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Effects Of Different Extrinsic Regulators On The Growth And Ginsenosides Content Of The Panax Quinquefolium L. Hairy Roots

Posted on:2009-08-23Degree:MasterType:Thesis
Country:ChinaCandidate:Y HouFull Text:PDF
GTID:2144360242480093Subject:Fermentation engineering
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Panax Quinquefolium L. is a precious Chinese traditional medicinal material. Its main effective component is ginsenoside which mainly comes from cultivated Panax Quinquefolium L. with hight cost.And ginsenoside displays marked physiological activation.The hairy root of Panax Quinquefolium L. was induced from the root explants by Agrobacterium rhizogenes A4 with directly inoculating.The hairy root is characterized by rapid growth, hight live material content and stable hereditary form.In order to improve the effective component and satisfy the request of medication product,it should regulate the cultivated process,including nutrition factors, phytohormones, precursors and elicitors.We want to change the metabolic pathway to improve the secondary metabolite by adding extrinsic matters to culture medium. The main contents and conclusions are as follows:(1)Got the method of drawing total ginsenoside: dry hairy roots of Panax Quinquefolium L.→rubbing(filtration of 40)→weighing up about 0.5g,adding saturated n-butyl alcohol 50ml→treating with ultrasonic 30min→heating and recycling for 1.5 hour,set for one night→volatilizing menstruum,adding 50ml water→adding petroleum ether(30~60℃) to draw→water flowing into column of macroreticular resin of D101 to wash and throwing away→adding a lot of ethanol (20%,50%)to wash and throwing away→getting last washing through 50% ethanol→making menstruum dry ,dissolving the powder with carbinol(10ml).(2) Choosing method of HPLC about ginsenoside Rb1 of Panax Quinquefolium L. hairy root: flowing part comprise acetonitrile(A) and 0.1% Phosphoric acid(B), flowing velocity is 1.0ml/min; checking wavelength is 203nm; volume of adding sample is 10ul;column temperature is 40℃. Washing program of grads is 0~25min, percent of acetonitrile is 19%~20%; 25~60min, percent of acetonitrile is 20%~40%; 60~70min, percent of acetonitrile is 40%~50%,computing by area of peak.(3)We adopt solid and liquid medium to cultivate the hair root.Hair root can elongate and increase ramus in solid medium and then it can be stronger in liquid medium.(4)Through studying ginsenoside content of the hairy root of Panax quinquefolium L. affected by phytohormones,we found that phytohormones is favourable to improve the growth speed,and can promote the ginsenoside content at feasible concentration.The lower 6-BA is valid to the accumulation of total ginsenoside content,while NAA is more favourable to improve the growth speed. The lower 6-BA is valid to the accumulation of ginsenoside Rb1 content.When adding 0.2mg/L 6-BA, yield of ginsenoside Rb1 is 2.5 times as much as the contrast.When adding 1.0mg/L NAA, yield of ginsenoside Rb1 is 4.5 times as much as the contrast.And NAA is more favourable to the root growth,at the same time the ginsenoside Rb1 content is so hight.(5) Through studying ginsenoside content of the hairy root of Panax quinquefoliumL. affected by precursors,we found that the low concentration of precursors displays stimulative but not clear.In addition,we should find the more valid direct and indirect precursors on basis of further studying.The suited concentration of Mg(Ac)2 is 0.5g/L,higher or lower is not good.Hight concentration of L- leucine displays an inhibitory effect,but the lower can increase the total ginsenoside content.Mg(Ac)2 could improve the accumulation of ginsenoside Rb1,especially 0.5g/L.The lower concentration of L-leucine is more effective,the max is 2.4 times as much as the contrast.In conclusion, Mg(Ac)2 is more valid to the total ginsenoside content,while L- leucine is more effective to ginsenoside Rb1.(6) Through studying ginsenoside content of the hairy root of Panax quinquefoliumL. affected by elicitors,we found that as a signal molecule, elicitors can bring self-defense reaction.Especially MJAis widely used in the production of secondary metabolites by plant cells.When adding MJA,biomass of hair root increases with time,but total ginsenoside content decreases with time.The reason may be that the secondary metabolite is excreted to the medium because of the elicitors.Elisitors can exerted an inhibitory effect to the growth speed,the higher concentration the more inhibitory . 10μmol/L MJA could promote markedly the saponin accumulation,and ginsenoside Rb1content is also the max,5 times as much as the contrast.SA is useful at the low concentration.In conclusion, MJA showed a significant effect, and more importantly, MJA changed the ratio of ginsenoside content. The content of ginsenoside Rb1 increased so much.The concentration of SA plays an important role in the combination of MJA and SA.So we should consider SA first. The combination exerted an inhibitory effect to the growth speed, while raise remarkably the total ginsenoside content and the proportion of ginsenoside Rb1 in the total saponin.The combination of 1μmol/L MJ and10μmol/L SA is so effective that the otal ginsenoside content is 3 times as much as the contrast.But we should notice the exceptive point.The conclusions in this thesis will play a positive role in producing the medicine with the hairy root and obtaining the ingredients of the Panax quinquefolium L.At the same time,it offered the reference on the theory and practice for research work of the present ginsenoside.
Keywords/Search Tags:Quinquefolium
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