Alkaloid distribution and catabolism in Cephalotaxus harringtonia

Cephalotaxus harringtonia produces a variety of antitumor alkaloids that are distributed throughout the tree. In a young plant grown in a controlled environment, the concentration of free alkaloids (homoerythrina alkaloids and cephalotaxine) did not increase with age, whereas the concentration of cephalotaxine esters (harringtonine, deoxyharringtonine, isoharringtonine and homoharringtonine) increased roughly 5-fold. Total alkaloid concentrations increased in the older leaves of the plant and decreased in the older stems. Physiological stress (pruning) causes hydrolysis of part of the stored alkaloid esters to free cephalotaxine within one week. In 4 out of 5 field-grown trees environmental factors caused complete ester hydrolysis and, in addition, the oxidation of cephalotaxine to 11-hydroxycephalotaxine and drupacine and of homoerythrina alkaloid to its epoxy derivative. This shows that the alkaloids in this perennial tree are not inert storage products, but are under metabolic control.     

Anticarcinogenic Alkaloids in the Cultured Cells of Cephalotaxus fortunei

Calli were induced from the leaves and stems of Cephalotaxus fortunei Hook. f. on MS medium supplemented with 0. 1 mg/L KT and 3 mg/L NAA, and from which the suspension culture cell line of this plant was established for the first time. Factors such as light, pH value of the medium, concentration of plant hormone, carbon resources and addition of substances to the medium, which affect the growth of suspension cells were investigated. The results showed that suspension cells grew appropriately at pH 5.8 with a low concentration of sucrose or glucose, and a low level of NAA. No difference effect on cell growth was seen between sucrose and glucose. Phenylalanine and protein hydrolysate were not suitable for cell growth in suspension cultures, and light inhibited cell growth. A sensitive and rapid high-performance liquid chromatographic method has been developed for detecting the alkaloids in cultured cells. The results revealed the following contents of cephalotaxine and its anticancer esters in cultured cells: harringtonine, isoharringtonine and homoharringtonine. The total alkaloid production in cell suspension cultures was doubled as that in solid cultures. The relative amounts of cephalotaxine, drupacine, harringtonine, homoharringtonine and isoharringtonine in suspension cells was 22%, 6%, 8%, 23% and 41% respectively. In addition, other alkaloid as deoxyharringtonine and some steroids, including ergdst-5-en-3-ol. stigmasta-5, 22-dien-3-ol, β-sitosterin and 2-naphthalenamine have also been detected in cell cultures using GC/MS combined technique.     

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.Key words: In vitro propagation, Pyridone alkaloid, Cerpegin, Callus, Ceropegia juncea     

Modified alkaloid pattern in developing tobacco callus

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.     

Enhanced Alkaloid Production in Callus Cultures of Heliotropium indicum L and Regeneration of Plantlets

Heliotropium indicum L (Boraginaceae) contains the anticancer pyrrolizidine alkaloid, indicine-N-oxide (INO). To study the yield of INO as a function of plant development, plantlets were regenerated in vitro from nodal and hypocotyl explants and also from hypocotyl callus, on Murashige and Skoog’s (MS) medium supplemented with 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), asparagine (Asp) and glutamine (Glu). The regenerated plantlets were rooted on MS supplemented with Glu or gibberellic acid (GA₃). While 5-week-old seedlings showed a high amount of INO (0.12% dry wt) that depleted gradually as the plants attained maturity and flowered, fast growing callus produced a much higher yield of INO (0.32% dry wt). As the callus cultures differentiated into shoots and subsequently into plantlets, the INO content decreased to about 0.2% dry wt. It appears that INO is the primary product of pyrrolizidine alkaloid biosynthetic pathway in rapidly growing meristematic tissue of Heliotropium indicum, later reduced to its alkaloidal base and reallocated to other tissues. The article includes an efficient micropropagation method for Heliotropium indicum.     

The effect of Cephalotaxus group alkaloids on elongation of the polypeptide chain in human ribosomes

Effects of Cephalotaxus alkaloids (homoharringtonine and cephalotaxine) on the translation of endogenous mRNA in a cell-free system of rabbit reticulocyte lysate and on poly(U)-directed poly(Phe) synthesis on human placenta ribosomes was studied. The effect of the alkaloids on the activity of human placenta ribosomes in a template-dependent aminoacyl-tRNA binding, N-acetyl-phenylalanyl-puromycin and diphenylalanine formation was also studied. Homoharringtonine was shown to have little effect of codon-dependent Phe-tRNA(Phe) binding but the alkaloid strongly inhibited (Phe)2 formation as well as N-Ac-Phe-puromycin synthesis from the complex N-Ac-Phe-tRNA(Phe).poly(U).80S ribosomes. It was concluded that the site of homoharringtonine binding overlaps or coincides with the acceptor site of the ribosomal peptidyltransferase center. The association constant of homoharringtonine to the ribosomes was estimated to be (4.8 +/- 1.0) x 10(7) M-1. Cephalotaxine had no effect on the elongation steps.     

