Cephaeline medium

In order to determine the rate of recovery, accurate amounts of emetine and cephaeline were added to the medium used for root culture where the adventitious roots of ipecac were cultured for 8 weeks and then extracted as follows twenty ml medium was mixed with 200 pi 10% ammonium hydroxide and 6 ml diethylether in a test tube for 5 min using vortex mixer. Four ml upper layer was accurately taken and concentrated under a stream of nitrogen gas. The extract was dissolved in an appropriate volume of methanol and quantitatively analyzed by HPLC. The HPLC system gave good recovery 104.4% for emetine and 104.8% for cephaeline (Table 6). 

Table 6. Recovery of emetine and cephaeline from the medium used for root culture of C. ipecacuanha...

The alkaloid contents of adventitious shoots in liquid medium were quantified by HPLC, Fig. (20). They produced alkaloids, especially cephaeline (0.4 - 0.6% dry weight), and there was no obvious difference in alkaloid contents among different concentrations of BA. This result indicates that ipecac shoots, formed on intemodal segments, have the capabilities of alkaloid biosynthesis. 

Although the emetine and the cephaeline could be detected in the first passage calli, they were undetectable by the third passage (section 2.1). However, root culture, especially root culture in liquid medium, showed consistent levels of alkaloids even after the third culture passage. In order to increase the yield of the alkaloids in root culture, culture conditions, such as various ph5dohormones and basal media were investigated [15, 54]. 

The effects of lAA, NAA and 2,4-D on the growth and alkaloid yields of root cultures in MS liquid medium are shown in Fig. (31). The cultured roots produced much more cephaeline than emetine, although the rootlets of the parental plant cultivated in a greenhouse contained almost the same amounts of emetine and cephaeline, i.e. 1.6% and 1.9% dry weight, respectively (Table 4). The roots grew well in a liquid medium in the presence of 5 mg/1 NAA, while the roots formed calli and swelled in the presence of 10 mg/1 lAA, and NAA and 0.5 mg/1 2,4-D and were not suitable for the alkaloid formation. Therefore, organized structures such as roots may be important for the biosynthesis of emetine and cephaeline. 

A one-year-old Cephaelis ipecacuanha plant propagated through shoot-tip culture (section 1.1) in a greenhouse accumulated ca. 2 mg emetine and ca. 2 mg cephaeline, respectively, in its roots ca. 0.2 g total dry weight). On the other hand, the root culture of ipecac cultured for 7 weeks in 50 ml MS medium containing 0.01 mg/1 5,6-Cl2-IAA yielded almost the same amounts of alkaloids, 0.6 mg emetine and 2.4 mg cephaeline. Thus the root culture of ipecac is a realistic economic source of the isoquinoline alkaloids. 

Cultural conditions are the same as Fig. (54). Bars represent standard deviations of the mean. Left column protoemetine, center column cephaeline, right column emetine. Upper column alkaloid in roots, lower column alkaloid in medium. 

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