Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

Distribution of phenolic compounds in Middleeuropean taxa of the Achillea millefolium L. aggregate

Achillea millefolium L. s.l. is a cytogenetically, morphologically, and chemically polymorphic aggregate. Besides the sesquiterpenes that possess chemotaxonomic relevance and mediate the antiphlogistic activity, the plant contains phenolic compounds such as dicaffeoylquinic acids and flavonoids causing choleretic and spasmolytic effects. To evaluate their contribution to the chemotaxonomy of European taxa of the A. millefolium group, we developed a SPE-HPLC/UV method that allows quantification of the phenolic constituents in the different taxa. The investigated species displayed differences in the quantitative and qualitative composition of phenolic acids and flavonoids. Hence, they seem to be of chemotaxonomic significance, especially for the distinction of the diploid taxa. Combining the obtained results with the data of the sesquiterpene analyses gives a comprehensive insight into the distribution of those pharmacologically relevant plant constituents in the A. millefolium group.     

In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine—BA; kinetin—Kin; and thidiazuron—TDZ) and auxins (indole-3-acetic acid—IAA; α-naphthaleneacetic acid—NAA; and 2,4-dichlorophenoxy acetic acid—2,4-D). Maximum multiple shoots (12.7?±?0.6) were produced from nodal explants on MS?+?7.5 μM BA. Shoots were rooted in vitro on MS containing 15 μM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 μM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.     

Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in hairy root cultures of Psoralea corylifolia L. Untreated hairy roots (control) accumulated 1.55% dry wt of daidzein and 0.19% dry wt of genistein. In precursor feeding experiment, phenylalanine at 2 mM concentration led to 1.3 fold higher production of daidzein (1.91% dry wt) and genistein (0.27% dry wt). In biotic elicitors, chitosan (2 mg/L) was found to be the most efficient elicitor to induce daidzein (2.78% dry wt) and genistein (0.279% dry wt) levels in hairy roots. Salicylic acid at 1 mM concentration stimulated the maximum accumulation of daidzein (2.2% dry wt) and genistein (0.228% dry wt) 2 days after elicitation. In case of polyamines, putrescine (50 mM) resulted in highest accumulation of daidzein (3.01% dry wt) and genistein (0.227% dry wt) after 5 days of addition. Present results indicated the effectiveness of elicitation and precursor feeding on isoflavones accumulation in hairy roots of P. corylifolia. This is the first report of elicitation on isoflavones production by hairy roots of P. corylifolia.     

Alkaloid production by plant cell cultures of Holarrhena antidysenterica: II. Effect of precursor feeding and cultivation in stirred tank bioreactor

Precursor feeding strategy for increasing the yield of conessine, a steroidal alkaloid of Holarrhena antidysenterica, was established in cell suspension culture. A total of 50 mg/L added cholesterol was converted into 43 mg/L of alkaloid, 90% of which constituted the conessine. By applying the precursor feeding policy to the cell suspension culture in modified Murashige and Skoog (MS) medium, a total of 143 mg/L of alkaloid was produced in 8 days. In this way the alkaloid content of the cells was increased more than six times compared to that obtained in the standard MS medium. The steps leading to biotransformation of cholesterol into alkaloids were unaffected by phosphate. The shake flask data were successfully transferred to a bench scale 6-L stirred tank bioreactor in which the specific biosynthetic rate of alkaloid production was 110 mg/100 g dry cell weight per day, about 160 times higher than that of whole plant.     

