Phlorotannins as Radical Scavengers from the Extract of Sargassum ringgoldianum

To screen algal phlorotannins with antioxidative activities, 50% ethanol extracts of 25 Japanese marine algae were evaluated. Scavenging activity against superoxide anion radicals was frequently found with a high content of total phenolic compounds. Among these, the extract from the brown seaweed, Sargassum ringgoldianum, showed the strongest scavenging activity. The active fraction contained a mixture of high molecular weight polyphenols, phlorotannins that were found to be polymerized bifuhalol, as analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The scavenging activity of the fraction against superoxide anion radicals was estimated to be 1.0 μg/ml (IC₅₀), which were approximately five times stronger than that of catechin.     

Local and chemical distribution of phlorotannins in brown algae

The local and chemical distribution of phlorotannins among the Japanese Laminariaceae, Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, was investigated. As a result of light microscopy observations with vanillin-HCl staining, phlorotannins were found to be accumulated within the vegetative cells of the outer cortical layer of the thalli, regardless of the species, stage of growth or organ. Crude phlorotannins comprised about 3.0% of the algal powder for each of the algae. High-performance liquid chromatography (HPLC) showed that the phlorotannins of E. bicyclis were composed of phloroglucinol (0.9%), phloroglucinol tetramer (4.4%), eckol (7.5%), phlorofucofuroeckol A (21.9%), dieckol (23.4%), and 8,8'-bieckol (24.6%), plus some other unknown phenolic compounds (17.3%). The composition of the phlorotannins differed little among the Laminariaceae, except for a significantly larger amount of the tetramer, MW 478, in E. bicyclis.     

Influence of extract purification on the recovery of citrinin in the HPLC analysis

Various analytical methods of citrinin determination are known from publications which cannot be applied without any difficulties. Low fluctuating recovery rates of standard additions, an insufficient extract purification for rye, and a rather low sensitivity based on improper HPLC conditions are being observed if the published methods are applied. An easily applicable, reliable and sensitive HPLC method in the lower ppb range on the basis of a solid phase extraction combined with a HPLC gradient which allows for a sensitive fluorescence detection, is under development. The results already achieved are described in the following.     

Tea prepared from Anastatica hirerochuntica seeds contains a diversity of antioxidant flavonoids, chlorogenic acids and phenolic compounds

HPLC-PDA-MS(2) was used to identify phenolic and polyphenolic compounds in an herbal tea made from seeds of Anastatica hirerochuntica, a plant found in the Sahara-Arabian deserts and used to treat a variety of ailments. Twenty compounds comprising a series of flavone C- and O-linked glycosides, phenolic acids, chlorogenic acids and flavonols were identified or partially identified on the basis of co-chromatography with reference compounds and MS(2) and MS(3) fragmentation patterns. The flavones were the principal components, occurring as luteolin, apigenin and diosmetin conjugates. The antioxidant potential of individual compounds in Anastatica was assessed using HPLC with an on-line ABTS·(+) detection system. This revealed that 14 compounds exhibited antioxidant activity. The highest contribution to the antioxidant capacity of the tea, 56%, came from 3,4-dihydroxybenzoic acid and caffeoyl- and dicaffeoylquinic acids while 6-C-glucosides of luteolin and apigenin contributed 41%.     

Characterisation of polyphenols by HPLC-PAD-ESI/MS and antioxidant activity in Equisetum telmateia

The antioxidant activity of an aqueous extract (infusion) and respective ethyl acetate fraction of Equisetum telmateia Ehrh. (Equisetaceae), a plant used in traditional medicine for its anti-inflammatory and diuretic properties, has been evaluated by DPPH, TEAC and TBARS assays. A high and significant antioxidant activity was detected in the ethyl acetate fraction. Analysis of the aqueous extract and the ethyl acetate fraction by HPLC-PAD-ESI/MS allowed the identification of the major phenolic compounds as flavan-3-ol, kaempferol and phenolic acid derivatives. Among the flavan-3-ols, A-type proanthocyanidins and afzelechin derivatives were detected as well as the more common B-type procyanidins, B2 and C1, whose identification was further confirmed by HPLC using detection involving chemical reaction with p-dimethylamino-cinnamaldehyde. The results suggest that the anti-inflammatory activity of E. telmateia could be due, at least in part, to the presence of compounds with antioxidant activity.     

