Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.     

The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57 μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8 μM) or TDZ (thidiazuron) (0.2, 0.5, 1 μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1 μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35 mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55 mg/g dry wt). Verbascoside accumulation was also stimulated by 1 μM TDZ; its level was about six times higher than that found on the control medium (6.2 mg/g dry wt vs. 1.03 mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8 μM BAP.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

l-NAME prevents GM1 ganglioside-induced vasodilation in the rat brain

Monosialoganglioside (GM1) is a glycosphingolipid present in most cell membranes that displays antioxidant and neuroprotective properties. It has been recently described that GM1 induces vasodilation. However, the mechanisms underlying GM1-induced vasodilation were not evaluated to date. Therefore, in this study we investigated whether the nonspecific NOS inhibitor l-NAME prevents GM1-induced vasodilation in rats. The systemic injection of GM1 (50 mg/kg, i.p.) increased the outer diameter of pial vessels by 50% in anesthetized animals at 30 min, and this effect was fully prevented by the administration of the nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME, 60 mg/kg, i.p. 15 min before GM1 injection).A 30 min exposure of cerebral cortex slices to GM1 (100 μM) increased the content of nitrite plus nitrate (NOx) by 50%. Addition of l-NAME (100 μM) to the incubation medium fully prevented GM1-induced NOx increase. Conversely, a 60 min exposure of slices to GM1 (100 μM) decreased NOx content, revealing a biphasic effect of GM1. Our results suggest that NO plays an important role in the vasodilation induced by GM1.     

In vitro study on the effect of cytokines and auxins addition to growth medium on the micropropagation and rooting of Paulownia species (Paulownia hybrid and Paulownia tomentosa)

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.     

Indirect organogenesis from various explants of Hildegardia populifolia (Roxb.) Schott & Endl. – A threatened tree species from Eastern Ghats of Tamil Nadu,India

Hildegardia species are an important resource for fiber industry. This investigation was conducted to develop a plant regeneration protocol for Hildegardia populifolia (Roxb.) Schott & Endl. via indirect organogenesis Callus was obtained from leaf, internode and petiole explants, among these explants internode explant gave best result on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). The highest percentage (100%) of regeneration was obtained with benzyladenine (BA) (2.0 mg/l) + indole-3-acetic acid (IAA) (0.1 mg/l) + glutamine (25 mg/l) + thidiazuron (TDZ) (0.5 mg/l) from internode explants. Shootlets were highly rooted on MS medium supplemented with 3.0 mg/l indole-3-butyric acid (IBA). In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facilitate further development of reliable procedures for this genus.     

Responses of non-protein thiols to Cd exposure in Cd hyperaccumulator Arabis paniculata Franch

We investigated the responses of phytochelatins (PCs), glutathione (GSH) and other non-protein thiols in Cd hyperaccumulator Arabis paniculata after Cd exposure. Applying γ-glutamylcysteine synthetase (γ-ECS) inhibitor, l-buthionine-sulfoximine (BSO), the roles of PCs in Cd tolerance and Cd accumulation in A. paniculata were evaluated. Plants were exposed to four Cd concentrations (0, 50, 100 and 250 μM) for different times (2w or 3w) with and without BSO. Overall, Cd exposure had little impact on plant biomass after 2w or 3w of growth except at the highest Cd level. A. paniculata tolerated ≤100 μM Cd with up to 1127 mg kg^?1 Cd in the shoots and 5624 mg kg^?1 Cd in the roots after 3w of Cd exposure. Cd exposure induced formation of PCs and three unknown thiols in the roots, but none were detected in the shoots. BSO had no significant effect on Cd sensitivity in plants though it reduced Cd accumulation in the roots. In addition, the molar ratio of PCs:Cd, which ranged from 0.7 to 1.3 after exposing to 50–100 μM Cd without BSO in the roots, was close to the value expected for PC-mediated Cd sequestration in plants. Those data indicate that GSH and PCs did not contribute to Cd tolerance in the shoots and Cd transport from the root to shoot in A. paniculata, but they may play an important role in Cd accumulation and Cd complexation in the roots of A. paniculata.     

