| Summary: | 碩士 === 朝陽科技大學 === 應用化學系生化科技碩士班 === 102 === Saussurea involucrata Kar.et Kir (Family Asteraceae) is a perennial herb. The herb is also known as snow S. involucrata, since it mainly grows on snowy mountains. The plant is about 15 to 30 cm in height; leaf in clusters, elongated oval, with serrated edges, enclosing the bud; flower with a tubular purple corolla. S. involucrata contain polysaccharides lignans, guaianolides, and flavonoids, such as quercetin and rutin and syringetin. These flavonoids have been reported to be anti-rheumatic, anti-inflammatory, dilate blood vessels, lower blood pressure, prevent cardiovascular disease, and enhance immunity, anti-aging, anti-cancer and anti-fatigue. In recent years, due to excessive collection of S. involucrata plants in the natural habitat, and their slow growth in wild, the species has become endangered, resulting in short supply of plant materials and scarcity of medicine. Owing to this, the direct use of herbs to extract the active ingredients is a serious limitation. Hence, it is very important to apply tissue culture technique to obtain the active compounds. In the present study, different plant parts (leaf, petiole and root) of S. involucrata were used as explants to induce callus. Experiments were carried out to optimize the callus induction and its proliferation using different physical (temperature, container closure type) and chemical factors (different basal media, plant growth regulators, gelling substances and elicitors).
Induction of callus in leaf, petiole and roots explants of S. involucrate varied among the five different basal media. The maximum leaf explants (80%) induced callus on 1/2 MS basal medium supplemented with 0.4% Gelrite, 0.5 mg/L BA, 0.5 mg/L 2,4-D or 0.4% Gelrite, 1.0 mg/L BA, 0.5 mg/L 2,4-D. The highest callus proliferation was achieved in S. involucrata leaf callus culture on 1/2 MS basal medium supplemented with 0.5 mg/L BA, 0.5 mg/L NAA, 0.4% Gelrite. In the experiment with temperature, the maximum callus proliferation was recorded at 20℃. Sealing of culture vessels with 3 layers of Dispense papers supported the maximum callus proliferation (1.036 g) on medium supplemented with 0.5 mg/L BA and 0.5 mg/L NAA. In the experiment on effect of elicitors on syringetin and rutin content in callus, we obtained the maximum syringetin (13.524 mg/g) and rutin (0.374 mg/g) contents on control and medium with phenylalanime (3.0 mg/L) respectively, when cultures were incubated at 20℃. The HPLC analysis of different in vitro plant materials and commercially available crude drug had different levels of syringetin and rutin contents in them. The highest syringetin (11.235 mg/g) and rutin (2.556 mg/g) content were recorded in petioles of two month old in vitro plantlets and leaves of crude drug, respectively. Interestingly, leaf callus in an experiment with elicitors induced a still higher syringetin content (13.524 mg/g) in control (on 1/2 MS basal medium supplemented with 0.5 mg/L BA, 0.5 mg/L NAA, 3% sucrose, 0.4% Gelrite and without any elicitor.).
In summary, the present results demonstrate that tissue culture technique can be used successfully to obtain the syringetin and rutin compounds in Saussurea involucrate without sacrificing the plants in the wild.
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