Biscoclaurine alkaloids in callus tissues of Stephania cepharantha

Although callus tissues derived from tubers of Stephania cepharantha cannot synthesize the main alkaloids of the original plant, cepharanthine and isotetrandrine, they are able to synthesize biscoclaurine alkaloids, berbamine and aromorine, the latter not being found in the original plant. These results suggest that enzymes controlling specific methylation and methylenedioxy group formation are absent from the callus. The maximum content of total alkaloid in the callus tissues subcultured for 9 months was more than 3 times that of original plant. Alkaloid content was affected by addition of various auxins, IAA being most effective.     

Stepharine production in morphogenic cell cultures of <Emphasis Type="Italic">Stephania glabra</Emphasis> (ROXB.) Miers

Young leaves of Stephania glabra (ROXB.) Miers. (Menispermaceae) were used to establish callus cultures with the goal of studying their ability to produce the valuable alkaloid stepharine. The establishment of callus cultures from this plant is extremely difficult because primary calli are not viable and must be transferred to a liquid culture medium for subsequent subculturing. All obtained cultures possessed high morphogenic or rhizogenic potential. The methanolic extracts of two examined cell lines were analyzed by HPLC with high-resolution mass spectrometry to determine the alkaloid composition. Eleven alkaloids were detected in both cultures, including stepharine, magnoflorine, menisperine, roemerine, palmatine, corydalmine, N-methylcorydalmine, columbamine, tetrahydropalmatine, jatrorrhizine and tetrandrine. The predominant alkaloid was stepharine, the structure of which was identified through ¹H and ¹³C NMR, UV, HRMS and tandem MS. Quantitative HPLC-UV analysis showed that stepharine was produced at high levels (0.9?% dry cell weight) in the morphogenic callus culture S2-L cultivated in liquid media. To our knowledge, this is the highest level of stepharine production identified in plant cell cultures. The overall production parameters of the culture have remained stable over a long-term cultivation period for 3 years.     

In vitro organogenesis and alkaloid accumulation in Datura innoxia

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.     

Trends in accumulation of ephedrine in callus cultures of <Emphasis Type="Italic">Ephedra major</Emphasis> Host

Ephedra major Host, a medicinal plant, belongs to the family of Ephedraceae. Ephedrine is the main alkaloid in Ephedra, which has different medicinal properties. However, the amount of ephedrine in plant material is low and callus culture can be a way to increase the alkaloid content. The aim of this research was to compare Murashige and Skoog (MS) and Gamborg’s B5 culture media for callus induction and ephedrine production. For this purpose, stem explants were cultured on MS or B5 media containing 0.0, 0.5, 1.0, 2.0, or 3.0 mg L^?1 of kinetin (Kin) either alone or in combination with 0.0, 0.5, 1.0, or 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and/or naphthalenacetic acid (NAA), in five replicates. MS medium containing 1.0 or 2.0 NAA and 0.5 mg L^?1 Kin were the most effective for callus induction. The highest percentage of callus induction (100%) on B5 culture medium was obtained with 2.0 2,4-D and 0.5 mg L^?1 Kin treatments. The results showed that there was no significant difference between MS and B5 media for callus induction, and fresh and dry weight production. High-performance liquid chromatography was conducted for the identification and quantification of ephedrine in the obtained callus. The highest level of ephedrine (7.38 mg g^?1 DW) was found in callus grown on MS medium containing 0.5 mg L^?1 of 2,4-D. The results revealed that ephedrine can accumulate in callus cultures to levels much higher than in E. major wild plants.     

Growth and alkaloid production of callus culture induced from Securinega suffruticosa

The growth of, and production of alkaloids by, callus derived from budding stem explants of the germinated seeds of Securinega suffruticosa has been studied. The major alkaloids produced were securinine and allosecurinine with the latter being present in the greatest amount. The effects of pH, growth hormones, sucrose concentration and light and dark on callus growth and alkaloid production have been examined in detail. The pattern of alkaloid production in the callus culture appeared to be similar to that in the root of the securinega plant.     