In vitro propagation and secondary product production by Merwilla plumbea (Lindl.) Speta

Merwilla plumbea is a popular but threatened traditional medicinal plant sold at herbal markets in South Africa. It produces bioactive compounds and contributes to Traditional African Medical systems for the treatment of various diseases. Due to high demand, the plant is recommended for commercial cultivation. We investigated in vitro propagation and secondary compound production in in vitro cultures of M. plumbea, cultivated on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with various plant growth regulators [PGRs: adenine sulphate (Ads), benzyladenine (BA), 2-isopentenyladenine (2iP), meta-topolin (mT), meta-topolin riboside (mTR), thidiazuron (TDZ), kinetin (Kin) and naphthaleneacetic acid (NAA)] and organic elicitors [casein hydrolysate (CH), glutamine (GM), haemoglobin (HB), mebendazole (MBZ), trimethoprim (TMP), yeast extract (YE) and yeast malt broth (YMB)]. A combination of 20?μM GM, 0.45?μM TDZ and 0.054?μM NAA produced the highest number of adventitious shoots per explant (30.6 shoots/explant) after 12?weeks of culture. The regenerated shoots were rooted and the plantlets successfully acclimatized. The total phenolic, flavonoid, gallotannin and condensed tannin contents were determined. The highest levels of flavonoids (50.97?μg CTE/g in shoots) and gallotannins (99.55?μg GAE/g in shoots) were recorded in combinations of 200?mg?l^?1 YE, 0.45?μM TDZ and 0.054?μM NAA and 100?mg?l^?1 YMB, 0.45?μM TDZ and 0.054?μM NAA. These increases were around 3- to 16-fold higher than in naturally-grown plants. This system offers the possibility to use in vitro culture techniques for mass propagation, secondary metabolite and pharmacological studies.     

Production of rhoifolin and tiliroside from callus cultures of Chorisia chodatii and Chorisia speciosa

The current work aims to stimulate the production of rhoifolin and tiliroside as two valuable phytochemicals from Chorisia chodatii Hassl. and Chorisia speciosa A. St.-Hil. callus cultures. A comparison between three explants from the in vitro germinated seedlings of both species for callus induction and accumulation of both flavonoids was carried out. Highly efficient calluses were induced from the leaves, stems and roots of C. chodatii seedlings on Gamborg’s B5 (B5) and Murashige and Skoog (MS) media containing 2.0 mg/l β-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenin (BA) or kinetin (Kn), while those of C. speciosa seedlings efficiently produced calluses on both media supplemented with 0.5 or 1.0 mg/l NAA and 0.5 mg/l BA. Besides, the highest contents of rhoifolin (1.927 mg/g DW) and tiliroside (1.776 mg/g DW) from C. speciosa cultures were obtained from the calluses of seedlings’ roots and stems maintained on B5 medium containing 1.0 mg/l NAA and 0.5 mg/l BA, respectively. On the other hand, the maximum rhoifolin content (0.555 mg/g DW) from C. chodatii cultures was obtained from the calluses of seedlings’ stems grown on B5 medium supplemented with 2.0 mg/l NAA and 0.5 mg/l BA, whereas the highest tiliroside content (0.547 mg/g DW) was provided by the root explants on B5 medium containing 2.0 mg/l NAA and 0.5 mg/l Kn. Both flavonoids were bioaccumulated in greater amounts than the wild and cultivated intact plants, which provides a promising tool for their future commercial production under a controlled environment, independent of climate and soil conditions.     

Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn.—An endangered plant

Leptadenia reticulata (Retz.) Wight. & Arn. is an important medicinal plant, belongs to the family Asclepiadaceae. This plant is known for its medicinal uses since 4500 BC. Presently this is an endangered species (Arya et al., 2003). Six shoots (2–4 cm long) per node differentiated on MS medium + 5.0 mg/l of BAP + additives. Incorporation of additives in the culture medium promoted growth of cultures. The shoots differentiated per explant were repeatedly transferred on to fresh MS + 1.0 mg/l of BAP + 0.1 mg/l of NAA and additives. The regenerated shoots were subcultured for further multiplication on MS + 1.0 mg/l BAP + 0.5 mg/l Kin + 2-iP (0.5 mg/l) and 0.1 mg/l of NAA + additives regularly after an interval of 3 weeks. Addition of ammonium sulphate in the medium resulted in increase in shoot number and promoted elongation also growth of cultures was sustained even if subculturing was delayed (26 ± 2 days). Success was also achieved in defining protocol for in vitro regeneration of shoots from petiole derived callus. Shoots regenerated in vitro by both processes were rooted in vitro on 1/4 strength of MS medium + 3.0 mg/l of IBA after 15–20 days. Cent percent of the shoots rooted ex vitro, if the in vitro regenerated shoots were treated with 200 mg/l of IBA. The in vitro–ex vitro rooted plantlets were hardened under different regimes of temperature and humidity in a greenhouse. The hardened plantlets were transferred to soil in polybags. More than 95% plants survived in field conditions. Total dry biomass harvested per year was 2800 kg/acre.     

