Aspergillus niger elicitor for cell suspension Yanhuanglian synthesis of alkaloids in
Author: Cheng Hua Zhang Yuqin Jiang Yu Ming Ao Zhang
【Abstract】 Objective To improve the cell suspension culture of Corydalis saxicola Bunting alkaloid production. Ways to different concentrations of Aspergillus niger elicitor treatment Yanhuanglian suspension cultured cells. The results of alkaloid production in induction time with the extension of the rapid increase to the first 6 days to reach the maximum; add 50 mg / L induced by neutrons alkaloids highest yield reached 89.2 mg / L, compared with 2.5 times higher than the control group, when the induced concentration increased further, the alkaloid production but reduced; with the
elicitor concentration from 0 mg / L increased to 150 mg / L, cell biomass, but reduced gradually. Conclusion Aspergillus niger elicitor Yanhuanglian cells can promote the synthesis of alkaloids, the best elicitor concentration of 50 mg / L; high
concentration of elicitor is not conducive to cell growth and alkaloid synthesis, cell biomass reduction is the result alkaloid production in an important reason for the decline. The treatment provides a simple and effective means to enhance Yanhuanglian alkaloid production in cell culture.
Key words Aspergillus suspension cell cultures Yanhuanglian elicitor
Abstract: ObjectiveTo improve the production of alkaloids in Corydalis saxicola Bunting suspension cell
cultures.MethodsElicitor prepared from Aspergillus niger was
used to treat the suspension cells on day 12.ResultsThe results showed that the production of alkaloids increased with the extension of time after treatment and the greatest value of alkaloid production was obtained on day 6. Cultures treated
with 50 mg / L elicitor generated the highest alkaloid production, 89.2 mg / L, which was about 2.5-fold of that in
the control.Alkaloid production significantly decreased in response to increased elicitor concentration from 50 mg / L to 150 mg / L. When elicitor concentration increased from 0 to 150 mg / L, biomass of cultures inversely
decreased.ConclusionThis elicitor is effective to enhance alkaloid biosynthesis in cell suspension cultures of C. saxicola and the optimal concentration for alkaloid
biosynthesis is 50 mg / L. Higher concentration of the elicitor is unfavorable to not only cell growth but also alkaloid production. This decrease of biomass induced by higher concentration of elicitor is a main reason for the suppression of alkaloid production. This treatment provides a simple and effective approach to enhance alkaloid biosynthesis of C. saxicola cells.
Key words: Aspergillus niger; Cell suspension cultures; Corydalis saxicola Bunting; Elicitor
Fungal elicitor of plant cells has been widely used to promote the synthesis of metabolites in a variety of plant cell suspension cultures induced to display a high efficiency has become in recent years, cell culture in vitro production of useful natural products research focus [1 ~ 3]. Aspergillus
niger (Aspergillus niger) is a widely distributed in soil and air in the fungus, prepared from Aspergillus niger elicitor was shown to promote a variety of plant cell cultures in the synthesis of specific metabolites, Huang et al  the use of
source in Aspergillus niger elicitor enhanced Taxus cell culture anti-cancer drug - Taxol content, Namdeo et al  the
use of Aspergillus niger elicitor increased Catharanthus roseus cell cultures in the A do alkali production. The use of
fungal elicitors activate the metabolic pathways of plant cells to improve plant cell culture has become the target product of the effective means of production .
Yanhuanglian (Corydalis saxicola Bunting), Corydalis Corydalis genus Branch [6,7], perennial herb. All grass-
containing dehydrogenation Ka Weiding (dehydrocavidine), berberine (berberine) and other active ingredients ; has significant anti-bacterial, anti-inflammatory, analgesic and
strong stability and role in civil society as a treatment for
a cure for hepatitis B . We have established Yanhuanglian suspension cell culture system , in order to improve alkaloid production in culture, this paper Aspergillus niger elicitor treatment Yanhuanglian suspension cultured cells to
study its alkaloid synthesis and cell growth and found that black Aspergillus niger elicitor can effectively promote the synthesis of alkaloids in cell suspension.
1 Materials and methods
1.1 Materials Yanhuanglian cell culture reference 
method. Suspension culture using 250 ml flask culture, Mei Pingzhuang medium 100 ml, after inoculation (25 ? 1) ?
constant temperature condition (130 ? 5) r / min shaker
culture, cell inoculation density of 10 g/100 ml (FW / V), dark culture. Elicitor Yanhuanglian suspension cell cultures in the medium-term (12 days) to join the control group by adding an equal volume of distilled water.
1.2 Preparation of elicitor Aspergillus fungal elicitor preparation of reference  method. With sugar concentration
as the concentration of elicitor used anthrone determination of sugar content in the reserve.