Chemotaxonomic significance of flavonoids and phenolic acids in the Hieracium rohacsense group (Hieracium sect. Alpina; Lactuceae, Compositae)

Five apomictic taxa from the Hieracium rohacsense group were studied for their phenolic constituent composition. The following substances represent dominant compounds in the leaves: chlorogenic acid, 3,5-dicaffeoylquinic acid, luteolin 7-O-β- -glucopyranoside, luteolin 4′-O-β- -glucuronopyranoside and apigenin 4′-O-β- -glucuronopyranoside. Within the group only quantitative differences were found, luteolin 7-O-glucoside being the most important chemotaxonomic marker. Each taxon has its own specific quantitative pattern, invariable within the taxon. Based on these characteristic profiles, H. rohacsense can be distinguished from a closely related and still undescribed taxon from Mt. Pip Ivan. The proportion of luteolin 7-O-glucoside to apigenin 4′-O-glucuronoside also clearly separates the individuals of two morphologically close species—H. ratezaticum and H. pseudocaesium, which corresponds to a few slight but recognisable morphological and phenological characteristics. The ontogenetic stage of leaf development and seasonal variation are also important factors, which must be taken into consideration, as the quantity of the substances changes during leaf ontogeny and with season.     

Characterisation of the phenolic profile of Boerhaavia diffusa L. by HPLC-PAD-MS/MS as a tool for quality control

Phenolic acids and flavonols of nine leaf and three root samples of Boerhaavia diffusa L., collected at different locations and subjected to several drying procedures, were characterised by reversed-phase HPLC-PAD-ESI/MS for the first time. Ten phenolic compounds were identified: 3,4-dihydroxy-5-methoxycinnamoyl-rhamnoside, quercetin 3-O-rhamnosyl(1-->6)galactoside (quercetin 3-O-robinobioside), quercetin 3-O-(2"-rhamnosyl)-robinobioside, kaempferol 3-O-(2"-rhamnosyl)-robinobioside, 3,5,4'-trihydroxy-6,7-dimethoxyflavone 3-O-galactosyl(1-->2)glucoside [eupalitin 3-O-galactosyl(1-->2)glucoside], caffeoyltartaric acid, kaempferol 3-O-robinobioside, eupalitin 3-O-galactoside, quercetin and kaempferol. Quantification was achieved by HPLC-PAD and two phenolic patterns were found for the leaves, in which quercetin 3-O-robinobioside or quercetin 3-O-(2"-rhamnosyl)-robinobioside was the major compound. Caffeoyltartaric acid was only present in the root material where it represented the main phenolic constituent. The results obtained demonstrated that the geographical origin (particularly the nature of the soil), but not the drying process, influences the phenolic composition.     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Improved Taxol production in suspension cultures of Taxus chinensis var. mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor

Taxus chinensis var. mairei was grown in two-liquid-phase culture in a 2.5 l airlift external loop reactor for production of Taxol. A mixture of oleic acid and dibutyl phthalate (1:1, v/v) in a volume ratio of 8:92 to the culture medium gave higher production of Taxol (up to 12 mg l^–1) with an aeration rate of 1 vvm. By combination of the in situ extraction, precursor feeding and additional sugar introduction, Taxol production (16.7 mg l^–1) was 5 times higher than that in the case without any additives (3.4 mg l^–1) and 1.4 times of that in the case with only thein situ extraction (12 mg l^–1). Taxol was extracted from the organic solvents with water and methanol (1:1:1.4, by vol.) and lyophilized giving a total yield of ca. 90%.     

Phenolic compounds in Norway spruce as affected by boron nutrition at the end of the growing season

The increase in concentrations of phenolic compounds in boron (B) deficiency has been demonstrated in many herbaceous plant species, but information on woody plants is scarce. It has been suggested that accumulation of phenolic compounds plays a role in the development of cold hardiness in herbaceous plants but also that B deficiency decreases winter hardiness. Here we study the effects of B nutrition on phenolic compounds in Norway spruce (Picea abies L.) in the course of cold acclimation. Phenolic compounds were analysed in Norway spruce seedlings from three different B-fertilisation treatments in two harvests: non-acclimated and cold-acclimated seedlings. Norway spruce phenolic compounds consisted mainly of condensed tannins. During B deficiency, condensed tannins and monocoumaroyl–astragalin der. 1 increased in non-acclimated seedlings. The increase in tannins was 21%, which was nearly significant. However, the effect of B on phenolic compounds was almost absent in cold-acclimated seedlings. The condensed tannin concentration increased much more with time in the simulated autumn than due to B deficiency, and we conclude that the B effect was probably not large enough to be important for the hardening of the seedlings. The total phenolic concentrations more than doubled during the course of cold hardening suggesting that phenolics have a role in the winter hardiness in Norway spruce.     

