Phenolic glucosides as feeding cues for willow-feeding leaf beetles

The effects of individual phenolic glucosides and total glucoside fractions on the feeding behaviour of three willowfeeding leaf beetles (Coleoptera: Chrysomelidae) were tested in the laboratory. Feeding preferences of the tested leaf beetles were strongly influenced by certain phenolic glucosides which are typical secondary compounds of willows (Salicaceae:Salix). Two of the tested leaf beetles,Galerucella lineola andLochmaea capreae showed rather similar responses to glucoside treatments. Both of them were strongly stimulated by total glucoside fractions fromSalix triandra and by its major glucoside salidroside. The third species,Phatora vitellinae, was attracted most by the fractions fromS. myrsinifolia andS. pentandra, and by two related salicylate glucosides, tremulacin and salicortin. Food selection pattern of the tested beetles in the laboratory concords fairly well with their distributions in the field and with the occurrence of phenolic glucosides in their host willows. Phenolic glucoside extracts stimulated more feeding than individual pure glucosides. This indicates that different compounds have synergistic effects in the feeding behaviour of leaf beetles. Our results clearly show that willow leaf beetles select their food based on phenolic glucosides of their host plants.     

Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures

After elicitation, cell suspension cultures of Catharanthus roseus accumulate phenolic compounds. The major phenolic compound produced was isolated and identified as 2,3-dihydroxybenzoic acid (DHBA). The accumulation of this compound is a rapid response to the addition of elicitor; within 6 h after the addition of elicitor, DHBA concentration reached 6.3 mg/l cell suspension. DHBA was not detected in non-elicited cells. The formation of DHBA in elicited cells was correlated with the induction of the enzyme isochorismate synthase (ICS). Shoot cultures of C. roseus also presented a strong induction of ICS after elicitation. Due to its biological activity, DHBA could play a role in the defence mechanism of C. roseus.     

RNAi-mediated suppression of the phenylalanine ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis

Medicinal Salvia miltiorrhiza contains two main groups of active pharmaceutical ingredients: lipid-soluble tanshinones and water-soluble phenolic acids, including rosmarinic acid and salvianolic acid B. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway and is assumed to be closely related to the accumulation of rosmarinic acid and its derivatives. We selected a 217-bp fragment, located at the 3′ end of the coding region of PAL1, to establish an RNA interference construct that was introduced into S. miltiorrhiza via Agrobacterium tumefaciens-mediated transformation. PAL-suppressed plants exhibited several unusual phenotypes such as stunted growth, delayed root formation, altered leaves, and reduced lignin deposition. The total phenolic content was decreased by 20–70% in PAL-suppressed lines, and was accompanied by lower PAL activity. Down-regulation of PAL also affected the expression of C4H, 4CL2, and TAT, which are related genes in the rosmarinic acid pathway. Moreover, rosmarinic acid and salvianolic acid B were markedly reduced in PAL-suppressed lines, as detected by HPLC analysis. Our results indicate that PAL is very important for the synthesis of major water-soluble pharmaceutical ingredients within S. miltiorrhiza.     

Strategies for the improvement of salidroside production in cell suspension cultures of Rhodiola sachalinensis

Strategies of elicitation and precursor feeding were applied to improve salidroside production in cell suspension cultures of Rhodiola sachalinensis. Of the seven elicitors examined, that extracted from Aspergillus niger was the most effective, increasing the salidroside content by five-fold when added on the day of inoculation 40 mg carbohydrate is medium. Three possible precursors for salidroside synthesis, l-phenylalanine, l-tyrosol and l-tyrosine were added to the cultures. A high content of salidroside (1.440%) was attained with an initial l-tyrosol concentration of 0.5 mm in the medium. Combined application of the two strategies resulted in a significantly high salidroside content of 1.734%, corresponding to a salidroside yield of 200 mg/l. Received: 5 November 1996 / Revision received: 17 February 1997 / Accepted 11 April 1997     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

Effect of inhibition of phenylalanine ammonia-lyase activity on growth of alfalfa cell suspension culture: Alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins and polyamines

The effect of inhibition of phenylpropanoid biosynthesis on the growth of Medicago sativa L. suspension culture was studied. 2-Aminoindan-2-phosphonic acid (AIP), a potent inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), caused a marked reduction in the amount of hydroxycinnamic acid derivatives in a few hours after cell inoculation into AIP medium. The treatment of alfalfa suspension culture with this inhibitor increased the extractable PAL activity and elevated ethylene production during the growth cycle. The addition of AIP (10 μ M ) stimulated cell division activity during the growth cycle, although the onset of cell division was slightly delayed. The maxima of cytokinin content as well as of the mitotic index were postponed in AIP-treated cells, however, the unchanged content of cytokinins did not correlate with increased mitotic activity of treated cells. The decreased level of hydroxycinnamic acid derivatives, which represent the phenolic conjugation partners of free polyamines (PAs), influenced the rate of PA conjugation. Consequently, the balance between free and conjugated PAs was shifted in favor of the free PA form. A potential role of the reduction of the pool of phenolic acids in the enhancement of cell division of alfalfa cell suspension culture is discussed.     

