Changes in the content of phenolic substances during the growth ofNicotiana tabacum cell suspension culture

The dynamics of changes in the content of four groups of phenolic substances was investigated during the growth cycle of the cell suspension culture ofNicotiana tábacum by means of fractionation. The relative contents of free phenolic acids, their esters, phenolic glycosides, and phenole acids non-extractable with methanol changed in dependence on the growth phase of the culture. A sharp increase, especially in the content of ester- and glycoside-bound phenolics and to a lesser extent also of phenolics belonging to the other two groups, occurred at the end of the lag phase. Then, after a temporary decrease at the early linear phase, the level of phenolics in the three fractions representing bound forms considerably increased again at the late linear and early stationary phases. The synthesized phenolic substances were partially released from the cells into the cultivation medium, which contained 15 to 30 % of the total content of the phenolics in the culture at different phases of the growth cycle. Likely causes of these changes are discussed.     

Variation in the growth and biosynthetic activity of cloned cell cultures of Capsicum frutescens and their response to an exogenously supplied elicitor

Twenty clones established from single cells of a suspension culture of Capsicum frutescens were maintained as callus and in suspension over a sixteen week culture period. These clones exhibited marked differences in growth, chlorophyll and chloroform-soluble phenolic content which became more apparent with increasing time in culture. Clones in suspension exhibited a more rapid change in morphology and biosynthetic activity than those cultured as callus. Elicitation increased PAL activity, reduced the incorporation of L-[U-¹⁴C] phenylalanine into the chloroform-soluble fraction of the culture medium and increased incorporation into the methanol-soluble fraction of the cells in ten suspension clones. Differences to elicitation were observed among clones; in particular the faster growing isolates incorporated more radioactive label into soluble phenolics that remain in the cells than those that are released into the medium. The implications of these results are discussed.Abbreviations SH Schenk & Hildebrandt - PAL phenylalanine ammonia-lyase - RGR relative growth rate - TCC total chlorophyll content - HPLC high performance liquid chromatography     

Changes in the content of phenolic compounds and IAA-oxidase activity during the growth of tobacco crown gall suspension culture

Phenolic content and IAA-oxidase (IAA-o) activity have been assayed in cells and medium of tobacco crown gall suspension culture in several stages of culture cycle. The highest content of total phenolics in the cells were found prior to cell division and in the middle stage of intensive growth. The beginning of intensive growth is accompanied by temporary reduction in phenolic level in the cells as well as their intensive secretion to the medium. In the second part of the culture cycle, when the phenolic production was weaker, the majority of these compounds were maintained in the cells. The highest activity of IAA-o in the cells was detected in the middle stage of intensive growth, simultaneously with high phenolic content; following it a considerable decrease of IAA-o activity is correlated with maximum of chlorogenic acid (ChA) content (at reduced amount of total phenolics). IAA-o activity increased again at the end of the stage of intensive growth when the level of phenolics was low including ChA. These data suggest that IAA-o in relation to phenolic level determines cell growth in the culture. In the culture medium — fairly distinct negative correlation between IAA-o activity and phenolic content suggests that the latter participates in enzyme activity regulation. During intensive growth IAA-o activity is strongly inhibited. The results prove that phenolic level. IAA-o activity and auxin level are closely correlated and may constitute essential elements of a mechanism of regulation crown gall cell growth in culture.     

Metabolism of phenolic compounds in <Emphasis Type="Italic">Vitis riparia</Emphasis> seeds during stratification and during germination under optimal and low temperature stress conditions

We studied the alterations in phenolic compounds in grape seeds during their stratification and germination under optimal conditions (+25 °C) and at low temperature (+10 °C). Biological materials in the study were seeds of Vitis riparia. Phenolic compounds were extracted from defatted seeds using 80 % methanol or 80 % acetone. The content of total phenolics was determined with the Folin-Ciocalteau reagent, while the content of tannins was determined by vanillin assay and the protein (BSA) precipitation method. The RP-HPLC method was used to determine phenolic compounds (phenolic acids, catechins) in the extracts. High amounts of tannins, catechins, gallic acid and lesser amounts of p-coumaric acid were found in the seeds. The content of total phenolics in acetone extracts was higher than that obtained using methanol. The amounts of phenolic acids and tannins found in V. riparia seeds after stratification were much lower. It may confirm a possible role of these compounds in dormancy of V. riparia seeds. After 72 h of low temperature treatment, inhibition of grape root growth and biochemical changes in seeds were detected. The chilling stimulated increased accumulation of some phenolic compounds (free gallic acid and catechins) in the seeds. These substances can protect plants against some abiotic stressors.     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

