Devanand P. Fulzele

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 μg 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 μg g−1 DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2‐fold increase in alkaloid and yielded 52 and 56 μg 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 μg 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.

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.

Rapid method for the detection and determination of artemisinin by gas chromatography

Abstract Artemisinin, and antimalarial principle of Artemisia annua, is thermally unstable and decomposes on the column to give two peaks under various gas chromatographic conditions. A simple and rapid method was developed for its indirect detection and determination, at nanogram levels, both in plants and in tissue cultures developed from the plants. It is based on the linear relationship obtained between the concentration of artemisinin and the respective peak areas for either of the two thermally degraded products.

Production of terpenoid from Artemisia annua L. plantlet cultures in bioreactor

Abstract Seeds of Artemisia annua obtained from different locations in Europe, Michigan, Washington, Hoechst (India), and Lucknow (India) were grown at the Experimental Field Station at Trombay, Bombay. The plants synthesized various levels of terpenoids of which camphor, 1,8-cineol and β-caryophyllene were the major constituents. The Lucknow variety synthesized the highest level 1.99 mg% fresh weight (FW) of camphor, the Michigan variety produced 1.08 mg% FW of 1,8-cineol, and the Washington variety contained 0.38 mg% FW of β-caryophyllene. Callus cultures interspersed with shoot buds were obtained from all the plant varieties on Murashige and Skoogs medium containing 1 mg l −1 6-benzyladenine and l mg l −1 napthaleneacetic acid (Murashige and Skoog, 1962). Multiple shoot cultures were established from the Europe variety on MS medium supplemented with 0.1 mg l −1 6-benzyladenine and l mg l −1 indole-3-acetic acid. Plantlet formation occurred on medium containing l mg l −1 napthaleneacetic acid and 0.1 mg l −1 kinetin. The plantlets produced camphor (0.81 mg% FW), 1,8-cineol (0.07 mg% FW) and β-caryophyllene (0.025 mg% FW). Multiple shoot and plantlets were grown in 1-1 capacity bioreactors. The plantlet cultures from the bioreactors produced 1.08 mg% FW camphor followed by 0.14 mg% FW 1,8-cineol and 0.65 mg% FW β-caryophyllene. Addition of gibberellic acid and ethephone to the culture medium in the bioreactor stimulated the synthesis of terpenoids. The cultures produced 3.41 mg% FW camphor after 15 d of cultivation in the bioreactor with ethephone addition.

SOMATIC EMBRYOGENESIS, PLANT REGENERATION, AND THE EVALUATION OF CAMPTOTHECIN CONTENT IN NOTHAPODYTES FOETIDA

SummarySomatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoogs (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.

Large-scale cultivation of Catharanthus roseus cells: Production of ajamalicine in a 20-l airlift bioreactor

Bioreactor systems have been developed for the production of ajmalicine, an alkaloid used in the treatment of hypertension. Cell cultures of Catharanthus roseus produced higher levels of ajmalicine (323 micrograms g-1 dry weight) in a production medium enriched with tryptophan. The cell cultures were grown in medium prepared in tap water and market sugar with a view to minimise the costs of production. Large-scale cultivation of cell suspension was performed in a 20-l airlift bioreactor under controlled conditions. An ajmalicine production of 315 micrograms g-1 dry weight was achieved in the bioreactor after 14 d of cultivation.

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.

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 Skoogapos;s (MS) medium with a high concentration of IAA (11.42 μM) and a low concentration of BA (2.22 μM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 μM) and a high concentration of BA (8.90 μM), 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 μM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2‐fold more in IAA (11.42 μM) supplemented medium compared with the high concentration of BA (8.90 μM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 μM) 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.

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 NH4+ and NO3− and their growth and production of isoflavones were assessed. Biomass and productivity increased when MS medium was supplemented with NH4+ and NO3− 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 PO43−. 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.

HPTLC Densitometric Evaluation of Tissue Culture Extracts of Nothapodytes foetida Compared to Conventional Extracts for Camptothecin Content and Antimicrobial Activity

Tissue culture technique is becoming popular because of its well-known ability to enhance the content of secondary metabolites in plants. Callus tissue cultures of Nothapodytes foetida were developed using 250 different medium compositions to optimize this procedure. Methanolic extracts of callus (MEC) and of various parts of N. foetida were comparatively analyzed for camptothecin content, and a high performance thin layer chromatography method was developed for its quantitation. Chloroform-ethylacetate-methanol (4 : 5 : 0.5 v/v) was used as the mobile phase. The method was validated for linearity, precision (interday and intraday), repeatability, limit of detection (LOD), limit of quantitation (LOQ), and accuracy. The relationship between the concentration of standard solutions and the peak response was linear within the range of 80 to 480 ng/spot with a correlation coefficient of 0.998 +/- 0.020. Instrumental precision was evaluated as 0.54 (% CV). Repeatability of sample and standard were estimated to be 1.08 and 1.01 (% CV), and LOD and LOQ were found to be 40 and 80 ng/spot, respectively. The accuracy of the method was checked out by a recovery study and the average percentage recovery was calculated as being 99.13 %. The methanolic extract of callus grown in tissue culture with medium composition picloram + thidiazuron + gibberellic acid (1 : 1 : 4; MEC-PTG) showed a higher percentage of camptothecin (5.74 % w/v) than the methanolic extract of fruits (3.56 % w/w), leaves (1.56 % w/w), stem (1.19 % w/w), and root (1.11 % w/w). The results of the antimicrobial screening indicate that MEC-PTG exhibited maximum activity against all microorganisms. Among the fungi tested, MEC-PTG showed maximum activity against A. niger and C. albicans (MIC value 10 microg/mL) whereas among bacteria strains, its activity was highest against B. subtilis and S. lutea (MIC 20 microg/mL).