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15or indirectly function in the L-type Ca channel and in the KATP in myocardial cells.H. attenuatuminhibitsthe inflow of Ca2+ions and regulatesthe Ca2+ion concentration in myocardial cells by inhibiting the height of the opening of L-type Ca channels. Moreover, H. attenuatumrestrains the occurrence of tachyarrhythmia by facilitatingthe opening of KATP. Further studyisrequired to clarify the mechanism of flavonoidsofH. attenuatumagainstmyocardial ischemia-reperfusion arrhythmia.
5.Acknowledgements
This work was supported by National Natural Science Foundation of China(81173185); Key Project of Natural Science Foundation of Heilongjiang Province(ZD201111); Heilongjiang Province Graduate Students ResearchKey Project (YJSCX2012-334HLJ); and Outstanding Talents Support Program of Heilongjiang University of Chinese Medicine(2012).
6.References
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Studies on Improving Diosgenin Content of Dioscorea zingibenisis with Solid
Fermentation of Endophytic Fungi C39
Ying Xu, Siqi Gu, Dan Yu, Xiaowei Du
Pharmacognosy,HeilongjiangUniversity of Chinese Medicine, Harbin,China
Abstracts
Objectives: The aim of the study was to use solid fermentation of C39 isolated from dioscorea nipponica improving the content of diosgenin in D.zingibenisis C.H.Wright to find the
new resources of diosgenin production. Methods: After C39 was activated, the strain C39 was cultivated toPD liquid medium. And then it was added into medicinal culture medium for solid fermentation. Results: Endophytic fungi C39 isolated from D.nipponica Makino can improve the content of diosgenin in D.zingibenisis C.H.Wright. The growth rate was 129.21%. Conclusions: Endophytic fungi C39 can improve the yield of diosgenin in D.zingibenisis C.H.Wright not just in D.nipponica Makino. Consequently, we can apply the fementation technology of C39 to other Dioscorea plants which have similar substance to open up new resource of diosgenin production.
Keywords diosgenin, endophytic fungi, solid fermentation, D.zingibenisis C.H.Wright
1.Introduction
Diosgenin as a steroidal sapogenin, is one of the most important precursors to synthetize 300 kinds of steroid hormones and contraceptives[1,2]. And it has been reported to possess many biological activities, such as anticancer, cardiovascular action, and antimalarial activities[3-5].Diosgenin is mainly exists in dioscorea plants and it has higher content in D.zingibenisis
C.H.Wright and D.nipponica Makino[6]. But with the development of the pharmaceutical industry and the increasing demand of domestic and international market for diosgenin, the wildlife resources have become more failure. Additionally, the limiting of the area of artificial cultivating, the long cultivation cycle of dioscorea tubers herbs and the biological breeding methods such as tissue culture and cell culture not nearly be applied to actual production due to the constrains results in the shortage of the materials from which we can extract diosgenin. It is difficult to meet the growing market demand. Therefore, finding and establishing new methods to obtain high yields of diosgenin, improving the utilization of limited resources, expanding the range of materials to product diosgenin is an essential way to ensure its clinical medicine and pharmaceutical industry product supply.
Microbial transformation is an important method to transform and modify the structure of the compound. Microbial, especially fungal, has strong ability of decomposite and transform compound and can produce a wealth of secondary metabolites, meanwhile reduce the use of acid to decrease environmental pollution. So far it has been successively isoslated a variety of endophytes from hundreds of species medicinal plants[7]. And using the fermentation of endophytes with medicinal plants or herbs promotes the production of some important natural active ingredients. Our laboratory isolated C39 from D.nipponica Makino and then fermented it with herbs, which significantly improve the content of the diosgenin. D.zingiberensis has the similar components with
D.nipponica Makino. After fermentation by C39, the content of diosgenin was improved. Sowe found the new resourses for the production of diosgenin.
2.Materials and methods
2.1.Chemicals and materials
2.1.1.Medicinalmaterials: Rhizomes of D.zingiberensis were obtained from Bazhong (Sichuan Province, China, 2011) and identified by Professor Xiaowei Du.
