Kalevi Pihlaja

Molecular size distribution and spectroscopic properties of aquatic humic substances

The number- and weight-averaged molecular weights of different isolated humic fractions and unfractionated organic matter from natural water samples were measured using high-performance size-exclusion chromatography (HPSEC). The chromatograms obtained, with sodium acetate as the mobile phase on a TSK G3000SW high-speed column, provided illustrative information about the nature of the dissolved organic matter that was not available with phosphate buffer eluents. The calibration of the column system merely with protein standards generated misleading information about the molecular weight and size distribution of humic solutes. Instead, the present results support the utilization of polystyrene-sulphonates (PSS) either alone or together with certain other model compounds for the calibration of HPSEC in studying molecular weights of humic solutes. The data obtained with the HPSEC system indicate that the average molecular weight of humic solutes is distinctly lower than is generally assumed. Besides, aquatic humic solutes also contain some very large-sized though minor constituents, leading to a fairly polydisperse mixture. Number-averaged molecular weights obtained using the HPSEC system were 4–5-fold greater than those from vapour-pressure osmometry. Bulk spectroscopic properties such as molar absorptivity at 280 nm and the quotient, E2E3, correlated strongly with the total aromaticity and averaged molecular weights of all the humic solutes. This observation suggests that bulk spectroscopic properties can be applied, as a first approximation, for estimating the size of humic solutes and their aromaticity in natural surface waters.

Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula Retz. fruit

A 70% methanol extract of Terminalia chebula fruit, was studied for its effects on growth in several malignant cell lines including a human (MCF-7) and mouse (S115) breast cancer cell line, a human osteosarcoma cell line (HOS-1), a human prostate cancer cell line (PC-3) and a non-tumorigenic, immortalized human prostate cell line (PNT1A) using assays for proliferation ([(3)H]-thymidine incorporation and coulter counting), cell viability (ATP determination) and cell death (flow cytometry and Hoechst DNA staining). In all cell lines studied, the extract decreased cell viability, inhibited cell proliferation, and induced cell death in a dose dependent manner. Flow cytometry and other analyses showed that some apoptosis was induced by the extract at lower concentrations, but at higher concentrations, necrosis was the major mechanism of cell death. ATP assay guided chromatographic fractionation of the extract yielded ellagic acid, 2,4-chebulyl-beta-D-glucopyranose (a new natural product), and chebulinic acid which were tested by ATP assay on HOS-1 cell line in comparison to three known antigrowth phenolics of Terminalia, gallic acid, ethyl gallate, luteolin, and tannic acid. Chebulinic acid (IC(50) = 53.2 microM +/- 0.16) > tannic acid (IC(50) = 59.0 microg/ml +/- 0.19) > and ellagic acid (IC(50) = 78.5 microM +/- 0.24), were the most growth inhibitory phenolics of T. chebula fruit in our study.

Characterization, differentiation and classification of aquatic humic matter separated with different sorbents: synchronous scanning fluorescence spectroscopy

Aquatic humic solutes were separated by the non-ionic macroporous XAD-8 and DAX-8 resins and a weakly basic DEAE cellulose anion exchanger from seven different fresh water sources. Synchronous fluorescence spectroscopy was applied for characterization, differentiation and classification of the different humic-solute aggregates. Fluorescence properties verified that humic-solute fractions isolated parallelly with the non-ionic XAD-8 and DAX-8 resins resembled very closely each other speaking strongly for their structural similarities. DAX-8 resin separated ca. 19% more aquatic humic matter than did the analogous XAD-8 resin. It was possible to tentatively differentiate the untreated water samples according to their fluorescent materials. Several distinct classes of chromophores were detected in both DOM and isolated humic fractions by the synchronous technique: lambda(ex)/lambda(em) 280/298, 330/348, 355/373, 400/418, 427/445, 460/478, 492/510 and 516/534 nm.

