|dc.description.abstracteng||Nature has been known to be a rich source of pharmacologically essential compounds over the years. Phytochemical studies have revealed a large number of natural products with a unique chemical structure and important medical potentials. Hyoscyamus niger L. is considered as one of the important medicinal plants during the history and Sesamum indicum, which has been known for a long time as an oil crop Lately, several studies proved their biomedical activity. This study aimed to prove new pharmacological uses of these two medicinal plants and to develop in vitro protocols for improving the yield and quality of the secondary metabolite and accurate system for examining their biomedical activity.
Germination efficiency and dormancy in black henbane and Sesame were tested in the laboratory for 7 varieties of H. niger and 25 varieties of S. indicum using different gibberellic acid concentrations in mg/L. The results showed that GA3 is one of the most important factors that affect breaking seed dormancy. Seed germination was more pronounced in the dark, and there was an increase in seed germination percentage with increasing GA3 doses up to 250 mg/L, which gave the highest germination percentage. The hairy root culture accumulates phytochemicals is most similar in quantity than the intact plants with stable genotype and biosynthesis. Hairy root cultures of 7 varieties of H. niger and 25 varieties of S. indicum were generated using Agrobacterium rhizogenes ATCCA15835 and A4 strains. Two different sterilization protocols were used to examine the viability of the explants and the response capability; aseptic protocol and non-aseptic protocol. For the aseptic protocol, 100% transformation frequency was recorded, while the highest response for the non-aseptic protocol was 38%.
The transformation frequency (TF) of both A. rhizogenes strains was examined for all varieties of both plant species. The highest TF using A. rhizogenes ATCCA15835 for both plants were 100%, and the lowest TF was 88% and 31% for H. niger and S. indicum respectively while for A. rhizogenes A4 strain the highest TFs were 57% and 68% for H. niger and S. indicum respectively, and the lowest TF was 13% for S. indicum. The hairy root suspension culture was established for all plant varieties, the highest hairy root biomass yield for H. niger using A. rhizogenes ATCC15835 and A4 strain was 10 g/L and 7g/L respectively, while biomass yield for S. indicium using A. rhizogenes ATCC15835 and A4 strain was 9 g and 7 g respectively. Bubble column bioreactor has been designed and used for hairy root initiation. The highest dry weight biomass obtained from bioreactor propagation was 14 g/L for H. niger and 12 g/L for S. indicum which was significantly different compared with HRCs grown in a shaking flask culture with fixed air and agitation which was 9 g/L for H. niger and 8 g/L for S. indicium.
Different protocols have been used to examine the biomedical activity of H. niger, and S. indicum varieties, one of the most accurate method is using Caenorhabditis elegans nematodes as an advanced model. The methanolic extract of the plant varieties was prepared from lyophilized seed, root, and hairy root culture. Moreover, essential oil and thermal release were examined. The highest mortality from H. niger essential oil was 99% at 500 µg/mL, and the LC50 ranged from 33.6 to 58.1 µg/mL for the varieties KRI Pinjwen, and Iran Isfahan respectively.
Hairy root methanol extract for both H. niger and S. indicum shows significant response against C. elegans. Nine concentrations of 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500 µg/mL were used to conduct this study. The lethal dose LC50 for H. niger ranged between 39.7, and 48.2 µg/mL, and for S. indicum ranged between 299.4, and 367.1 µg/mL. While for H. niger root extract ranged between 136.6, and 319.5 µg/mL but for S. indicum was considered as undefined. Regarding both plant species seeds, two types of the extract were prepared, crude extract and fixed oil extract. The crude extract from H. niger showed a significant response and LC50 ranged between 41.0 and 92.2 µg/mL, while for S. indicum was considered as undefined, moreover, the fixed oil showed significant mortality at LC50 for H. niger ranged between 57.0, and 66.9 µg/mL, and for S. indicum 427.7 µg/mL for Indian 7 varieties.
The thermal release was examined as a protocol for the antinematode activity of H. niger and S. indicum seeds against C. elegans nematodes. Several heating degrees were examined for one hour at 0, 50, 60, 70°C, 80, 90, and 100°C. The results were surprising, in which highly significant mortality 100% was recorded for all varieties at 100oC, and 94% at 90oC and the LT50 ranged from 77.4, 77.3, 72.9, 80, 82.6, 79.8, and 78.11°C for the varieties KRI Hawraman, KRI Hasanawa, KRI Pinjwen, KRI Daray Mar, Iran Takhte, Iran Isfahan, and Germany 1 respectively, while S. indicum showed no a significant response.
Secondary metabolite profiling of H. niger and S. indicum varieties were studied using HPLC-ELSD chromatograms, for chemical analysis of hairy root exudates from both bioreactors and flask culture. The functionality of the bioreactor was demonstrated by the growing hairy roots of both plants; then, the exudates were collected from hairy roots grown in bioreactors and shaking flask cultures then analyzed by HPLC-ELSD. Differences among metabolic profiles for different varieties of the same species revealed substantial chemical diversity, verifying the need to examine multiple plant varieties while optimizing metabolite production in hairy root cultures. Different classes of lignans as sesamin, sesamolin, sesaminol, pinoresinol, and sesamol were detected in S. indicum using HPLC-MS. The results show that all three different plant tissues for all varieties contain lignans. Furthermore, the production of the hairy root Sesamin was higher than the normal root.
The basic level of biodiversity is genetic diversity, which means the variability of genotypes in plant species. The genetic diversity of H. niger and S. indicum was examined using DNA sequencing; for this reason, four specific primers, four genes were amplified from H. niger and S. indicum. The genes were HNTR 1, HNTR 2, and FAD2, SAD, respectively. The multiple sequence alignment results showed that most variations were detected among the samples of H. niger when compared to the reference strain. However, the amplified genes in the S. indicum in different isolates showed fewer variations.||de