In vitro flowering in Oldenlandia umbellata L.

BACKGROUND: Oldenlandia umbellata L. (Indian madder) is an antique Ayurvedic Indian herb and a source of various anthraquinone derivatives. The red dye from its roots has been used in diverse applications since ancient times. OBJECTIVES: To establish reliable and effective protocols for in vitro flowering of O. umbellata. MATERIALS AND METHODS: For in vitro flowering, organogenic calli were subcultured onto Murashige and Skoog (MS) medium supplemented with various concentrations of Naphthalene acetic acid (NAA) (0.15-1.0 mg/l) and Benzyladenine(BA) (0.5-1.5 mg/l) with and without 0.4% of coconut milk (CM). RESULTS: The highest number of in vitro flowers (22.8%) and best response (92.73%) was achieved on MS medium supplemented with 0.7 mg/l NAA + 1.5 mg/l BA with 0.4% CM. It was found that MS medium devoid of BA promoted best root development (47.3 per calli) as well as response (100%). It was also observed that when embryogenic calli grown in depletion of required nutrition transferred to fresh media induced more flowering. In vivo and in vitro floral comparative analysis revealed that in vitro flower induction was required for short time duration (20.67 days) than in vivo flower. CONCLUSION: To the best of our knowledge, this is the first report on in vitro flowering and this study will help to overcome problems associated with flower development and seed production. As a result, this study may be a potent conservation tool to restore innate population size in its natural habitat.

Toxicity study of dibutyl phthalate of Rubia cordifolia fruits: in vivo and in silico analysis.

Natural toxins from plant sources with wide ranges of biological activities reflect the upswing of drug design in the pharmaceutical industry. Rubia cordifolia L. is one of the most important red dye yielding plants. Most of the former researches have focused on the bioactive compounds from the roots of R. cordifolia, while no attention was paid towards the fruits. For the first time, here we report the presence of dibutyl phthalate in the fruits of R. cordifolia. Structural characterization was carried out using Ultraviolet-Visible spectrophotometer (UV-Vis), Fourier transform infrared (FTIR), gas chromatography-mass spectrophotometer (GC-MS), Nuclear magnetic resonance (NMR). Acute toxicity of the crude ethanolic extracts of the R. cordifolia fruits was examined in Swiss albino mice. No mortality was observed in all treated mice with 100, 500, 1000 mg/kg body weight of crude extract of R. cordifolia fruit and it indicates that the LD50 value is higher than 1000 mg/kg body weight. This study exhibited a significant change in the body weight. Alanine transaminase (ALT), total protein, triglycerides, glucose, and also the histopathological analysis of liver for all treated mice showed difference from the control group. The dibutyl phthalate was further evaluated for the toxicity study through in silico analysis. Together, the results highlighted that the toxic potential of R. cordifolia fruits extracts and also the toxicity profile of the fruit should be essential for the future studies dealing with the long term effect in animals. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1059-1067, 2016.

Protective effect of Scutellaria species on AAPH-induced oxidative damage in human erythrocyte.

BACKGROUND: Scutellaria baicalensis is a well-known plant in traditional Chinese medicine. Recently, several Scutellaria species with therapeutic potential have been recognized worldwide. Scutellaria colebrookiana and Scutellaria violacea, native to the Western Ghats of India, are reported to possess free radical scavenging efficacy. At present, the protective effect of these Scutellaria spp. against 2,2' azobis (2-amidinopropane) hydrochloride (AAPH)-induced oxidative damage in human erythrocytes has been analyzed. METHODS: Oxidative stress in erythrocyte was induced by AAPH. The inhibition of hemolysis, membrane lipid peroxidation, and protein damage by chloroform extracts of Scutellaria spp. was assessed biochemically. Phytochemicals of the extracts were analyzed by Fourier transform infrared spectrophotometer (FTIR). RESULTS: Approximately 95% of erythrocytes were lysed by AAPH over 3 h of incubation. Significant reduction in hemolysis was observed by the extracts, and the IC50 values were 18.3 and 23.5 µg/mL for S. colebrookiana and S. violacea, respectively. Both the extracts were found to inhibit AAPH-induced lipid peroxidation in ghost membrane with IC50 92±2.8 and 70±5.6 µg/mL. In the analysis of the membrane proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the AAPH-induced degradation of actin was found reduced by both the extracts. The FTIR spectrum revealed the presence of polyphenols, carboxylic acids, alkanes, and aromatic compounds in extracts. In quantitative analysis, the total polyphenolic content estimated was 380±0.23 and 203.7±1.4 mg of gallic acid equivalent per gram extract of S. colebrookiana and S. violacea. CONCLUSIONS: Results indicate that S. colebrookiana and S. violacea are capable of protecting erythrocytes from oxidative damage. This cytoprotective effect of the extract is possibly by its antioxidant property.

Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia.

Transformed hairy roots of Psoralea corylifolia were established by infection with Agrobacterium rhizogenes LBA 9402. The aim of this work was to elucidate the effects of media constituents on production of the phytoestrogenic isoflavones daidzein and genistein. A. rhizogenes strain LBA 9402 harboring Ri plasmid was used to transform stem segments of in vitro seedlings. The resultant hairy roots were confirmed by polymerase chain reaction (PCR) and exhibited Ri T-DNA. Transformed hairy root clones were cultured in Murashige and Skoog's (MS) medium altered with different concentrations of NH(4) (+) and NO(3) (-) and their growth and production of isoflavones were assessed. Biomass and productivity increased when MS medium was supplemented with NH(4) (+) and NO(3) (-) at a ratio of 20:10. Increased yield of daidzein was obtained when sucrose level in the culture medium increased, whereas decreased level of sucrose favored genistein production. The hairy roots produced the highest levels of daidzein (2.06% dry wt.) and genistein (0.37% dry wt.) in the presence of low concentrations of PO(4) (3-). Hairy roots secreted trace amounts of daidzein and genistein into the culture medium. The present results demonstrated that the productivity of daidzein was 2.2-fold more than that of untransformed roots.

Studied enhancement strategies for phytoestrogens production in shake flasks by suspension culture of Psoralea corylifolia.

This study proposed secondary metabolites incremental yield due to manipulation of nutrient components into the culture medium. To validate this, the effects of nutrients such as carbon, phosphate and nitrogen on growth and production of phytoestrogens daidzein and genistein by suspension cultures of Psoralea corylifolia was investigated for the first time. The maximum production of daidzein and genistein was achieved when sucrose and maltose used as a sole source of carbon. Suspension cell cultures enriched with sucrose (3%) stimulated accumulation of isoflavones daidzein (1.76% dry wt) and genistein (0.25% dry wt) compared to glucose, fructose and maltose. Sucrose feeding strategy significantly stimulated biomass growth and isoflavones (2.79% dry wt of daidzein and 0.32% dry wt of genistein) production rate. Reduced concentrations of phosphate (0.625 mM) promoted daidzein (1.89% dry wt) and genistein (0.26% dry wt) production by suspension cell cultures, whereas high amount (5mM) in medium was inhibited isoflavones production. It was observed that medium fortified with NH(4)(+) and NO(3)(-) alone inhibited production of isoflavones. The maximum production obtained of daidzein (2.20% dry wt) and genistein (0.29% dry wt) when medium comprised with NH(4)(+)/NO(3)(-) at ratio 20:40 mM as a nitrogen source. Similar nutrient components ratio when altered NH(4)(+)/NO(3)(-); 40:20mM) resulted in approximately 3-fold decrease in production. HPLC analysis revealed that suspension cells cultures leached out trace amount of daidzein and genistein into the culture medium.

Distribution of anticancer drug camptothecin in Nothapodytes foetida.

The topoisomerase I-DNA inhibitor alkaloid camptothecin has been evaluated from the various parts of Nothapodytes foetida. The bark contained 0.27% dry wt of camptothecin and 0.11% dry wt 9-methoxycamptothecin followed by the root, stem, and leaves. Immature seeds contained higher concentrations of camptothecin (0.32% dry wt) and 9-methoxycamptothecin (0.16% dry wt) compared to mature seeds. Various parts of mature and immature seeds were analysed to determine the content of the major alkaloids. Zygotic embryos of immature seeds contained 0.11% dry wt of camptothecin and 0.04% dry wt of 9-methoxycamptothecin. The highest concentration of camptothecin (0.42% dry wt) and 9-methoxycamptothecin (0.18% dry wt) were accumulated in the cotyledons of immature seeds.

Antimicrobial activity of Gymnema sylvestre leaf extract.

The ethanolic extract of Gymnema sylvestre leaves demonstrated antimicrobial activity against Bacillus pumilis, B. subtilis, Pseudomonas aeruginosa and Staphylococcus aureus and inactivity against Proteus vulgaris and Escherichia coli.

Studies on production of ajmalicine in shake flasks by multiple shoot cultures of Catharanthus roseus.

The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 microM) and a low concentration of BA (2.22 microM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 microM) and a high concentration of BA (8.90 microM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 microM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 microM) supplemented medium compared with the high concentration of BA (8.90 microM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 microM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus.

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.