Alterations in phenylpropanoid content in soybean roots during low temperature acclimation

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, growth and phenolic acid content during low temperature acclimation in soybean (Glycine max. (L.) Merr.) roots were investigated. Elongation of soybean roots was inhibited after the transfer of 3-d-old seedlings grown at 25 to 10 °C. Extractable PAL activity as well as the total amount of phenolics increased 24 h after plant transfer to low temperature. The high pressure liquid chromatography analyses revealed the presence of six phenolic acids in soybean roots: p-hydroxybenzoic, vanillic, syringic, anisic, p-coumaric and ferulic. Analyses of different fractions of phenolic acids showed that during 24 h of low temperature exposure, an increase in the relative level of ester-bound-soluble phenolic acids occurred. The highest increase in this fraction was observed for ferulic acid (26 %). At the same time, a decrease in phenolic glycosides took place. The amount of phenolic acids released after alkaline treatment of the cell wall material was strongly inhibited (3-fold), which may suggest an alteration of the physical properties of the wall in acclimation to low temperature. The possible role of phenolics in acclimation to low temperature in roots is discussed.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.     

Caffeic acid derivatives from a hairy root culture of Lactuca virosa

In a search for biologically active phenolics, a hydroalcoholic extract from the hairy roots of Lactuca virosa was fractionated by chromatographical methods. The procedure led to the isolation of a substantial amount of 3,5-dicaffeoylquinic acid (3,5-DCQA)—a potent free radical scavenger. An analytical RP-HPLC separation of the hydroalcoholic extract from the hairy roots allowed identification of further hydroxycinnamates: caftaric acid (CTA), chlorogenic acid (5-CQA) and cichoric acid (DCTA), as well as small amounts of unbound phenolic acids. A time course of growth and caffeic acid derivatives accumulation in the hairy root culture was also investigated. The highest contents of the compounds in the examined roots were detected at the logarithmic phase of growth. The average content of 3,5-DCQA in the roots (ca. 2.5% DW) was at least one order of magnitude higher than that found in roots of Lactuca species and callus culture of L. virosa.     

Effect of Plant Growth-Promoting Rhizobacteria and Culture Filtrate of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> on Phenolic and Salicylic Acid Contents in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea (Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident. Received: 1 April 2002 / Accepted: 4 May 2002     

Phenylalanine ammonia-lyase,phenolic acids and ethylene in alfalfa (Medicago sativa L.) cell cultures in relation to their embryogenic ability

Phenylalanine ammonia-lyase (PAL) activity, contents of phenolic acids and ethylene production during the lag-phase, and contents of phenolic acids at the late exponential phase, showed significant differences in embryogenic (EC) and non-embryogenic (NEC) suspension cultures of Medicago sativa L. Maximum PAL activity at 6 h after inoculation was followed by an increase in the level of phenolic acids from 9.6 g g^–1 fresh mass to 21 g g^–1 fresh mass in NEC at 12 h. Thereafter the level of phenolic acids decreased to 5.2 g g^–1 fresh mass at 72 h. The decline was caused predominantly by the decrease of ester-bound cinnamic acid derivatives, the decrease ranging from 83 to 20% of total phenolics. Two maxima of ethylene production were observed in NEC: the first one immediately after inoculation and the second at 6 h, coinciding with the peak of PAL activity. In NEC, most of the phenolic acids occurred in esterified form. Ability to form somatic embryos (EC) was associated with the absence of the second peak of ethylene production as well as of the peak of PAL activity at 6 h. The level of phenolic acids during the lag-phase remained low (7.2 g g^–1 FM) and did not change. The proportion of cinnamic acid derivatives was very low (18% of total phenolics), mostly due to the extremely low level of ferulic acid. In EC, phenolic acids bound to methanol insoluble material formed the major fraction. Loss of embryogenic potential of the embryogenic culture (ECL) was associated with qualitative and quantitative changes in the contents of phenolic acids insignificantly increased PAL activity after inoculation was followed by a moderate increase in the contents of phenolic acids from 9.35 g g^–1 fresh mass to 12.42 g g fresh mass. A high rate of ethylene production was observed only immediately after the transfer of the culture to fresh medium. The loss of embryogenicity correlated also with changes in the relative amounts of the investigated fractions of phenolic acids. A distinct increase in the level of methoxy-substituted phenolic acids is a characteristic feature of the ECL culture.Abbreviations NEC non-embryogenic suspension culture - EC embryogenic suspension culture - ECL embryogenic suspension culture after the loss of embryogenic potential - AA anisic acid - CA cinnamic acid - CaA caffeic acid - pCA p-coumaric acid - FA ferulic acid - pHBA p-hydroxybenzoic acid - SA syringic acid - SaA salicylic acid - SiA sinapic acid - VA vanillic acid - PhA phenolic acids - HPLC High-Performance Liquid Chromatography - GC Gas Chromatography - 2,4-D 2,4-dichlorophenoxyacetic acid - kin kinetin - NAA 1-naphthaleneacetic acid - BL-medium medium of Blaydess - FM fresh mass     