High‐performance liquid chromatography with diode array detection coupled to electrospray time‐of‐flight and ion‐trap tandem mass spectrometry to identify phenolic compounds from a Cistus ladanifer aqueous extract

Introduction – Cistus ladanifer is an aromatic shrub that is widespread in the Mediterranean region. The labdanum exudate is used in the fragrance industry and has been characterised. However, there is not enough information about the phenolic content of the raw plant, the aerial part of it being a very rich source of bioactive compounds. Objective – Characterisation of the bioactive compounds of the raw plant and its aerial parts. Methodology – High‐performance liquid chromatography with diode array and electrospray ionisation mass spectrometric detection was used to carry out the comprehensive characterisation of a Cistus ladanifer shrub aqueous extract. Two different MS techniques were coupled to HPLC: time‐of‐flight mass spectrometry and tandem mass spectrometry. Results – Many well‐known compounds present in Cistus ladanifer were characterised, such as flavonoids, phenolic acids, ellagitanins, hexahydroxydiphenoyl and derivatives, and other compounds. Conclusion – The method described simultaneously separated a wide range of phenolic compounds and the proposed characterisation of the major compounds of this extract was carried out. It is important to highlight that, to our knowledge, this is the first time that a Cistus ladanifer aqueous extract from the raw plant has been characterised. Copyright © 2009 John Wiley & Sons, Ltd.     

Antimicrobial activity and phytochemical screening of Ocimum americanum L extracts against pathogenic microorganisms

The aim of the present study is to assess the antimicrobial activities of various leaf extracts of Ocimum americanum were tested against pathogenic microorganisms. Preparation of different extracts viz., aqueous, acetone, ethyl acetate and methanol through soxhlet extraction method. Various extracts were investigated against MTCC strains of Bacillus cereus, Clostridium penfrigens, Klebsilla pnemoniae, Salmonella paratyphi, Candida albicans and Aspergillus niger by agar well diffusion and disc diffusion methods. Minimum inhibitory concentration (MIC), Minimum Bactericidal/Fungicindal Concentration (MBC/MFC) were determined through micro dilution method. Elucidation of phytochemicals and functional groups were observed by HPLC and FT-IR respectively. Ethyl acetate leaf extract of O.americanum showed significant antimicrobial activity against the all tested pathogens in agar well diffusion method in which B.cereus (17 mm) was observed high zone of inhibition. Whereas lowest inhibition was observed in aqueous extract against C.pentrigens (7 mm). The ranges of MIC values from 0.78 μg/ml to 50 μg/ml and MBC/MFC 1.56 μg/ml to 50 μg/ml were observed. Phytochemicals such as alkaloids, steroids, saponins, flavonoids, tannins, terepenes, phenolic compounds cardiac glycosides were detected. Saponinns, flavonoids, tannins, phenolic compounds were observed in only ethyl acetate leaf extracts. Functional group of the leaf extracts was exhibited by FTIR and HPLC analysis of the ethyl acetate leaf extract was elutated at six peaks. Based on the results we concluded that ethyl acetate leaf extract of O.americanum has proved to be potentially effective than the other extracts. Therefore, ethyl acetate leaf extract of O.americanum could act as antimicrobial agent and further studies are recommended for isolation of compounds and toxicological studies.     

Red Mexican grapefruit: a novel source for bioactive limonoids and their antioxidant activity

Citrus limonoids have shown to inhibit the growth of cancer in colon, lung, mouth, stomach and breast in animal and cell culture studies. For the first time in the present study, an attempt has been made to isolate antioxidant fractions and five limonoids from red Mexican grapefruit seeds. Defatted seed powder was successively extracted with hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH) and MeOH/water and the extracts were concentrated under vacuum. Radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total phenolic content were also measured for comparison with the antioxidant capacity in the phosphomolybdenum method for the above extracts. Acetone and MeOH extracts, respectively, showed the highest (85.7%) and lowest (53.3%) radical scavenging activity, at 500 ppm. The total phenolic contents were found to be highest in the acetone extract (15.94%) followed by the MeOH extract (5.92%), ethyl acetate extract (5.54%) and water extract (5.26%). Antioxidant capacity of the extracts as equivalents to ascorbic acid (micromol/g of the extract) was in the order, EtOAc extract > acetone extract > water extract > methanol extract. Furthermore, the EtOAC and acetone extracts were loaded onto silica gel columns to obtain four limonoid aglycons. MeOH fraction was loaded onto a dowex-50 and sepabeads resin column to obtain a limonoid glucoside. The purity of the isolated five compounds was analyzed by HPLC using a C18 column and UV detection at 210 nm. Finally, the structures of the compounds were identified as obacunone, nomilin, limonin, deacetylnomilin (DAN) and limonin-17-beta-D-glucopyranoside (LG) using 1H and 13C NMR studies.     