Lead,zinc, cadmium hyperaccumulation and growth stimulation in Arabis paniculata Franch

Metal hyperaccumulation is of great interest in recent years because of its potential application for phytoremediation of heavy metal contaminated soils. In this study, a field survey and a hydroponic experiment were conducted to study the accumulation characteristics of lead (Pb), zinc (Zn) and cadmium (Cd) in Arabis paniculata Franch., which was found in Yunnan Province, China. The field survey showed that the wild population of A. paniculata was hyper-tolerant to extremely high concentrations of Pb, Zn and Cd, and could accumulate in shoots an average level of 2300 mg kg^?1 dry weight (DW) Pb, 20,800 mg kg^?1 Zn and 434 mg kg^?1 Cd, with their translocation factors (TFs) all above one. Under the hydroponic culture, stimulatory effects of Pb, Zn and Cd on shoot dry biomass were noted from 24 to 193 μM Pb, 9 to 178 μM Cd and all Zn supply levels in nutrient solution, while the effects were not obvious in the roots. Chlorophyll concentrations in Pb, Zn and Cd treatments showed an inverted U-shaped pattern, consistent with the change of plant biomass. Pb, Zn and Cd concentrations in the shoots and roots increased sharply with increasing Pb, Zn and Cd supply levels. They reached > 1000 mg kg^?1 Pb, 10,000 mg kg^?1 Zn and 100 mg kg^?1 Cd DW in the 24 μM Pb, 1223 μM Zn and 9 μM Cd treatment, respectively, in which the plants grew healthy and did not show any symptoms of phytotoxicity. The TFs of Zn were basically higher than one and the amount of Zn taken by shoots ranged from 78.7 to 90.4% of the total Zn. However, the TFs of Pb and Cd were well below one, and 55.0–67.5% of total Pb and 57.8–83.5% of total Cd was accumulated in the shoots. These results indicate that A. paniculata has a strong ability to tolerate and hyperaccumulate Pb, Zn and Cd. Meanwhile, suitable levels of Pb, Zn and Cd could stimulate the biomass production and chlorophyll concentrations of A. paniculata. Thus, it provides a new plant material for understanding the mechanisms of stimulatory effect and co-hyperaccumulation of multiple heavy metals.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

Studies on the accumulation of phenolic acids and flavonoids in different in vitro culture systems of Schisandra chinensis (Turcz.) Baill. using a DAD-HPLC method

Different in vitro culture systems of the East-Asian origin medicinal plant species − Schisandra chinensis, were tested in order to investigate their potential for the accumulation of two groups of phenolic compounds. In vitro cultures were maintained on the Murashige and Skoog (MS) medium supplemented with 3 mg/l BA and 1 mg/l NAA in an agar system (30- and 60-day growth cycles), and also in two different liquid systems: stationary and agitated. Stationary liquid cultures were grown in batch (30- and 60-day growth cycles) and fed-batch modes. Of the twenty compounds, seven free phenolic acids and of the eleven compounds, five flavonoids were quantified in methanolic extracts from lyophilized biomass and in the growth media using the RP-HPLC-DAD method. For comparison purposes, phytochemical analyses of leaf and fruit extracts from the parent plant were also conducted. The estimated compounds were not detected in the growth media. The highest total amounts of phenolic acids (71.48 mg/100 g DW) and flavonoids (29.36 mg/100 g DW) were found in extracts from the biomass of agar cultures harvested after 30 days of cultivation. The main metabolites in all the tested systems were: protocatechuic acid (max. 35.69 mg/100 g DW), chlorogenic acid (max. 13.05 mg/100 g DW), and quercitrin (max. 27.43 mg/100 g DW).     