Phenolic compounds profiling in shake flask and bioreactor system cell cultures of <Emphasis Type="Italic">Scrophularia striata</Emphasis> Boiss

Variation in levels of phenolic acids and flavonoids in Scrophularia striata Boiss. cells cultured in both shake flask and bioreactor in vitro systems, was studied at different growth phases. Four phenolic acids (cinnamic, salicylic, coumaric, and caffeic acid), one stilbenoid (resveratrol), and seven flavonoids (diosmin, rutin, kaempferol, catechin, myricetin, quercetin, and luteolin) were analyzed by high-performance liquid chromatography with photodiode array detection. Production of phenolics in the bioreactor was higher than in shake flasks. Catechin was the most abundant flavonoid in both culture systems, while quercetin, which was detected only in the bioreactor, was the lowest amount represented (32.82 μg g^?1 DW). Resveratrol accumulation in bioreactor cultures was 59.84-fold higher than that in shake flasks. Moreover, hierarchical clustering analysis based on Pearson’s correlation coefficient confirmed a positive correlation between the growth phase and some metabolites. The flavonoid accumulation increased with the cells’ physiological age in the bioreactor. Principal component analysis showed that the time course of induction of phenolic acids, flavonoids, and a stilbenoid (resveratrol) was significantly correlated. These findings highlight the capacity of S. striata for large-scale production of desired phenolics using a bioreactor system.     

Peroxidase activities and contents of phenolic acids in embryogenic and nonembryogenic alfalfa cell suspension cultures

Contents of phenolic acids, peroxidase activities and growth curves showed significant differences in embryogenic (EC) and nonembryogenic (NEC) suspension cultures ofMedicago sativa L. NEC gave a typical growth curve while in EC the distinct phases were absent. The total content of phenolic acids was higher in NEC (related to EC), changed during the growth cycle and most of the acids occurred in ester-bound methanol soluble form. The level of phenolic acids in EC was significantly lower and did not change during 12-d cultivation. The major fraction was formed by phenolic acids ester-bound to the cell wall. The cytoplasmic peroxidase activity in NEC increased continuously during the growth and reached the maximum value at the end of exponential phase. In EC the extremely low cytoplasmic peroxidase activity did not change during cultivation. Ionically bound peroxidases in NEC represented 14 to 30% of the total extracted activity in dependence on the growth phase while in EC formed about 50% of the total activity and did not change during studied period. A possible participation of ionic peroxidase in the incorporation of phenolics into the cell wall is discussed.     

Phenolic substances in tissue cultures ofCentaurium erythraea

Phenolic compounds l,2,3-trihydroxy-5-methoxyxanthone, l-hydroxy-3,5,6,7, 8-pentamethoxyxanthone, and l,8-dihydroxy-2,3,4,6-tetramethoxyxanthone predominate in the callus tissue ofCentaurium erythraea, their content increasing with culture age. By contrast, the contents of the derivatives of cinnamic, chlorogenic, and ferulic acids decrease or do not change. In the cell suspension culture ofC. erythraea a larger amount of xanthones is synthesized than in the callus from which the suspension culture was derived. The content of phenolic acids is lower in the suspension culture than in the callus, but a larger number of low-molecular-mass phenolic substances occurs in the suspension culture than in the callus tissue.     

