Preliminary Studies On Metabolic Mechanism Of Cantharides By Photosynthetic Bacteria

Cantharides has become a hot research of modern anti-cancer drug because of its unique curative effect on various cancer.Cantharidin is the main effective component in cantharides,however,the application of cantharides was limited for the strong toxicity of cantharidin.Our research group taking advantage of the biotransformation capacity from photosynthetic bacteria(PSB),the cantharides were transformed by PSB.Prophase research proved that the biotransformation of cantharides by PSB can achieve the effect of reducing toxicity,but the mechanism is not clear.Based on the prophase research,the paper makes a preliminary study on basic mechanism of the biotransformation of cantharides by PSB through analyzing the enzyme activity,isozyme spectrum and the main active components of PSB.Main study results in this paper are displayed as follows:1.The p H value of culture medium and the determination of protein concentration of PSB before and after metabolism: The p H value of the culture solution was measured and the growth and survival of PSB before and after metabolism was investigated by adopting dilution plate.p H change shows that the formation of acidic metabolites leads to a decrease in p H(0-3 days),the formation of alkaline metabolites leads to p H elevation(4-10 days).The results of the protein concentration of PSB shows that the protein concentration of PSB increased after metabolism,which may be due to the growth environment changes-the participation of cantharides,PSB was forced to make emergency response.2.Analysis of Co-Q10 and oxygenase activity of PSB before and after metabolism: In order to optimize the ultrasonic assisted extraction of Co-Q10,Box-Behnken response surface methodology was used.The HPLC method was also used to determine the content of Co-Q10 and the consumption of indole in the experiment of oxygenase activity.The optimal extraction conditions are: ultrasonic power 250 W,ultrasonic extraction time at a time 10 min and ultrasonic radiation time at a time 8 s.Due to the participation ofcantharides,the concentration of Co-Q10 in PSB was increased.The amount of substrate converted by oxygenase was indirectly evaluated through determination of indole conversion,for further understanding the process of PSB transforming cantharides.3.Quality control of cantharides transformation products and cantharidin: In order to further understand the process of PSB transforming cantharides,the content changes of cantharides transformation products and cantharidin were measured before and after metabolism,so that having certain quality control on them.On the one hand,it indicated that PSB played a role on the cantharides,and the direction of effect tended to reduce toxicity.On the other hand,our previous experiments showed that the antitumor effect of transformation liquid didn't change obviously.It may be indicated that the other antitumor substances generated or their content rose.The in vitro antitumor activity of experimental group showed that compound A,compound B and compound C all had obvious inhibitory effect on lung cancer A549 and cervical cancer Hela cells,and HPLC was used to detect changes of the content of these compounds.4.Isozyme activity: The esterase(EST)activity in PSB before and after transformation decreased gradually,and the EST activity in the experimental group was significantly higher than that in the blank group(P<0.05).The peroxidase(POD)activity in the blank group and the experimental group all firstly rose and then declined,and the POD activity of the experimental group was higher than that of the blank group,which showed a significant difference(P<0.01).The superoxide dismutase(SOD)activity in the blank group and the experimental group all firstly increased and then decreased,and the SOD activity of the experimental group was higher than that of the blank group,which showed a significant difference(P<0.01).Therefore,EST,POD and SOD may be play a role in the process of the biotransformation of cantharides by PSB.5.Changes of isozymograms of PSB: The enzyme activity of EST,POD and SOD before and after metabolism showed on isozymograms were consistent with the above results.EST and SOD isozymograms showed that there was a new EST band and SOD band respectively appearing in the the experimental group compared with control group.Some EST and SOD in PSB began to express in the induction of components of cantharides,which may be involved in the biotransformation.According to POD isozymograms,only the color of POD band was deepened or changed between blank group and experimental group,no new bands appearing or disappearing.Therefore,Effect of cantharides on expression of POD bands of PSB was not obvious.