CAPÍTULO I. FUNDAMENTACIÓN TEÓRICA
1.5 Palas. Elementos de las palas
Defined mixtures of herbal substances (table 38), were analyzed with the qPCR assay (see figure 43 and figure 44). Figure 44 shows the Cq values obtained when the defined mixtures were investigated. As shown in the Cq-diagram, all five components of the mixture could be reliably verified by qPCR. While in the before mentioned cloning approach (4.5.1) and the multiplex PCR approach (4.5.2) not all components of the DMHS were detected, with the qPCR approach all plants of all mixtures (DMHS 1 to DMHS 5) were traceable.
For the component A. clematitis, Cq values in the range of 25.78 and 26.26 were obtained for mixtures DMHS 1 to DMHS 4, while the Cq value for mixture DMHS 5 was considerably higher with 27.86, which correlates to the low amount of only 4% of the plant in this mixture (table 38). J. regia in general had very low Cq values, especially in mixture DMHS 5, which corresponds to the high abundance of 230 mg of plant material in this mixture. DMHS 4 shows the highest Cq values for J. regia with a Cq of 22.54, conforming to the low percentage of 7% of plant material of J. regia in mixture DMHS 4. Compared to the Cq values and DNA concentrations from the dilution series (table 41), the Cq value for J. regia in mixture DMHS 4 corresponds to a DNA concentration of about 2 ng DNA, which is approximately 10-fold lower than in the other four investigated mixtures. For M. recutita Cq values between 22.57 for mixture DMHS 5 and 24.97 for DMHS 1 were obtained. These values correspond to the amounts of plant material, used for the DNA isolation of the samples. The highest Cq value, meaning the lowest DNA concentration obtained, corresponds nearly to the highest DNA concentration of 20 ng DNA used for the dilution series. As described in table 38, the amount of plant material of S. miltiorrhiza used for the different mixtures DMHS 1 to DMHS 5 does not vary very much. This correlates to the Cq values obtained for S. miltiorrhiza which are in a range of 24.96 (DMHS 1) and 27.04 (DMHS 4). In case of Q. robur, the Cq value for mixture DMHS 5 is not considered, as the performance of the qPCR cannot be evaluated, as shown in the amplification curve for Q. robur in DMHS 5 (see figure 43). The Cq values for Q. robur, are in a range of 22.33 (DMHS 1) to 25.01 (DMHS 4), whereby the high Cq value of mixture DMHS 4 correlates with the low abundance of Q. robur plant material used for this mixture (13 %, see table 38).
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a) b)
c) d)
e)
Figure 43: Amplification curves obtained from the analysis of defined mixtures of herbal substances with multiplex qPCR (DMHS 1 to DMHS 5, according to table 10). Figures a) to e) show the amplification curves obtained at the different fluorescences according to the reporter dye used respectively. Detection of a) M. recutita, b) A. clematitis, c) Q. robur, d) J. regia, e) S.
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Figure 44: Cq values of the five plant components of defined mixtures of herbal substances (DMHS 1 to DMHS 5, according to table 10) investigated with the established multiplex qPCR assay (Results are depicted on an interrupted y-axis, as the major changes occur with Cq-values starting at a Cq value of about 19; samples investigated in triplicates).
4.5.4.1 Multiplex qPCR investigation of the herbal medicinal product Imupret®
The herbal medicinal product Imupret® was already investigated by the cloning method in part 4.5.1. It is composed of the seven medicinal plants Matricaria recutita, Althaea officinalis, Equisetum arvense, Achillea millefolium, Juglans regia, Taraxacum officinale and Quercus robur. Consequently, the established multiplex qPCR should only detect the three components J. regia, Q. robur and M. recutita.
As shown in the Cq-diagram in figure 46, the latter three samples were detected in all four Imupret® samples investigated, while S. miltiorrhiza and A. clematitis were negative. All samples shown here were run with one master mix. The positive control (PC; figure 46) shows signals for the five components of the mixture, while A. clematitis and S. miltiorrhiza are missing in the analyzed Imupret® samples. J. regia, Q. robur and M. recutita show relatively similar and constant levels of amplification in all Imupret® samples, whereby Q. robur exceeds the threshold above the background
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Despite the low quantities of the component Q. robur, all plant components in the Imupret® samples, that were detectable by appropriate primers in this approach, could be verified reliably by the established multiplex qPCR.
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a) b)
c)
d)
e)
Figure 45: Amplification curves obtained from qPCR investigation of DNA isolated from the finished herbal medicinal product Imupret®. Identification of a) A. clematitis, b) Q. robur, c) J.
regia, d) S. miltiorrhiza, e) M. recutita. Figures a) to e) show the amplification curves obtained at
the different fluorescences according to the reporter dye used respectively. Detection of a) M.
recutita, b) A. clematitis, c) Q. robur, d) J. regia, e) S. miltiorrhiza. (Each amplification curve
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Figure 46: Cq-values of four Imupret® samples investigated with the here established multiplex qPCR assay. PC = Positive Control; Imupret® 1, 2, 3 and 4 represent four independently taken samples from which DNA was isolated with the QIAGEN Plant Mini Kit (Imupret® 1, 2) and the Invitek Stool Kit (Imupret® 3, 4). (Results are depicted on an interrupted y-axis, as the major changes occur with Cq-values starting at a Cq value of about 19; samples investigated in triplicates).
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