Regeneration of Centella asiatica plants from non-embryogenic cell lines and evaluation of antibacterial and antifungal properties of regenerated calli and plants

Background

The threatened plant Centella asiatica L. is traditionallyused for a number of remedies. In vitro plant propagation and enhanced metabolite production of active metabolites through biotechnological approaches has gained attention in recent years.

Results

Present study reveals that 6-benzyladenine (BA) either alone or in combination with 1-naphthalene acetic acid (NAA) supplemented in Murashige and Skoog (MS) medium at different concentrations produced good quality callus from leaf explants of C. asiatica. The calli produced on different plant growth regulators at different concentrations were mostly embryogenic and green. Highest shoot regeneration efficiency; 10 shoots per callus explant, from non-embryogenic callus was observed on 4.42 μM BA with 5.37 μM NAA. Best rooting response was observed at 5.37 and 10.74 μM NAA with 20 average number of roots per explant. Calli and regenerated plants extracts inhibited bacterial growth with mean zone of inhibition 9-13 mm diameter when tested against six bacterial strains using agar well diffusion method. Agar tube dilution method for antifungal assay showed 3.2-76% growth inhibition of Mucor species, Aspergillus fumigatus and Fusarium moliniformes.

Conclusions

The present investigation reveals that non-embryogenic callus can be turned into embryos and plantlets if cultured on appropriate medium. Furthermore, callus from leaf explant of C. asiatica can be a good source for production of antimicrobial compounds through bioreactor.     

In vitro plantlet regeneration and assessment of alkaloid contents from callus cultures of Ephedra foliata (Unth phog), a source of anti-asthmatic drugs

Ephedra foliata, (Gymnosperm) is a pharmaceutically important plant known for the last 5,000 years and has a number of medicinal properties. We describe here for the first time, a method for plant regeneration from callus established from axillary buds as explant, with the aim of optimizing alkaloids production in vitro. The tissue cultures initiated are being maintained for the last 3 years on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 0.5 mg l^?1 each of 2, 4-D and Kin. Maintained callus cultures exhibited regeneration potential and maximum number (23.5 ± 0.44 shoots per culture vessel) of shoots with an average height (4.94 ± 0.23 cm) was achieved on MS medium containing combination of 0.25 mg l^?1 each of Kin, BA and 0.1 mg l^?1 of NAA. About 84.9 % regenerated shoots were rooted under ex vitro conditions on Soilrite^®, if their base was treated with 500 mg l^?1 of IBA for 5 min. The rooted plantlets were successfully acclimatized under greenhouse conditions with ≈80 % survival rate. We analyzed alkaloid contents of tissue culture raised plants/callus as affected by the different concentrations and combination of two additives, i.e., l-phenylalanine and IBA. The alkaloid production was higher in the in vitro grown cultures than field-grown plants. Highest alkaloid content was recorded in callus culture on M₅ medium having 0.5 mg l^?1 each of 2, 4-D and Kin, 100 mg l^?1 l-phenylalanine and 5 mg l^?1 IBA. The present protocol may be applicable for the large-scale cultivation of E. foliata and selection of cell line having higher secondary metabolite contents of this pharmaceutically important threatened plant species.     

Micropropagation of Corydalis ambigua through embryogenesis of tuber sections and chemical evaluation of the ramets

Corydalis ambigua, a perennial herb of the family Papaveraceae, was micropropagated through somatic embryogenesis starting from sliced tubers. Somatic embryos were proliferated on Linsmaier and Skoog medium of a half strength containing 2% sucrose and 0.1 M indole-3-acetic acid or indole-3-butyric acid solidified with 0.2% Gelrite. Somatic embryos were germinated and grew on plant growth regulator-free White's medium supplemented with 2% sucrose and 0.8% agar in the dark at 0–4°C for 6 months to give rise to microtubers that could be potted out of culture tubes. Three strains of micropropagated plants cultivated outdoors for 5 years gave different tetrahydroprotoberberine-type alkaloids pattern, respectively. The variation of tetrahydroprotoberberine-type alkaloid (corybulbine, corydaline and cavidine) content within a strain was not significantly different from that of the corresponding alkaloid in the wild plants.     