Phenolic composition and antioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis

Introduction – Polyphenolic phytochemicals in traditionally used medicinal plants act as powerful antioxidants, which aroused an increasing interest in their application in functional food development. Objective – The effect of extraction time (5 and 15 min) and hydrolysis on the qualitative and quantitative content of phenolic compounds and antioxidant capacity of six traditionally used medicinal plants (Melissa officinalis L., Thymus serpyllum L., Lavandula officinalis Miller, Rubus fruticosus L., Urtica dioica L., and Olea europea L.) were investigated. Methodology – The content of total phenols, flavonoids, flavan‐3‐ols and tannins was determined using UV/Vis spectrophotometric methods, while individual phenolic acids, flavones and flavonols were separated and detected using HPLC analysis. Also, to obtain relevant data on the antioxidant capacity, two different assays, (2,2‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid) (ABTS) radical scavenging and ferric reducing/antioxidant power (FRAP) assays were used. Results – The extraction efficiency of phenolics, as well as the antioxidant capacity of plant extracts, was affected by both prolonged extraction and hydrolysis. The overall highest content of phenolic compounds was determined in hydrolyzed extract of blackberry leaves (2160 mg GAE/L), followed by the non‐hydrolyzed extract of lemon balm obtained after 15 min of extraction (929.33 mg GAE/L). The above extracts also exhibited the highest antioxidant capacity, while extracts of olive leaves were characterized with the lowest content of phenolic compounds, as well as the lowest antioxidant capacity. The highest content of rosmarinic acid, as the most abundant phenolic compound, was determined in non‐hydrolyzed extract of lemon balm, obtained after 15 min of extraction. Although the hydrolysis provided the highest content of polyphenolic compounds, longer extraction time (15 min) was more efficient to extract these bioactives than shorter extraction duration (5 min). Conclusion – The distribution of detected phenolic compounds showed a wide variability with regard to their botanical origin. Examined medicinal plants showed to be a valuable supplement to a daily intake of bioactive compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

Distribution and radical scavenging activity of phenols in Ascophyllum nodosum (Phaeophyceae)

Phlorotannins have been purified and fractionated in the brown alga Ascophyllum nodosum using successively differential extraction, liquid-liquid separation and dialysis. Both the phenol content and the radical scavenging capacity of the resulting fractions were assayed by the Folin-Ciocalteu test and the DPPH method, respectively, whilst purity of the fractions was assessed by ¹H NMR analysis. The purification process resulted in the isolation of six fractions from each crude extract with only minor losses. High levels of phenols, up to 97-99%, were measured in semi-purified fractions containing phlorotannins more than 50 kDa in average molecular size, accounting for more than 95% of the ethyl acetate phenol pool. As a consequence, purity decreased in ethyl acetate fractions together with the molecular size of compounds. The importance of differential extraction based on the polarity of phenols is highlighted by the fact that most of these compounds were found in the ethyl acetate fraction after the first extraction step in 100% methanol, whilst two thirds of phenols extracted by 50% methanol remained in the aqueous phase. The radical scavenging activity of the fractions was correlated with the phenol content and was maximal in complete ethyl acetate fractions and in dialysis concentrates containing molecules more than 50 kDa in size. The specific activity of phenols was found to be maximal for molecules smaller than 2 kDa when isolated from the 100% methanol extract and 1-4 times smaller in the water phase separated from the same extract. The distribution of radical-scavenging potentials in the phenol pool of A. nodosum supports the idea that physiological roles and putative uses of phlorotannins are under the control of a polarity-molecular size complex.     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

The In vitro antioxidant properties of chinese highland lichens

Antioxidant properties of 46 lichen species collected from high-UV exposed alpine areas of southwestern China were evaluated for their therapeutic utilization. Anti-linoleic acid peroxidation activity, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity, reducing power and the total phenolic contents were assessed in methanol extract of the lichens in vitro. Extracts of Peltigera praetextata and Sticta nylanderiana exhibited potent activity in all antioxidant tests. Especially, extracts of S. nylanderiana exhibited 1.37 times higher anti-linoleic acid peroxidation activity than ascorbic acid used as a positive control. It also showed the strongest free radical scavenging activity among all the tested species with an inhibition of 90.4% at the concentration of 330microgram/ml. Potent reducing power was also detected in the lichen extract compared with BHA. Generally, the antioxidant lichens contained large amount of phenolic contents. The activity-guided bioautographic TLC and HPLC analysis are used to find out the compounds responsible for the potent antioxidant activities of S. nylanderiana extract. The analysis demonstrated that lecanoric acid is one of the effective antioxidant compounds in S. nylanderiana. The results suggested that several highland lichens can be used as a novel bioresource for natural antioxidants.     