2.1.2.Strains: C39 was isolated fromD.nipponica Makino by our laboratory.
2.1.3. Chemicalreagents: 95% ethanol, dichloromethane, hydrochloric acid, methyl alcohol (chromatographic grade), Wa Haha purified water.
2.2.Preparation of the medium
2.2.1.PDA medium
3
The potatoes (200g) was peeled, and cut into pieces about 0.5cm .Put them into 1500ml beaker with 1000ml distilled water. After the water boiling, timed 30 minutes. Then filtered it with eight layers of gauze, added glucose and agar 20g and 15g respectively .And then added water until 1000ml. The pH of it was natural.
2.2.2.PD medium
The preparation of PD medium was as same as PDA medium but not adding agar.
2.2.3.Medicinal matrix medium
20ml distilled water was added into 10g medicinal powder to wetting it before moist heat sterilization for 30 minutes.
2.3.The activation of C39 and the preparation of the C39 seed liquid
2.3.1.Activation of endophyte fungi
The endophytic fungi C39 preserved in the refrigerator at 4°C was inoculated into fresh PDA solid plate medium under sterile conditions and put in constant temperature incubator at 28° for 4 to 5 days until the strains grew to 3/4 of the plate.
2.3.2.Preparation of seed liquid of C39
Bacteria cake punched with the puncher from the edge of good growing strains was took into 250ml flask with 100ml PD liquid medium. Several 0.5cm diameter glass beads was added into the flask to make mycelium dispersed well when shaking. After 12 hours standing to adapt to the environment, the flask was placed in shaking incubator. The rotational speed was 120r/min. The fermentation period was 3 days at 28° .
2.4.Solid fermentation of C39
The dried rhizomes of D.zingiberensis crushed and 10g were weighted out and put into 500ml flask. 20ml distilled water was added into the flask to wet it for 12 hours. After moist heat sterilization for 30 minutes at 121° and cooled, the seed liquid of C39 2ml was inoculated on the medicinal matrix medium. And then it was cultured statically in constant temperature and humidity incubator for 15 days at 28°.
The medicinal power of D.zingiberensis without any strain was cultured 15 days in the same conditions as sterilization blank.
2.5.Dispose of the fermentation
At the end of incubation, the fermentation was dried at 50° for 48 hours in constant temperature drying oven. And then 100ml 95% ethanol was added for ultrasound (frequency 50Hz, temperature 20°) 50 minutes before it was filtered. 100ml 95% ethanol was added into the filter residue for ultrasound again and then filtered. The two filtrates were combined and concentrated with rotary evaporators to dryness. The residue was hydrolyzed and refluxed with 2.5mol/L hydrochloric acid under boiling water for 3 hours. It was extracted three times with an equal amount of dichloromethane after cooled. The dichloromethane layers were combined, washed with distilled water until neutral,and concentrated it to dryness. The residue was metered to the volume of 50ml with methanol.
The blank samples were disposed as the same as the fermentation samples.
2.6.Content assaying of diosgenin
2.6.1.Standard curve of diosgenin
Standard sample of diosgenin was weighted and added methanol to make the concentration of the solution 0.706mg/ml.
The chromatographic conditions was as follows: the eluent was methanol (91%) and H2O (9%) under isocratic elution. The column was eluted at 30°. The flow rate of the mobile phase was 1.0ml min-1 and the sample was detected at 203nm. The standard solution of the diosgenin was injected 5^L,10^L, 15^L, 20^L, 25^L, 30p,L, 35^L and the peak areas of diosgenin was measured respectively. The standard curve was drawn by defining the peak area as Y axis and the mass of the diosgenin as X axis.