Enhanced indole alkaloid production in suspension compact callus clusters of Catharanthus roseus: impacts of plant growth regulators and sucrose

A special culture system, compact callus clusters, was developed from Catharanthus roseus stem explants in a modified Murashige and Skoog (MS) liquid medium containing 5.37 µM -naphthaleneacetic acid and 4.65 µM kinetin. Morphological and anatomical studies showed that the globular compact callus cluster cultures consisted of many cohesive callus aggregates displaying some level of cellular/tissue differentiation, which was also in agreement with the results from peroxidase and esterase isoenzyme pattern analysis. The compact callus cluster cultures could synthesise about 2-fold more indole alkaloids than the dispersed cell cultures, and this was postulated to be associated with their differential status. Plant growth regulators and sucrose concentration, as well as shaking speed significantly affected properties of the compact callus clusters. In detail, 2,4-dichlorophenoxyacetic acid destroyed the compact structure and reduced alkaloid production of the compact callus cluster cultures; but a high concentration of cytokinins was necessary to maintain the compact structure and high alkaloid production of the special cultures. The optimum sucrose (5–6%) gave the greatest alkaloid and biomass production, as well as the greatest degree of compaction of the compact callus clusters.     

Tropane Alkaloids from Callus Cultures, Differentiated and Shoots of Hyoscyamus muticus L.

Alkaloid production has been observed in cotyledonary leaf derived callus tissues, and also in in vitro differentiated shoots, and roots of Hyoscyamus muticus. The callus tissue was developed form cotyledonary leaf explants on Murashige and Skoog medium enriched with 2 mg 1^-1 2, 4-D and 0.5 mg 1^-1 BAP. Cotyledonary leaf derived callus was proliferated in the same medium for 2 passages (1 passage 28-30 days). Green and compact callus was used for alkaloid extraction. Shoots and roots formed on MS medium containing 0.05 mg 1^-1 NAA and 0.5 mg 1^-1 BAP, and also compact, nodular and embryogenic calli from which these shoots and roots differentiated, were used for alkaloid extraction. Chromatographic studies performed with TLC showed the presence of hyoscyamine as the major alkaloid present in the callus tissues, differentiated shoots and roots. However, alkaloid content varied in different tissues. Differentiated roots were found to contain maximum amount of hyoscyamine.     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.     

Enhanced cephalotaxine production in Cephalotaxus mannii suspension cultures by combining glycometabolic regulation and elicitation

To study the combination effects of glycometabolic regulator NaF and elicitor methyl jasmonate (MJ) on cephalotaxine production in Cephalotaxus mannii suspension cultures, NaF of 10 mg/L, MJ of 100 μmol/L or both of them (NaF + MJ for short below) were added to the shake-flask cultures of C. mannii cell. It was found that NaF increased the activity of glucose 6-phosphate dehydrogenase (G6PDH), but had no significant effects on phenylalanine ammonium-lyase (PAL) activity and phenols formation. In contrast, MJ could activate PAL activity and led to phenols accumulation, but had no significant effects on G6PDH activity. To explore the effects of NaF and MJ on cephalotaxine biosynthesis, harringtonine and homoharringtonine, the two cephalotaxines, were analyzed in this work. The results obtained indicated that NaF + MJ treatment showed the strongest promotion of production in all tests. Harringtonine yield in NaF + MJ treated cells (7.245 mg/L) was 4.8-fold higher than that in control cells (1.506 mg/L), 1.7-fold that in NaF-treated cells (4.12 mg/L) and 1.6-fold that in MJ-treated cells (4.458 mg/L), respectively. No homoharringtonine was found besides in NaF + MJ treated cells (0.491 mg/L). With respect of the product release rates, they were 0%, 78%, 24% and 62% in control, NaF, MJ and NaF + MJ treatment, respectively. These results suggest that the combination of NaF and MJ had contributed to the synthesis and secretion of cephalotaxine in C. mannii cells.     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Indirect Organogenesis from the Leaf Explants of Medicinally Important Plant Curculigo orchioides Gaertn

Curculigo orchioides Gaertn is an important medicinal plant of commercial value. A protocol for indirect regeneration from the leaf explants was developed. The leaf explants on MS (Murashige & Skoog) basal medium fortified with 6-benzylaminopurine (9μM) gave rise to proliferating green callus. The calli regenerated shoots on subculturing in the MS medium with 1.1μM 6-benzylaminopurine which was the optimum concentration for multiplication. Rooting was observed in MS medium supplemented with α-napthaleneacetic acid (5.3μM), indole-3-butyric acid (1.2μM), and on MS basal medium free of plant growth regulators. The rooted plants were hardened and transferred to field with 80% survival.