Phytochemical Analysis and Antioxidant Activity of <i>Crocus speciosus</i> Leaves

The numerous studies indicate leaves of plants are a rich source of bioactive compounds that can be a valuable source of compounds used in the pharmaceutical and cosmetic industries. Aim of this study was to investigate the chemical composition and the antioxidant property of Crocus speciosus leaves. Primary phytochemical screening of C. speciosus leaves revealed the presence of some following compound categories such as phenolic compounds, aminoacids, saponins, proteins, tannins, triterpenoids, glycosides, polysaccharides. The total flavonoids and phenolic compounds content were determined spectrophotometrically and by HPLC-DAD and HPLC-MS. Antiradical activity was determined by ABTS radical-cation scavenging method, spectrophotometrically. The total amount of flavonoids in C. speciosus leaves was 1.07 ± 0.02 mg RE/g (p < 0.05), the total amount of phenolic compounds was 0.41 ± 0.01 mg GAE/g (p < 0.05). By HPLC-DAD-MS analysis the presence of the mangiferin, chlorogenic acid, isoorientin, kaempferol, hyperoside, and isoquercitin was established for the first time in Crocus leaves. The antiradical activity of C. speciosus leaves extracts was 150.08 ± 4.5 μmol/g (p < 0.05) and its was mainly attributed to phenolic compounds content. The high amounts of flavonoids and antiradical activity in C. speciosus leaves suggests promising phytochemical and pharmacological study of this Crocus species.     

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.     

Phenolic compounds accumulation in <Emphasis Type="Italic">Hypericum ternum</Emphasis> propagated in vitro and during plant development acclimatization

The phenolic compound content of Hypericum ternum was investigated after micropropagation establishment and during acclimatization over the phenological development of the plant. Plantlets cultured in vitro on full Murashige and Skoog medium without growth regulators displayed higher phenolic compound yields, were acclimatized, and field grown. Production of total phenolic compounds as well as hyperoside, chlorogenic acid, quercitrin, guaijaverin, isoquercitrin, and uliginosin B were quantified at vegetative, flowering and fructification stages, and different plant organs (roots, stems, leaves and reproductive parts) showing that reproductive parts at flowering stage and the leaves at fructification stage were the main repository site of secondary metabolites, except for uliginosin B. The stems were the least accumulative organ, while the roots accumulated only hyperoside and uliginosin B. Moreover, the accumulation of most of the flavonoids and uliginosin B in acclimatized plants surpassed the levels found in the wild plant, warranting further research with the species.     

Edible flowers — antioxidant activity and impact on cell viability

The phenolic compound composition, antioxidant activity and impact on cell viability of edible flower extracts of Allium schoenoprasum; Bellis perennis; Cichorium intybus; Rumex acetosa; Salvia pratensis; Sambucus nigra; Taraxacum officinale; Tragopogon pratensis; Trifolium repens and Viola arvensis was examined for the first time. Total phenolic content of the flowers of these plants fell between 11.72 and 42.74 mg of tannin equivalents/kg of dry matter. Antioxidant activity ranged from 35.56 to 71.62 g of ascorbic acid equivalents/kg of dry matter. Using the Human Hepatocellular Carcinoma cell-line (HepG2) and the Human Immortalized Non-tumorigenic Keratinocyte cell line (HaCaT), we assessed cell viability following a 3 day incubation period in media containing 25, 50, 75 and 100 μg/ml of total phenolic compounds using a colorimetric MTT assay. These three properties could make the herbs useful in treatment of various diseases like cancer. The tested extracts had significant effects on cell viability, but the effects were dependent not only on the phenolic compound concentration and the edible flowers species, but also on the phenolic compound and antioxidant profiles. In addition, responses differed between cell lines.     

Enzymes of phenylpropanoid metabolism in the important medicinal plant Melissa officinalis L.

Lemon balm (Melissa officinalis, Lamiaceae) is a well-known medicinal plant. Amongst the biologically active ingredients are a number of phenolic compounds, the most prominent of which is rosmarinic acid. To obtain better knowledge of the biosynthesis of these phenolic compounds, two enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL) and 4-coumarate:coenzyme A-ligase (4CL), were investigated in suspension cultures of lemon balm. MoPAL1 and Mo4CL1 cDNAs were cloned and heterologously expressed in Escherichia coli and the enzymes characterised. Expression analysis of both genes showed a correlation with the enzyme activities and rosmarinic acid content during a cultivation period of the suspension culture. Southern-blot analysis suggested the presence of most probably two gene copies in the M. officinalis genome of both PAL and 4CL. The genomic DNA sequences of MoPAL1 and Mo4CL1 were amplified and sequenced. MoPAL1 contains one phase 2 intron of 836 bp at a conserved site, whilst Mo4CL1 was devoid of introns.     