Qualitative analysis and simultaneous quantification of phenolic compounds in the aerial parts of Salvia miltiorrhiza by HPLC-DAD and ESI/MS(n)

Introduction – The increasing demands of roots and rhizomes of Salvia miltiorrhiza almost exhausted the wild Salvia sources in China. However, the content and composition of phenolic acids in the aerial parts of the plant and their potential to be used as a substitute has not been explored. Objective – To evaluate the potential of the aerial parts of Salvia miltiorrhiza as new natural sources of phenolic acids. Methodology – HPLC coupled with diode array detection (DAD) and electrospray ionization multistage mass spectrometry (ESI/MSⁿ) has been used for qualitative and quantitative analysis of phenolic compounds. Results – A total of 38 phenolic compounds were identified or tentatively characterized. A quantitative HPLC‐DAD method allowing the simultaneously quantification of six phenolic acids was optimized and validated for linearity, precision, accuracy, and limits of detection and quantification. Calibration curves showed good linear regression (r² > 0.9991) within test ranges; the recoveries ranged between 95.64 and 101.67% and the RSDs were less than 3.01%. Conclusion – The developed methods have been proved to be effective for the identification and quantification of phenolic acids in S. miltiorrhiza. The results obtained suggest that the aerial parts of the plant could be used as an alternative source of sage phenolics. Copyright © 2010 John Wiley & Sons, Ltd.     

Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC

A fast and reliable HPLC method for the determination of caffeic acid derivatives (caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid) in various species of the genus Echinacea has been developed. Extraction of root samples by magnetic stirring with 80% methanol aqueous solution at room temperature allowed the complete recovery of all compounds of interest. Root extracts were analysed on a reversed-phase column with gradient elution and photodiode array detection. Caffeic acid derivatives showed differential qualitative and quantitative distributions in Echinacea species. The total amount of phenolic compounds ranged from 33.95 to 0.32 mg/g. The highest contents of caffeic acid derivatives were found in E. paradoxa var. paradoxa, E. paradoxa var. neglecta and E. purpurea, followed by E. angustifolia var. angustifolia, E. simulata, E. pallida and E. laevigata, whilst E. tennesseensis, E. sanguinea and E. atrorubens had low amounts of phenolic compounds. The radical scavenging activities of methanolic extracts of roots of Echinacea species was evaluated in vitro using the DPPH* radical scavenging method. The EC50 values of the samples ranged from 122 to 1223 microg/mL. The radical scavenging activities of the root extracts were correlated with the content of phenolic compounds, with a correlation coefficient (r2) of 0.923.     

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.     

The detection of radical scavenging compounds in crude extract of borage (Borago officinalis L.) by using an on-line HPLC-DPPH method

The rapid evaluation of antioxidant activity of crude borage (Borago officinalis L.) extract was determined by using DPPH free radical method. This borage extract resulted in a rapid decrease of the absorbance and showed very high hydrogen-donating capacity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. A new HPLC-DPPH on-line method was applied for a screening of several radical scavenging components in this borage extract as well as for quantitative analysis. This on-line HPLC-DPPH method was developed using a methanolic solution of DPPH-stable radical. The HPLC-separated analytes reacted post-column with the DPPH solution in methanol. The induced bleaching was detected as a negative peak photometrically at 515 nm. The separation of antioxidative components was carried out by gradient HPLC with mobile-phase composition ranging from 2% to 80% acetonitrile with 2% acetic acid in water, UV detection was carried out at 280 nm. The HPLC analysis of borage extract revealed the presence of several radical scavenging components in the borage extract. The results obtained from the chromatograms suggest that some compounds present in the extract possess high radical quenching ability. The dominant antioxidative compound in the crude extract of borage leaves was identified as rosmarinic acid.     