Identification of novel acetylcholinesterase inhibitors: Indolopyrazoline derivatives and molecular docking studies

The synthesis of novel indolopyrazoline derivatives (P1-P4 and Q1-Q4) has been characterized and evaluated as potential anti-Alzheimer agents through in vitro Acetylcholinesterase (AChE) inhibition and radical scavenging activity (antioxidant) studies. Specifically, Q3 shows AChE inhibition (IC₅₀: 0.68 ± 0.13 μM) with strong DPPH and ABTS radical scavenging activity (IC₅₀: 13.77 ± 0.25 μM and IC₅₀: 12.59 ± 0.21 μM), respectively. While P3 exhibited as the second most potent compound with AChE inhibition (IC₅₀: 0.74 ± 0.09 μM) and with DPPH and ABTS radical scavenging activity (IC₅₀: 13.52 ± 0.62 μM and IC₅₀: 13.13 ± 0.85 μM), respectively. Finally, molecular docking studies provided prospective evidence to identify key interactions between the active inhibitors and the AChE that furthermore led us to the identification of plausible binding mode of novel indolopyrazoline derivatives. Additionally, in-silico ADME prediction using QikProp shows that these derivatives fulfilled all the properties of CNS acting drugs. This study confirms the first time reporting of indolopyrazoline derivatives as potential anti-Alzheimer agents.     

High seed Mn content does not affect germination of in vitro produced Centaurium pulchellum seeds

The effect of high Mn²⁺ content on Centaurium pulchellum seed germination has been investigated. Seeds containing extremely high Mn²⁺ content were produced by culturing single-node flowering explants for 2 months in the MS-media, supplemented with Mn in concentrations ranging from 1 to 10,000 μM. Although the seeds displayed the capacity to accumulate high amount of Mn, their germination was undisturbed. EPR spectroscopy was used to measure the ratio of free (aqueous) Mn to bound Mn and it was found that over 97% of total Mn was in the bound form. With elevating the external Mn supply, seed Mn concentration also increased, but the proportion of free Mn²⁺ fraction decreased from 3% in the control (1 μM Mn) to 0.35% and 0.15% in high Mn supply (1000 μM and 10,000 μM, respectively). These results suggest that an elevation of internal Mn concentration in seeds is associated with increased Mn binding pools, hence Mn remains bound during germination. Consequently, the action of potentially harmful Mn²⁺ ions, which may generate ROS and affect seed viability, is alleviated.     

Flavonoids and other bioactive constituents from Ficus thonningii Blume (Moraceae)

Continued interest in the chemistry of Ficus spp. led to the investigation of the figs and the roots of Ficus thonningii Blume. Two new flavonoids, thonningiol (1) and thonningiisoflavone (2) along with nineteen known compounds were isolated. β-Isoluteone (13) was isolated here for the first time from a natural source. Their structures were elucidated on the basis of spectroscopic evidence. Interestingly, thonningiisoflavone (2) and hydroxyalpinumisoflavone (21) showed strong DPPH radical scavenging activity with IC₅₀ = 65.50 μM and 68.20 μM respectively compared to the standard BHA with IC₅₀ = 44.20 μM. The methanolic extract of figs, taxifolin (14), conrauiflavonol (17) and shuterin (19) exhibited moderate antimicrobial activity against six micro-organisms with MIC below 1.5 mg/mL.     

Effect of arsenic on visible symptom and arsenic concentration in hydroponic Japanese mustard spinach