Production of salidroside and polysaccharides in Rhodiola sachalinensis using airlift bioreactor systems

Rhodiola sachalinensis is widely used in traditional Chinese medicine, and salidroside and polysaccharides are its important bioactive compounds. This study used airlift bioreactor systems to produce mass bioactive compounds through callus culture. Several factors affecting callus biomass and bioactive compound accumulation were investigated. Callus growth was vigorous in a bioreactor system, and the growth ratio was 2.8-fold higher in bioreactor culture than in agitated-flask culture. Callus biomass and polysaccharide content were favorable at 0.1 air volume per culture volume per min (vvm), whereas favorable salidroside content was observed at a high air volume (0.2 vvm). The maximum yields of salidroside (7.90 mg l^?1) and polysaccharide (2.87 g l^?1) were obtained at 0.1 vvm. Inoculum density greatly affected callus biomass and bioactive compound accumulation, and the highest biomass and contents or yields of salidroside and polysaccharide were determined at a high inoculum density of 12.5 g l^?1. The level of hydrogen ion concentration (pH) at 5.8 improved callus biomass accumulation. Acidic medium (pH 4.8) stimulated salidroside synthesis but higher pH level (7.8) promoted polysaccharide accumulation. The highest yields of both bioactive compounds were obtained at pH 5.8. Methyl jasmonate (MeJA) participated in synthesis promotion of bioactive compounds, and the contents and yields of salidroside [4.75 mg g^?1 dry weight (DW), 58.43 mg l^?1] and polysaccharides (392.41 mg g^?1 DW, 4.79 g l^?1) were at maximum at 125 and 150 μmol of MeJA. Therefore, bioreactor systems can be used to produce R. sachalinensis bioactive compounds, and callus culture in a bioreactor can be as an alternative method for supplying materials for commercial drug production.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

Insect feeding preferences and plant phenolic glucosides in the system Gonioctena linnaeana–Salix triandra

The aim of this study was to investigate the preference of a leaf beetle to different Salix clones, and to relate this preference to plant chemistry. The preference of Gonioctena linnaeana Schrank (Coleoptera: Chrysomelidae) was tested in cafeteria experiments using its host Salix triandra L. (Salicaeae) from three Swedish isolated populations and one Russian population from the main distribution range of the host. The leaves from the different host clones were used to analyse the content of phenolic glucosides and the amount of condensed tannins. The larvae did not show any feeding preferences for host clones from the population they originated from, but all Swedish host populations were preferred over the Russian host population. This suggests a preference for regional hosts. We analyzed whether leaf chemistry parameters may explain host plant preferences. Chemical analysis of the leaves showed that the quantities, but not the quality, of the phenolic compounds differed between populations. A Principal Component Analysis of the chemical data also highlighted a difference between the Swedish and Russian host plant populations. The two most important compounds separating Russian from Swedish clones were gallocatechin and salidroside. However, the difference in preference could also be a result of, for example, nutritional quality, water content, or leaf texture. The relative importance of these different factors should be evaluated in future experiments.     

菊芋悬浮细胞对盐胁迫的生理响应

为探讨菊芋（Helianthus tuberosus）悬浮培养细胞对盐胁迫的生理响应,在0、50、100、150、200 mmol·L^-1NaCl处理下测定了细胞的生物量、相对细胞活力、抗氧化酶活性以及过氧化氢、丙二醛、脯氨酸、可溶性糖、可溶性蛋白质的含量,测定了总酚含量并鉴别、定量分析了14种酚类化合物。结果表明：盐胁迫显著减弱了菊芋悬浮细胞的活力,抑制了细胞的增长,200 mmol·L^-1NaCl处理下细胞生长基本上停止。盐胁迫诱发了细胞氧化胁迫,丙二醛含量显著增加,抗坏血酸过氧化物酶、过氧化物酶活性以及总酚含量、部分酚类化合物含量均随NaCl浓度升高而增加,酚类物质和抗氧化酶系统共同参与了应对氧化胁迫的抗氧化作用。脯氨酸在菊芋悬浮细胞应对NaCl渗透胁迫的渗透调节作用中扮演了重要的角色,而可溶性糖发挥的作用不大。     

Biochemical characteristics and antioxidant activity of crude and purified sulfated polysaccharides from Gracilaria fisheri

Sulfated polysaccharides (SPs) from Gracilaria fisheri of Thailand, which were extracted in low-temperature (25 °C) water showed the highest content of phenolic compounds compared with those extracted at high temperature (55 °C). Crude SP antioxidant activity was evaluated by measuring the DPPH free radical scavenging effect which is directly related to the level of phenolic compounds. The sulfate content, total sugar, and SPs yield were also directly related to the extraction temperature. All extracts contained galactose as a major monosaccharide. High antioxidant activity of crude SP, positively correlated with the phenolic compound contents (R² = 0.996) contributed by the existence of sulfate groups and phenolic compounds. In purified SP, F1 fraction exhibited strong radical scavenging ability, but it was not significantly different compared to crude SP extracted at 25 °C. This indicated that the appropriate density and distribution of sulfate groups in the SP extract showed the best antioxidant activity.