Exploring the influence of organ differentiation on biosynthesis and accumulation of camptothecin in Ophiorrhiza rugosa var. decumbens.

Genus Ophiorrhiza has recently emerged as one of the promising sources of Camptothecin (CPT), an antitumour monoterpene indole alkaloid. It possesses CPT in its every part and has a relatively short life span. To determine whether differentiation plays any role in the synthesis and/or accumulation of CPT, the concentration of CPT was analyzed across various tissues of Ophiorrhiza rugosa var. decumbens obtained through both direct as well as indirect modes of regeneration. The results revealed that the plants obtained from both types of regeneration showed similar levels of CPT. It was also observed that with differentiation, the accumulation of CPT increases, as the callus, being an undifferentiated mass of cells, had only traces of CPT. In contrast, the completely differentiated in-vitro plant obtained from it showed a significantly higher percentage of CPT in shoots (0.22% dry weight) and roots (0.247% dw). The CPT when analyzed after hardening, varied among different organs of the plant. It was also observed that the inflorescence accumulated the highest concentration of CPT (0.348% dw) once the flowering began, accompanied by a decrease in remaining organs. This decrease may result from CPT being mobilized to the inflorescence as a chemical defense mechanism. These findings allowed us to determine the ideal plant harvesting age for CPT extraction. The findings could be used to decide the right stage of plant harvest, which is just before the onset of blooming. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03999-4.

Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization.

Azadirachtin is one of the most potent biopesticides so far developed from a plant sources. Influence of different culture media and elicitation on growth and production of azadirachtin by hairy root cultures of Azadirachta indica was studied. Out of the three media tested, namely Ohyama and Nitsch, Gamborg's and Murashige and Skoog's basal media, hairy roots cultured on Ohyama and Nitsch's basal medium produced maximum yield of azadirachtin (0.0166% dry weight, DW). Addition of biotic elicitor enhanced the production of azadirachtin by approximately 5-fold (0.074% DW), while signal compounds such as jasmonic acid and salicylic acid showed a approximately 6 (0.095% DW) and approximately 9-fold (0.14% DW) enhancement, respectively, in the production of azadirachtin as compared to control cultures on Ohyama and Nitsch medium. Extracts from hairy roots were found to be superior to those from the leaves for antifeedant activity against the larvae of Spodoptera litura.

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.

Comparison of techniques for the extraction of the anti-cancer drug camptothecin from Nothapodytes foetida.

Extraction methods using stirring extraction, Soxhlet extraction, ultrasonic extraction and microwave-assisted extraction (MAE) were evaluated for the percentage extraction of camptothecin (CPT) and 9-methoxycamptothecin (9-Me-CPT) from Nothapodytes foetida. The extracts were analyzed by high performance liquid chromatography (HPLC). Methanol (90%, v/v) extracted high percentage extraction of CPT and 9-Me-CPT compared to ethanol (90%, v/v). The results shows that the percentage extraction of CPT and 9-Me-CPT from N. foetida by MAE was more efficient in short time followed by Soxhlet extraction, ultrasonic and stirring extraction methods. Maximum percentage extraction of CPT (2.67%, w/w) was obtained by MAE technique. MAE has need of 3 min, whereas ultrasonic extraction, Soxhlet extraction and stirring extraction techniques require 30, 120 and 30 min, respectively to leach higher percentage extraction of CPT and 9-Me-CPT. The times taken by the microwave extraction process was 40 times less than the Soxhlet extraction for percentage extraction of alkaloids. The present results show that the extraction efficiency and considerable saving of time by MAE was more competent than the other extraction techniques.

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.