Responses of Japanese mustard spinach (JM-spinach; Brassica rapa L. var. pervirdis) were investigated at elevated levels of arsenic (As). Plants were grown hydroponically in the greenhouse under 0, 6.7, 33.5 and 67 μM As (equal to 0, 0.5, 2.5 and 5 mg L^?1 As, respectively) for 14 days. Arsenic was used as sodium meta-arsenite (NaAsO₂). Toxicity symptom was solely shown as shoot growth repression at 33.5 and 67 μM As exposures. Dry weight (DW) enhanced by 19.4% in shoot and 38.9% in root in the 6.7 μM As level as compared to control but decreased by 48.1% and 72.1% DW in shoot and 24.1% and 61.1% DW in root in the 33.5 and 67 μM As levels, respectively. This result indicated that As at lower concentration might have slight stimulating effect on JM-spinach growth, but toxicity increased with increasing As. Based on the regression lines between growth and As concentration in the plant tissues, the critical toxicity level (CTL) of As in JM-spinach shoot was 7.85 μg g^?1 DW considering 10% DW reduction. The CTL for the root was almost 2110 μg As g^?1 DW, indicating that shoot of JM-spinach was more sensitive to As-toxicity than that of root. Arsenic concentrations increased in plant parts with increasing As in the medium. Arsenic concentrations were also compared in DW and fresh weight (FW) basis. The JM-spinach concentrated unaccepted level of As in shoots for human consumption in the higher As levels without showing visible toxicity symptom. In spite of decreasing iron (Fe) concentration in shoot in the highest As level, chlorophyll index did not decrease accordingly. Phosphorus (P) concentration also decreased. Phosphorus concentration decreased much more than Fe concentration. Low P might help to mobilize Fe in shoots, resulting in higher chlorophyll index at 67 μM As level. Phosphorus might compete with Fe in shoot tissues of As-stressed JM-spinach.     

Changes of phenylethanoid and iridoid glycoside distribution in various tissues of shoot cultures and regenerated plants of Harpagophytum procumbens (Burch.) DC. ex Meisn

Harpagophytum procumbens is a medicinal plant containing several compounds with pharmaceutical activity. Previously, we established shoot culture and in vitro regenerated plants of H. procumbens. In this study, HPLC and LC-ESI-MS were used to identify harpagoside, harpagide, verbascoside and isoverbascoside in various tissues (stems, leaves and callus) of shoots multiplied on Schenk and Hildebrandt (SH) solid medium supplemented with 0.57 μM indole-3-acetic acid (IAA) and 8 μM 6-benzylaminopurine (BAP), as well as in stems, leaves and root tubers of in vitro propagated plants grown in the greenhouse for 3, 6 and 12 months. The content of the compounds was also determined by HPLC. For comparison, control H. procumbens plants initiated from seeds were analyzed. H. procumbens shoots grown under in vitro conditions accumulated lower amounts of iridoids and phenylethanoids than the plants derived from them. The levels of analyzed compounds were higher in the organs of 3- or 6-month-old plants than in those of 12-month-old plants. Differences in the distribution of secondary metabolites were also observed between organs. The aerial parts (stems, leaves) of 3-month-old in vitro regenerated plants were characterized by the highest amounts of phenylethanoids, which significantly exceeded those detected in control plants. Total iridoid content, calculated as the sum of harpagoside and harpagide, was highest in the root tubers of 6-month-old plants. In these organs the level of harpagoside was comparable to that in root tubers of 6-month-old seed-propagated plants, but the level of harpagide was much lower.     

Effects of triterpene derivatives from Maytenus rigida on VEGF-induced Kaposi's sarcoma cell proliferation

Betulinic acid (BA) is a naturally occurring lupane-type triterpene which exhibits a variety of biological activities including potent cytotoxic properties. On the basis of the structural similarity to BA, two lupane derivatives namely lup-20(29)-ene-3β,30-diol (1) and lup-20(29)-ene-3β,28-diol (2), along with two friedelane derivatives, namely friedelan-3-one (3) and friedelan-3β-ol (4), isolated from the Brazilian plant Maytenus rigida, have been evaluated for their anti-proliferative effect. Similarly to BA, compounds 1 and 3 at 1 μM concentration significantly inhibited the VEGF-induced Kaposi's sarcoma (KS) cell proliferation by 50%. In contrast, this effect was not found in control endothelial cells (EC).Moreover, compounds 1 and 3 showed a dose-dependent effect on the apoptotic cell death, as detected by FACS analysis and caspase-3 assay. Specifically, at 10 μM concentration, apoptosis was significantly induced (from 45% to 55% of hypodiploid cells vs control cells) and showed the same potency order observed for the anti-proliferative effect at 1 μM, i.e., compound 3 > BA > compound 1.Taking into account the interest given rise by BA as anticancer agent, the comparable anti-proliferative activity shown by compounds 1 and 3 and BA, can give an impulse to further investigate lupane and friedelane derivatives as cytotoxic agents.