SEASONAL VARIATION IN THE CONTENT OF HYDROLYZABLE TANNINS, FLAVONOID GLYCOSIDES, AND PROANTHOCYANIDINS IN OAK LEAVES

Oaks have been one of the classic model systems in elucidating the role of polyphenols in plant–herbivore interactions. This study provides a comprehensive description of seasonal variation in the phenolic content of the English oak (Quercus robur). Seven different trees were followed over the full course of the growing season, and their foliage repeatedly sampled for gallic acid, 9 individual hydrolyzable tannins, and 14 flavonoid glycosides, as well as for total phenolics, total proanthocyanidins, carbon, and nitrogen. A rare dimeric ellagitannin, cocciferin D2, was detected for the first time in leaves of Q. robur, and relationships between the chemical structures of individual tannins were used to propose a biosynthetic pathway for its formation. Overall, hydrolyzable tannins were the dominant phenolic group in leaves of all ages. Nevertheless, young oak leaves were much richer in hydrolyzable tannins and flavonoid glycosides than old leaves, whereas the opposite pattern was observed for proanthocyanidins. However, when quantified as individual compounds, hydrolyzable tannins and flavonoid glycosides showed highly variable seasonal patterns. This large variation in temporal trends among compounds, and a generally weak correlation between the concentration of any individual compound and the total concentration of phenolics, as quantified by the Folin–Ciocalteau method, leads us to caution against the uncritical use of summary quantifications of composite phenolic fractions in ecological studies.

Variation of total phenolic content and individual low-molecular-weight phenolics in foliage of mountain birch trees (Betula pubescens ssp.tortuosa).

We studied seasonal and between-tree variation in the composition and content of total and individual low-molecular-weight phenolics (LMWP) in leaves of mountain birch trees (Betula pubescens ssp.tortuosa). The major phenolic compounds were chlorogenic acid, quercetin-3-O-β-D-glucuronopyranoside, myricetin-3-O-(5-acetyl)-L-rhamnopyranoside, and 1-O-galloyl-β-D-(2-O-acetyl)-glucopyranose. The content of total phenolics, as well as the sum of individual LMWP, varied only slightly among trees while variation in contents of individual LMWP was large. Concentrations of almost all phenolics decreased during the growing season but pairwise correlations between individual phenolics remained similar over the whole season indicating tree-specific LMWP profiles over the season. Among flavonoids, the between-tree component of variation was 2.6 times as large as the seasonal component, while for variation of nonflavonoids the between-tree component was larger than the seasonal one. To explain the significant correlations within both flavonoid and nonflavonoid compounds, we discuss the biogenesis of LMWP in birch leaves, as well as their ecological role.

Covariation of fluctuating asymmetry, herbivory and chemistry during birch leaf expansion

Abstract Fluctuating asymmetry (FA) is used to describe developmental instability in bilateral structures. In trees, high FA of leaves has been assumed to indicate the level of environmental or genetic stress, and for herbivores leaves from such trees have been shown to be in some cases (though not invariably) of higher quality compared to trees with symmetrical leaves. We demonstrated that FA of birch leaves correlated positively with growth rate of leaves, and with the amount of leaf biomass consumed by larvae of the geometrid Epirrita autumnata. Since asymmetry per se cannot define leaf quality for a herbivore, we determined the biochemical compounds which covary with the degree of foliage FA, in order to elucidate relationships between leaf FA, chemistry and herbivory. High foliar FA was characteristic of birches with high initial concentrations, and rapid seasonal decline in the concentrations of gallic acid and hydrolysable tannins, and with rapid seasonal changes in the concentrations of flavonoid-glycosides and sugars. In contrast, leaf FA was not related to concentrations of proanthocyanidins, protein-bound amino acids or soluble phenylalanine, the precursor of proanthocyanidins and proteins with aromatic amino acids. The positive correlation between leaf FA and consumption by E. autumnata was presumably related to the previously demonstrated compensatory consumption of E. autumnata to high concentrations of foliar gallotannins. Furthermore, sugars are well-known feeding stimulants. We propose that the variable results in studies correlating leaf FA and herbivory may stem from variable chemical associations of FA in different plants and of species-specific effects of compounds on insects.