Alternations in phenolic acids content in developing rye grains in normal environment and during enforced dehydration

A series of high pressure liquid chroamtography analyses revealed the presence of five phenolic acids in rye caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic), three of which (p-coumaric, ferulic and sinapic) were found in the free phenolic fraction. Ferulic acid was predominant, both among free acids and total phenolic acids (i.e. free, liberated from soluble esters and glycosides). The highest content of the free phenolic acids in rye caryopses was observed at the beginning of development, when on 22 DAF it was estimated at 11.55 μg·g⁻¹ DW. During dehydratation the total level of free phenolic acids in rye caryopses decreased in all investigated samples. Although total phenolic acids contents in all samples of unripe rye caryopses always decreased after dehydration, in rye sample collected in full ripeness (57 DAF), the amount of these compounds increased after the enforced dehydration. It should be added that in ester-bound-soluble phenolic acids fraction (the largest part in the total phenolic acids fraction), irrespective of the total amount decrease, much increase of sinapic acid content in this fraction was observed after dehydratation treatment in all investigated samples of caryopses of various ripeness. During the development and ripening of rye caryopses, a gradual increase in the precocious germination ability of the grain was observed. The enforced dehydration stimulated the process of precocious germination of developing and ripening rye caryopses. A possible role of phenolics in preventing precocious germination and acclimation to dehydration of developing and ripening rye grains is discussed.     

Interactive effects of leaf maturation and phenolics on consumption and growth of a geometrid moth

Phenolic compounds are commonly regarded as the main chemical defenses of deciduous woody plants against insects. To examine how indices of leaf maturation (water content, toughness, and sugar/protein ratio) modified larval consumption and growth relative to phenolics and phenolic-related leaf traits, we measured consumption and growth of fourth-instar Epirrita autumnata (Bkh.) (Lepidoptera: Geometridae) larvae on three different days on young, normal, and mature leaves, respectively, from the same mountain birch (Betula pubescens ssp. czerepanovii (Orlova) Hämet-Ahti) trees. The larvae achieved the same growth rates on young and normal leaves, but had to consume 40% more on the latter. On more mature leaves, larval growth was poorer and was positively correlated with sugar/protein ratios (although the ratio peaked at that time). Indices of leaf maturation correlated with several phenolics in data pooled over the three study days, but poorly in any individual day. Similarly, in the pooled data, larval consumption and growth correlated with several leaf traits, but correlations between leaf and insect traits were few on any of the three days, and no trait was significant on each of the three days.We next examined whether variation in the maturation indices modified the associations of phenolics with insect consumption and growth. When interactions between phenolics and leaf maturation indices were taken into account, the number of phenolic compounds displaying significant associations with insect traits more than doubled. The relative importance of interactive versus direct associations increased with leaf maturation: on young leaves five phenolics showed direct and eleven interactive associations with insect traits, while in mature leaves we found two phenolics to display direct and thirteen phenolics interactive associations. Leaf water content, either alone or together with toughness and sugar/protein ratio, generally explained more of the variance in Epirrita growth (up to 59%) than any phenolic or phenolic-related trait alone (highest value 20%). Including interactive effects between phenolics and indices of leaf maturation in the model increased the proportion explained of variance in larval growth between 49 and 73%. Maturation indices explained 0 to 23% of variance in consumption, and the phenolic compound with the highest (positive!) correlation alone up to 28%, but taking into account interactions between phenolics and maturation indices raised the degree of explanation much (namely, 32 to 53%) over that explained by indices of leaf maturation alone. This indicates strong interactive effects on consumption between phenolics and indices of leaf maturation.     

Partitioning of 14C-labeled photosynthate to allelochemicals and primary metabolites in source and sink leaves of aspen: evidence for secondary metabolite turnover

Theories on allelochemical concentrations in plants are often based upon the relative carbon costs and benefits of multiple metabolic fractions. Tests of these theories often rely on measuring metabolite concentrations, but frequently overlook priorities in carbon partitioning. We conducted a pulse-labeling experiment to follow the partitioning of ¹⁴CO₂-labeled photosynthate into ten metabolic pools representing growth and maintenance (amino acids, organic acids, lipids plus pigments, protein, residue), defense (phenolic glycosides, methanol:water and acetone-soluble tannins/phenolics), and transport and storage (sugars and starch) in source and importing sink leaves of quaking aspen (Populus tremuloides). The peak period of ¹⁴C incorporation into sink leaves occurred at 24 h. Within 48 h of labeling, the specific radioactivity (dpm/mg dry leaf weight) of phenolic glycosides declined by over one-third in source and sink leaves. In addition, the specific radioactivity in the tannin/phenolic fraction decreased by 53% and 28% in source and sink leaves, respectively. On a percent recovery basis, sink leaves partitioned 1.7 times as much labeled photosynthate into phenolic glycosides as source leaves at peak ¹⁴C incorporation. In contrast, source leaves partitioned 1.8 times as much ¹⁴C-labeled photosynthate into tannins/phenolics as importing sink leaves. At the end of the 7-day chase period, sink leaves retained 18%, 52%, and 30% of imported ¹⁴C photosynthate, and labeled source leaves retained 15%, 66%, and 19% of in situ photosynthate in metabolic fractions representing transport and storage, growth and maintenance, and defense, respectively. Analyses of the phenolic fractions showed that total phenolics were twice as great and condensed tannins were 1.7 times greater in sink than in source leaves. The concentration of total phenolics and condensed tannins did not change in source and sink leaves during the 7-day chase period. Received: 31 July 1998 / Accepted: 8 February 1999     