1.3 Determination of alkaloids in alkaloid content determination method of Reference . Weigh fresh cells, 1 g, by adding 20 ml 75% acidic ethanol, ultrasonic extraction 3
times, 1 h / times, leaching, take filtrate 1 ml, adding 0.1 mol / L citric acid - sodium citrate buffer (pH = 5.4) 5 ml, with 2 ml 0.05% bromocresol green as the color reagent, add 10 ml chloroform extraction 30 min, taking the organic phase,
adding proper amount of anhydrous Na2SO4 dehydration, with the spectrophotometer at 416 nm was measured the light absorption value; the amount of take 1 ml culture medium, and then the same approach, take organic phase measured optical density.
With berberine alkaloid content as a standard product label, the measured content of alkaloids in cells and culture medium, and the alkaloid content.
1.4 Determination of cell growth to cell dry weight as indicators of cell growth, determination, op. Culture through
the cloth-type funnel leaching with distilled water to clean the residue of the culture medium re-leaching get cell fresh
weight (FW), and then proceeds cell set 50 ? oven drying to
constant weight (about 48 h), weighing get cell dry weight (DW). Repeat three times experiments.
2 Results and analysis
2.1 elicitors on alkaloid synthesis in the result shown
in Figure 1.
Figure 1 alkaloid synthesis time curve (omitted)
Figure 1 shows the Add 50 mg / L cell culture induced by neutrons Yanhuanglian the synthesis of alkaloids. From the figure we can see that the output of alkaloid induction time
as the extension of the rapid increase to the first 6 days and reached the maximum at this time 2.5 times higher than the control group, then slowly lower alkaloid production. Alkaloid
production in the control group throughout the training
process gradually increased until the end of training (9 days) the highest.
2.2 elicitor concentration on the alkaloid yield the results shown in Figure 2.
Figure 2-induced concentration of the alkaloid yield (omitted)
2.3 elicitors on cell growth of Yanhuanglian adding elicitors on cell growth Yanhuanglian a significant impact. Can be seen from Figure 3, add the elicitor, the cell growth was inhibited. Compared with the control group, elicitor treatment group reduced the cell biomass. As the elicitor
concentration from 50 mg / L increased to 150 mg / L, decreasing the amount of cell biology. Elicitor concentration of 50mg / L, the biomass in the first 3 days after treatment to reach the highest, 12.8 g / L. Biomass in the control group
reached the maximum in the first 6 days than the same period to add 50, 100,150 mg / L induced by sub-group were 15%
higher, 30% and 40%. Reposted elsewhere in the paper for free download http://
? 0 mg / L elicitor; ? 50 mg / L elicitor; ? 100 mg /
L elicitor; 150 mg / L elicitor
Figure 3 elicitors on cell growth Yanhuanglian effects (abbreviated)
In plant cell cultures to induce the child has been
widely used to enhance the production of secondary
metabolites. Derived from the fungal cell wall components, polysaccharides, simple sugars, sugar, protein and other ingredients can be used as an elicitor . In this study, the elicitor, derived from Aspergillus niger, the main
component of cell wall polysaccharides, the elicitor can stimulate the rock synthesis of alkaloids in Coptis cells, thereby enhancing their productivity. The results with Zhang et al [12 ~ 14] in the Taxus cell culture consistent with the
In Yanhuanglian suspension cell culture, high
concentrations of elicitors led to a lower biomass, did not arouse a higher alkaloid production. That a suitable elicitor concentration of alkaloid synthesis and cell growth is
essential, high concentrations of elicitors is not conducive to cell growth and alkaloid synthesis, cell biomass reduction is the result in a significant decline in alkaloid production in reasons. Matsumura et al  The study found that treatment with the elicitor of rice suspension culture cells, causing a large number of H2O2 generation, medium alkalization, leading to cell death. Lan et al  that the elicitor-induced cell death in cultured cells of Taxus suspension does not contribute to the role of synthesis of
Taxol. Piffanelli, etc.  study found that barley suspension cells, biological and abiotic stress can activate the role of cell death and defense response and regulation of genes related to MLO. Zhang et al  with the elicitor treatment of tobacco suspension cells were also found to lead to cell death, and think that this elicitor activated WIPK pathway. In our experiment, the best elicitor concentration of 50 mg / L, although Yanhuanglian cell biology has led to some reduction in the amount, but will alkaloid production in
culture increased by 2.5 times, this treatment provides a simple and effective way to improve Yanhuanglian alkaloid production in cell culture, and to study the Coptis cells, elicitors on the induction mechanism of rock lay the
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