1H and 13C NMR Study of 1‐Hydrazino‐2,3‐dihydro‐1H‐pyrazolo[1,2‐a]pyridazine‐5,8‐diones and ‐1H‐pyrazolo[1,2‐b]phthalazine‐5,10‐diones and Their Ring‐Chain Tautomerism

Treatment of 1-hydroxy-2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-5,8-diones and -1H-pyrazolo[1,2-b]phthalazine-5,10-diones with hydrazides produces corresponding acylhydrazino derivatives. In this work, ten new hydrazino derivatives were synthesized, and their properties were studied by 1H and 13C NMR spectroscopy. These compounds exhibited several types of structural variation, including ring-chain tautomerism, cis-trans isomerism with respect to the substituents in the pyrazole ring, and (E)/(Z) rotamerism with respect to the nitrogen−carbon hydrazide bond with partial double-bond character. In [D6]DMSO, 2,3-dihydro-1H-pyrazolo[1,2-a]pyridazine-5,8-dione derivatives were found mainly as linear tautomers, whereas 2,3-dihydro-1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives favored cyclic tautomers. For the latter compounds, a six-component equilibrium was found, consisting of a linear and of cis and trans cyclic tautomers, all of which had two rotamers, (E) and (Z). (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Isolation and characterization of natural organic matter from lake water: Two different adsorption chromatographic methods

Aquatic humic solutes were isolated from lake water by two different methods: non-ionic sorbing solid (XAD technique) and weakly basic anion exchanger (DEAE cellulose). The DEAE procedure removed from the fresh water sample at natural acidity 80% of the DOC as humic solutes. This was practically equal to the total amount of the three organic acids obtained directly by the XAD-8 resin at preadjusted acidity (pH 2) and of the acidic solutes obtained by a specific anion exchanger in connection with the XAD-8 column. The acidic gross character of the humic solutes obtained by the DEAE procedure was similar to the combined character of the four distinct acidic fractions obtained at pH 2 by different adsorbents. The structural composition of humic solutes obtained by the DEAE isolation procedure appears to be a given combination of the various hydrophobic and hydrophilic acidic solutes obtained at pH 2 by the XAD technique. It is likely the acidic treatments connected to the isolation and fractionation of organic solutes by the XAD technique can be to some extent coincidental, especially from the structural chemistry point of view. However, aquatic humic solutes (especially so-called fulvic and humic acids) modelled by the XAD technique are in some degree real, rather than accidental products of the isolation method.

High-performance liquid chromatographic separation and identification of phenolic compounds from leaves of Betula pubescens and Betula pendula

Abstract The following major phenolics with non-flavonoid structure were identified from leaves of Betula pubescens (white birch) and Betula pendula (silver birch): 1-O- galloyl -β- d -(2-O- acetyl )- glucopyranose , 1-(4″-hydroxyphenyl)-3′-oxopropyl-β- d -glucopyranose, gallic, chlorogenic, neo-chlorogenic, cis- and trans-forms of 3- and 5-p-coumarolyloquinic acids. Chromatographic (analytical and preparative HPLC), chemical (hydrolysis) and spectroscopic (UV, 1H and 13C NMR, MS) techniques were applied for separation, isolation, purification and identification of these phenolics. Moreover, 33 low-molecular-mass phenolics were detected and quantitated and their occurrence was compared in leaves of white and silver birches.

Total phenolics concentration and antioxidant potential of extracts of medicinal plants of Pakistan.

Abstract Thirty-seven plant organs, traditionally used as drugs, collected in Pakistan, were extracted with 70% acetone and analyzed for their total phenolics concentration and antioxidant potential. Seven extracts showed more than 85% inhibition of lipid peroxidation in vitro as compared with blank. Butylated hydroxytoluene (BHT) (IC50 = 233.6 μg / 1 ± 28.3) was the strongest antioxidant in our test system. The IC50 results indicate that the extracts of Nymphaea lotus L. flowers, Acacia nilotica (Linn.) Delile beans, Terminalia belerica Roxb. fruits, and Terminalia chebula Retz. (fruits, brown) were stronger antioxidants than a-tocopherol, while Terminalia chebula Retz. (fruit coat), Terminalia chebula Retz. (fruits, black) and Ricinus communis L. leaves were weaker antioxidant extracts than a-tocopherol and BHT. Total phenolics concentration, expressed as gallic acid equivalents, showed close correlation with the antioxidant activity. High performance liquid chromatographic analysis with diode array detection at 280 nm, of the seven extracts indicated the presence of hydroxybenzoic acid derivatives, hydroxycinnamic acid derivatives, flavonol aglycones and their glycosides as main phenolics compounds. This information, based on quick screening methods, enables us to proceed towards more detailed chemical and pharmacological understanding of these plant materials.