The preparation of test solution:10g D.zingiberensis powder were weighted and 10ml 95% ethanol was added. Then it was extracted by ultrasonic for 50 minutes twice. The two extracting solution were combined and concentrated to dryness. The residue was hydrolyzed with 2.5mol/L hydrochloric acid for 3 hours. It was extracted three times with an equal amount of dichloromethane
after cooled. The dichloromethane layers were combined and concentrated to dryness. The residue was metered to volume of 25ml with methanol.
The research of methodological study was repeatability, precision, stability and recoveries
2.6.2. Content assaying of diosgenin in fermentation
Samples were filtered and analyzed on Shimadzu LC-2010A with a Diamonsil C18 column,5p,m,250mm*4.6mm.The chromatographic conditionswas as same as previously.
3.Results and discussion
3.1. Standard curve of diosgenin
The result of the standard curve of diosgenin was shown in Figure 1. The equation of linear regression was Y=315482X-608540 (R=0.9996), which indicated that diosgenin in the good linearity ranges from 3.53pg to 24.71pg.
8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0
Area
y = 315482x - 608540 R = 0.9996
10
Mass of the diosgenin (pg/mg)
15
20
25
30
0
5
Fig.1 Standard curve of diosgenin
3.2.Methodology study
3.2.1. Repeatability
5 copies of D.zingiberensis powder were weighted (1g each). The preparation of the sample solution was according to the test solution. Then the samples were injected 20^L and the content of diosgenin was determined. The standard deviation was 0.715% by calculating, which indicate that the repeatability of this determination method was good.
3.2.2.Stability test
1g D.zingiberensis powder was weighted and the preparation of the sample solution was according to the test solution. The sample was injected at 0h,6h 12h 18h and 24h, respectively. The injected volume of the sample was 20^L. Then the content of diosgenin was determined. The standard deviation was 0.542% by calculating, which indicated that the stability of this determination method was good.
3.2.3.Precision experiments
The standard solutions of diosgenin was taken precisely 20^L and injected 5 times. The area of the peak was determined. The standard deviation was 0.205% by calculating, which indicated that the precision of this determination method was good.
3.2.4.Recovery experiment
5 copies of D.zingiberensis powder were weighted precisely (0.4g each) and a certain amount of standard solution was added. The preparation of the sample solution was according to the test solution. The injected volume of the sample was 20^L. Then the content of diosgenin was
129.21
73.33
determined. The standard deviation was 0.955% and the average recovery was 100.004% by calculating, which indicated that the result of this determination method was accurate. 3.3.The content change of diosgenin before and after fermentation
In this experiment, we took D.nipponica Makino as a reference and took D.zingiberensis without C39 sample as blank to investigate the effect of C39 on diosgenin in D.zingiberensis .The results were shown in Table 1. From Table 1, the endophytic fungi C39 isolated from D.nipponica Makino can not only improve the content of diosgenin in D.zingiberensis but also make the growth rate 129.21%.
Table 1 Content change of diosgenin by solid fermentation of C39
Samples Average area of Content of Growth rate
Samples peak diosgenin (mg/g) (%)
Blank of D.zingiberensis 2356466 4.699
Fermentation of D.zingiberensis 6187296 10.771
Blank of D.nipponica 2612337 5.105
Fermentation of D.nipponica 4974133 8.848
4.Conclusion
In this study, the technology of biotransformation that endophyte fungi C39 can improve the content of diosgenin in D.nipponica Makino was apply to D.zingibenisis C.H.Wright which have the same or similar substrates to D.nipponica Makino and a new resourse of diosgenin production was opened up. D.zingibenisis C.H.Wright and D.nipponica Makino are both part of the genus of Dioscorea and their main ingredients are similar. C39 can transform some of substrates in the D.nipponica to diosgenin and also can increase the content of diosgenin in the D.zingibenisis C.H.Wright. This phenomenon explained that C39 transform some substrates in D.zingibenisis C.H.Wrigh which are similar to them in D.nipponica Makino. In the future studies, we need to study more about the mechanism of biotransformation by C39 and find the precursors of biotransformation in order to apply this technology to other herbs which have the similar substrates and expand the pathway of diosgenin production.
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