Effect of plant growth-promoting Rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea ( Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident.     

Free and bound phenolic acids of lucerne (Medicago sativa cv europe)

The distribution of free and covalently-bound phenolic acids was studied in various fractions obtained from fresh lucerne shoots. p-Hydroxybenzoic, vanillic, p-coumaric and ferulic acids were present, both free and bound, in all the fractions. Salicylic and sinapic acids occurred only in a bound, alkali-labile state and were found almost entirely in the ‘aqueous phase’ fraction after treatment of methanol-chloroform-water extract according to Bligh and Dyer. Many other common phenolics were absent. Amounts of the phenolic acids much larger than those extracted by methanol-chloroform-water were extracted subsequently by phenol-acetic acid-water and passed into the ‘diffusate’ fraction on dialysis of this extract against 70% acetic acid. Small, though significant, quantities of phenolic acids remained with the bulk protein in the ‘bag contents’ fraction. The extent to which the phenolic acids in these last two fractions are held to protein by covalent bonds or by secondary-valence attractions is discussed, particularly in relation to the isolation of N-feruloylglycylphenylalanine after partial hydrolysis. Suggestions are made for improving analytical procedures.     

Antiviral and virucidal activities against herpes simplex viruses of umesu phenolics extracted from Japanese apricot

Umesu phenolics were obtained from the salt extracts of Japanese apricot (Nanko‐mume cultivar of Prunus mume Sieb. et Zucc.) as purified phenolics. The antiviral activities of umesu phenolics obtained were then examined against herpes simplex virus type 1 (HSV‐1) and type 2 (HSV‐2), enveloped DNA viruses. The phenolics inhibited the multiplication of these viruses when added to the culture media of the infected cells. This inhibition occurred at phenolic concentrations at which they showed no severe cytotoxicity. One‐step growth experiments showed that the eclipse period in the HSV‐1 multiplication process was extended in the presence of umesu phenolics and that the addition of phenolics after the completion of viral DNA replication did not affect their multiplication. More drastic effects were observed on virucidal activities against HSV‐1 and HSV‐2; the infectivity decreased to 0.0001 when infected cells were incubated with 3 mg/ml phenolics at 30°C for 5 min. These results demonstrate the antiviral and virucidal activities of umesu phenolics and suggest a potential pharmacological use for these phenolics as a sanitizing or preventive medicine against superficial HSV infections.     

Clone-specific responses in leaf phenolics of willows exposed to enhanced UVB radiation and drought stress

The effects of enhanced UVB radiation and drought stress on willow secondary phenolics were studied using the leaves of 8‐week‐old micropropagated plantlets from interspecific hybrids (Salix myrsinites L. ×S. myrsinifolia Salisb.) and pure species (S. myrsinifolia). The plantlets were subjected for 4 weeks to two levels of UVB radiation (ambient, enhanced) and two levels of watering (well‐watered, drought‐stressed) according to a 2 × 2 factorial design. Enhanced UVB radiation increased the total concentration of flavonoids and phenolic acids in all plantlets, while the total concentration of salicylates remained unaffected. Drought stress reduced the total concentration of salicylates and phenolic acids in S. myrsinifolia plantlets, while in hybrids only phenolic acids were affected. The response of phenolic acids to enhanced UVB in drought‐stressed plantlets was different from that in well‐watered ones, indicating that drought stress limited the accumulation of phenolic acids under enhanced UVB radiation. Flavonoids increased in response to enhanced UVB radiation in drought‐stressed plantlets, although drought caused serious physiological stress on growth. There were significant differences between hybrid and S. myrsinifolia plantlets with respect to the composition of phenolics and between families and clones with respect to their concentration. In addition, the response of salicylates, flavonoids and phenolic acids to enhanced UVB and drought stress was clone‐specific, which may indicate that climatic changes will alter the genetic composition of northern forests.