavin and purpurogallin which are branching of the shikimate-pathway [9]. Compounds 1 - 5 all possess a similar benzene ring combined with a six-membered carbon chain which indicates they have the same biosynthetic origin, possibly derived from the polyketide pathway.

Acknowledgements

We are grateful to Dr. Gollmick, Dr. Berg and colleagues for recording spectral data. This work is part of a collaboration between the Hans-Knöll Institute for Natural Products Research (HKI) and the National Research Laboratory of Natural and Biomimetic Drugs, Peking University (Beijing) and was funded by the German Federal Ministry for Education and Research and Technology (BMBF 0312849A, CHN02/323 and CHN 02/322).

References

• 1 Miles D H, Kokpol U, Chittawong V, Santi T P, Tunsuwan K, Chi N. Mangrove forests - the importance of conservation as a bioresource for ecosystem diversity and utilization as a source of chemical constituents with potential medicinal and agricultural value.  Pure Appl Chem. 1998;  70 2113-22
  PubMedSearch in Google Scholar
• 2 Minjuan X, Zhiwei D, Min L, Jun L, Hongzheng F, Peter P, Wenhan L. Chemical constituents from the mangrove plant, Aegiceras corniculatum .  J Nat Prod. 2004;  67 762-6
  CrossrefPubMedSearch in Google Scholar
• 3 Subrahmanyan C, Rao B V, Ward R S, Hursthous M B, Hibbs D E. Diterpenes from the marine mangrove Bruguiera gymnorrhiza .  Phytochemistry. 1999;  51 83-90
  CrossrefPubMedSearch in Google Scholar
• 4 Misra S, Choudhury A, Dutta A K, Ghosh A. Sterols and fatty acids from three species of mangrove.  Phytochemistry. 1984;  23 2823-37
  CrossrefPubMedSearch in Google Scholar
• 5 Misra S, Datta A K, Chattopadhyay S, Choudhury A, Ghosh A. Hydrocarbons and wax esters from seven species of mangrove leaves.  Phytochemistry. 1987;  26 3265-8
  CrossrefPubMedSearch in Google Scholar
• 6 Achmadi S, Syahbirin G, Choong E T, Hemingway R W. Catechin 3-O-rhamnoside chain extender units in polymeric procyanidins from mangrove bark.  Phytochemistry. 1994;  35 217-9
  PubMedSearch in Google Scholar
• 7 National Committee for Clinical Laboratory S tandards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow Aerobically, Approved Standard. NCCLS Document M7-A4. 4th edn. National Committee for Clinical Laboratory Standards Villanova, PA; 1997
  Search in Google Scholar
• 8 Baldomero E, Maragarita F, Simon H O, Toscano R A, Ramamoorthy T P. Abietane and icetexane diterpeniods from the roots of Salvia aspera .  Phytochemistry. 1995;  39 139-43
  CrossrefPubMedSearch in Google Scholar
• 9 Dewick P M. The shikimate pathway: aromatic amino acids and phenylpropanoids. Medicinal Natural Products: a biosynthetic approach. 2nd edn 2002: 121-66
  Search in Google Scholar

Dr. Isabel Sattler

Hans-Knöll-Institute for Natural Products Research

Beutenbergstr. 11a

07745 Jena

Germany

Phone: +49-3641-656920.

Fax: +49-3641-656679.

Email: isabel.sattler@hki-jena.de

References

• 1 Miles D H, Kokpol U, Chittawong V, Santi T P, Tunsuwan K, Chi N. Mangrove forests - the importance of conservation as a bioresource for ecosystem diversity and utilization as a source of chemical constituents with potential medicinal and agricultural value.  Pure Appl Chem. 1998;  70 2113-22
  PubMedSearch in Google Scholar
• 2 Minjuan X, Zhiwei D, Min L, Jun L, Hongzheng F, Peter P, Wenhan L. Chemical constituents from the mangrove plant, Aegiceras corniculatum .  J Nat Prod. 2004;  67 762-6
  CrossrefPubMedSearch in Google Scholar
• 3 Subrahmanyan C, Rao B V, Ward R S, Hursthous M B, Hibbs D E. Diterpenes from the marine mangrove Bruguiera gymnorrhiza .  Phytochemistry. 1999;  51 83-90
  CrossrefPubMedSearch in Google Scholar
• 4 Misra S, Choudhury A, Dutta A K, Ghosh A. Sterols and fatty acids from three species of mangrove.  Phytochemistry. 1984;  23 2823-37
  CrossrefPubMedSearch in Google Scholar
• 5 Misra S, Datta A K, Chattopadhyay S, Choudhury A, Ghosh A. Hydrocarbons and wax esters from seven species of mangrove leaves.  Phytochemistry. 1987;  26 3265-8
  CrossrefPubMedSearch in Google Scholar
• 6 Achmadi S, Syahbirin G, Choong E T, Hemingway R W. Catechin 3-O-rhamnoside chain extender units in polymeric procyanidins from mangrove bark.  Phytochemistry. 1994;  35 217-9
  PubMedSearch in Google Scholar
• 7 National Committee for Clinical Laboratory S tandards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow Aerobically, Approved Standard. NCCLS Document M7-A4. 4th edn. National Committee for Clinical Laboratory Standards Villanova, PA; 1997
  Search in Google Scholar
• 8 Baldomero E, Maragarita F, Simon H O, Toscano R A, Ramamoorthy T P. Abietane and icetexane diterpeniods from the roots of Salvia aspera .  Phytochemistry. 1995;  39 139-43
  CrossrefPubMedSearch in Google Scholar
• 9 Dewick P M. The shikimate pathway: aromatic amino acids and phenylpropanoids. Medicinal Natural Products: a biosynthetic approach. 2nd edn 2002: 121-66
  Search in Google Scholar

Dr. Isabel Sattler

Hans-Knöll-Institute for Natural Products Research

Beutenbergstr. 11a

07745 Jena

Germany

Phone: +49-3641-656920.

Fax: +49-3641-656679.

Email: isabel.sattler@hki-jena.de