Simultaneous quantification of five triterpenoid saponins in Clematis L. spp. by high-performance liquid chromatography with evaporative light scattering detection

A simple and accurate method involving high-performance liquid chromatography with evaporative light scattering detection was developed for the simultaneous determination of five triterpenoid saponin components in Clematis L. spp. for the first time. The analysis was performed on a Zorbax SB-C(18) column and gradient elution with acetonitrile and water with 0.1% formic acid was utilised. All the calibration curves exhibited good linear characteristics with correlation coefficients in the range from 0.9979 to 0.9997. The limits of detection and limits of quantification were less than 0.152 and 0.506 microg, respectively. The overall recoveries for the five analytes were between 91.3 and 99.5%. A total of 10 samples from Clematis L. spp. were analysed under optimised conditions and the chemical profiles provided information for the identification of botanical origin, the development of new medicinal resources and chemotaxonomic investigation.     

Immunohistological localization of serotonin in the CNS and feeding system of the stable fly Stomoxys calcitrans L. (Diptera: Muscidae)

Serotonin, or 5-hydroxytryptamine (5-HT), plays critical roles as a neurotransmitter and neuromodulator that control or modulate many behaviors in insects, such as feeding. Neurons immunoreactive (IR) to 5-HT were detected in the central nervous system (CNS) of the larval and adult stages of the stable fly, Stomoxys calcitrans, using an immunohistological technique. The location and pattern of the 5-HT IR neurons are described and compared for these two different developmental stages. Anatomical features of the fly feeding system were analyzed in third instar larvae and adult flies using a combination of histological and immunohistological techniques. In third instar larvae, the cibarial dilator muscles were observed within the cibarial pump skeleton and innervated by 5-HT IR neurons in nerves arising from the brain. There were four pairs of nerves arising from the frontal surface of the larval brain that innervate the cibarial pump muscles, pharynx, and muscles controlling the mouth hooks. A strong serotoninergic innervation of the anterior stomatogastric system was observed, which suggests 5-HT may play a role in the coordination of different phases of food ingestion by larvae. Similarly, many 5-HT IR neurons were found in both the brain and the thoracico-abdominal ganglia in the adult, some of which innervate the cibarial pump dilator muscles and the stomatogastric muscles. This is tnhe first report describing neuromuscular structures of the stable fly feeding system. The results reported here suggest 5-HT may play a critical role in feeding behaviors of stable fly larvae and adults.     

Enhanced ethylene production by primary roots of Zea mays L. in response to sub-ambient partial pressures of oxygen

Ethylene production by primary roots of 72–h-old intact seedlings of Zea mays L. cv. LG11 was studied under ambient and sub-ambient oxygen partial pressures (pO₂) using a gas flow-through system linked to a photoacoustic laser detector. Despite precautions to minimize physical perturbation to seedlings while setting-up, ethylene production in air was faster during the first 6h than later, in association with a small temporary swelling of the roots. When roots were switched from air (20–8kPa O₂) to 3 or 5kPa O₂ after 6h, ethylene production increased within 2—3 h. When, the roots were returned to air 16 h later, ethylene production decreased within 2—3 h. The presence of 10kPa CO₂ did not interfere with the effect of 3kPa O₂. Transferring roots from air to 12–5kPa did not change ethylene production, while a reduction to 1 kPa O₂ induced a small increase. The extra ethylene formed in 3 and 5 kPa O₂ was associated with plagiotropism, swelling, root hair production, and after 72 h, increased amounts of intercellular space (aerenchyma) in the root cortex. Root extension was also slowed down, but the pattern of response to oxygen shortage did not always match that of ethylene production. On return to air, subsequent growth patterns became normal within a few hours. In the complete absence of oxygen, no ethylene production was detected, even when anaerobic roots were returned to air after 16 h.     

Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MS(n). In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol. Furthermore, the experimental results demonstrate that the stability of DDA depends on pH values of the buffer. Aconine as hydrolysate has been only found in pH 10.0 buffer, and the other hydrolysates and the pyrolyzates of DDA, such as benzoylaconine and deacetoxy aconitine, have been observed in all pH aqueous solutions. The decomposition pathways of DDA in buffers are related to the substituent on the C-3 position. The decomposition pathway of aconitine is similar to that of mesaconitine, but different from that of hypaconitine.     

Down-regulation of prostate specific antigen in LNCaP cells by flavonoids from the pollen of Brassica napus L.

The pollen of Brassica napus L. has been used in China to treat benign prostatic hyperplasia (BPH) for over decades. In this study, the pollen of Brassica napus L. was extracted successively with chloroform, ethyl acetate and ethanol. The ethyl acetate extract showed strong activity in decreasing the secretion of prostate specific antigen (PSA) in LNCaP cells as compared to two other extracts, measured by ELISA with finasteride as positive control in the assay. Five flavonoids were subsequently isolated from the active extract using bioassay-guided fractionation. They were Naringenin (1); Luteolin (2); Kaempferol (3); Kaempferol 3-(3-E-p-coumaroyl-alpha-L-rhamnopyranoside) (4); and Kaempferol 3-(2,3-di-E-p-coumaroyl-alpha-L-rhamnopyranoside) (5). All these compounds inhibited PSA secretion significantly, with IC50 values in the range of 5-50 microM. Compounds 2, 4 and 5 showed moderate cytotoxicity to LNCaP cells within the active concentration range, while compounds 1 and 3 showed no cytotoxicity. Further studies on the mechanism action of these compounds were performed by evaluating their activation of estrogen receptor (ER) and antagonistic activities on androgen receptor (AR) in cell-based reporter gene assays. All compounds described here were first isolated from the pollen of Brassica napus L.     

Simultaneous determination of three naphthoquinones in the leaves of Impatiens balsamina L. by reversed-phase high-performance liquid chromatography

Introduction – Naphthoquinones; lawsone ( 1 ), lawsone methyl ether ( 2 ) and methylene‐3,3′‐bilawsone ( 3 ) are the main active compounds of Impatiens balsamina leaves. Objective – To develop and validate an HPLC method for simultaneous quantitative determination of 1 – 3 in I. balsamina leaf extracts. Methodology – The method utilised a Supelco® C₁₈ column (5 µm, 4.6 × 150 mm) at 25°C with the mixture of 2% aqueous acetic acid : methanol (gradient elution as follows: 0–10 min, 25 : 75; 10–20 min, 32 : 68; 20–35 min, 55 : 45) as the mobile phase at a flow‐rate of 1 mL/min, and UV detection at 280 nm. The parameters of linearity, repeatability, reproducibility, accuracy specificity and sensitivity of the method were evaluated. Results – The recovery of the method was 96–101% and linearity (r² ≥ 0.9995) was obtained for all naphthoquinones. A high degree of specificity, as well as repeatability and reproducibility (RSD less than 5%), were also achieved. Copyright © 2010 John Wiley & Sons, Ltd.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var. amara

Reverse-phase HPLC coupled with photodiode array detection was used for the simultaneous separation and determination of naturally occurring adrenergic amines (octopamine, synephrine and tyramine) in fruits and dry extracts of Citrus aurantium L. var. amara and in herbal medicines derived therefrom. Synephrine was the main component in fruits (0.10-0.35%) and in dry extracts (3.00-3.08%) and was present in the range 0.25-0.99% in herbal medicines. Flavanones were analysed in the same samples using a reverse-phase HPLC technique which allowed the identification and quantification of neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, naringenin and hesperetin. C. aurantium fruits and derivatives contained mainly glycosylated flavanones: in particular, naringin and neohesperidin were found to be the major flavonoids and their concentrations ranged from 1.80 to 26.30 and from 3.90 to 14.71 mg/g, respectively. The levels of aglycones were very low in all samples tested.