Lipophilicity Determination of Highly Lipophilic Compounds by Liquid Chromatography

Different experimental strategies using short columns in both conventional liquid chromatography (HPLC) and ultra‐high pressure liquid chromatography (UHPLC) were evaluated to allow, for the first time with these techniques, the lipophilicity determination of compounds with log P>5. Various organic modifiers, stationary phases, and elution modes were tested on 14 rigid compounds with a CLogP between 5 and 8, and 38 compounds with log P_oct from 0 to 5. The best results in HPLC were obtained with the 20‐mm Discovery ^® RP Amide C16 stationary phase in isocratic mode using MeOH as organic modifier. To improve analysis time, the UHPLC approach was then evaluated. Consequently, a generic method was developed with a 30‐mm Acquity BEH Shield RP18 column in gradient mode using MeOH as organic modifier, allowing a fourfold gain of time compared to the HPLC method, for the highly lipophilic compounds tested. Finally, the most rapid and accurate results were obtained with a 10‐mm Hypersil^TM GOLD Javelin HTS stationary phase in UHPLC, enabling an eightfold gain of time compared to the HPLC method.     

Cytochrome P450 bio-affinity detection coupled to gradient HPLC: on-line screening of affinities to cytochrome P4501A2 and 2D6

Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column.     

HPLC method for the determination of carboplatin and paclitaxel with cremophorEL in an amphiphilic polymer matrix

Simple and rapid reversed phase HPLC methods for individual as well as simultaneous analysis of paclitaxel and carboplatin with cremophorEL (CrEL) in an amphiphilic polymer matrix were developed. Different analytical performance parameters such as linearity, accuracy, precision, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to ICH guidelines. All the analytical methods were developed by reverse phase HPLC on C-18 column with a mobile phase comprising of water-acetonitrile run on isocratic mode for the analysis of carboplatin and gradient mode for individual analysis of paclitaxel and for simultaneous analysis of the two drugs at a flow rate of 1 ml/min at 227 nm. The proposed methods for independent analysis of the drugs elute out carboplatin in 4.3 min and paclitaxel in 10.5 min while in simultaneous analysis carboplatin shows R(t) at 4 min and paclitaxel at 18 min with a continuous run for 17 more minutes to elute out CrEL. These methods were found to be specific as none of the components of the media, i.e. polymer, CrEL and buffer interfered with the drug peaks. The linearity of the calibration curves for each analyte in the desired concentration range was found to be good (r(2)>0.9995). The methods were accurate and precise with recoveries ranging from 98 to 101% for each drug and relative standard deviation (%RSD) <2%. Peaks corresponding to each of the drug showed positive value for the minimum peak purity index over the entire range of integrated chromatographic peak thus indicating the purity of the peaks. Stability analysis of the two drugs revealed that the drugs remain stable during the period of study.     

Variation in natural selection for growth and phlorotannins in the brown alga Fucus vesiculosus

Directional selection for plant traits associated with resistance to herbivory tends to eliminate genetic variation in such traits. On the other hand, balancing selection arising from trade-offs between resistance and growth or spatially variable selection acts against the elimination of genetic variation. We explore both the amount of genetic variation and variability of natural selection for growth and concentration of phenolic secondary compounds, phlorotannins, in the brown alga Fucus vesiculosus. We measured variation in selection at two growing depths and two levels of nutrient availability in algae that had faced two kinds of past growing environments. Genetic variation was low for growth but high for phlorotannins. The form and strength of selection for both focal traits depended on the past growing environment of the algae. We found strong directional selection for growth rate in algae previously subjected to higher ultraviolet radiation, but not in algae previously subjected to higher nutrient availability. Stabilizing selection for growth occurred especially in the deep growing environment. Selection for phlorotannins was generally weak, but in some past-environment-current-environment combinations we detected either directional selection against phlorotannins or stabilizing selection. Thus, phlorotannins are not selectively neutral but affect the fitness of F. vesiculosus. In particular, there may be a fitness cost of producing phlorotannins, but the realization of such a cost varies from one environment to another. Genetic correlations between selective environments were high for growth but nonexistent for phlorotannins, emphasizing the high phenotypic plasticity of phlorotannin production. The highly heterogeneous selection, including directional, stabilizing, and spatially variable selection as well as temporal change in selection due to responses to past environmental conditions, probably maintains a high amount of genetic variation in phlorotannins. Such variation provides the potential for rapid evolutionary response of phlorotannins under directional selection.