Authentication of herbal substances from TCM by ITS barcoding

4.2 Case study 2: Authentication of herbal substances from TCM

4.2.1 Authentication of herbal substances from TCM by ITS barcoding

already been started in former PhD studies (Daniel, 2009; Kersten, 2013; Orland et al., 2014). The results from bioinformatic data analysis using NCBI nucleotide blast alignment are displayed in table 26. If DNA isolation was successful, in most cases the labeled species could be confirmed by sequencing and BLAST-analysis results. Also five processed herbal substances were amongst the successfully identified samples, namely Coicis semen, Gardeniae fructus praeparata, Schisandrae chinensis fructus, Zanthoxyli pericarpium and Ziziphi spinosae semen.

The sample of Anemarrhenae rhizoma exhibited the highest similarity with Coix lacryma, but the maximum identity of 81% is not a significant value. This may indicate, that we obtained new sequence data, as there are no ITS sequence data for Anemarrhena aspheloides and even not for the whole genus Anemarrhena. There is one sequence in NCBI that should correspond to ITS data for A. aspheloides, but this sequence shows 100% congruence with the fungus Antherospora vindobonensis (Accession: JN104591 Name: Floromyces anemarrhenae, source host="Anemarrhena aspheloides"). This sequence entry seems to correspond to an adulteration with this fungus, thus, does not correspond to the plant A. aspheloides.

Regarding the low number of successfully sequenced samples it is obvious that identification of samples from TCM is difficult (Daniel, 2009; Kersten, 2013). In total,

73 60 samples were investigated, but only for 31 samples sequence data could be obtained, including five putative adulterations. However, 33 samples were processed, mostly including complex procedures with roasting, boiling, steaming or burning (see table 26, Pao Zhi treatments). Such processing procedures may have led to a degradation of DNA in these samples, thereby impeding successful PCR reactions and sequencing. This assumption is further supported by the fact, that mostly the DNA isolation and subsequent reactions failed for those samples that were processed (see table 26).

From table 26 it can be derived that all investigated Aconite species yielded no PCR result, and a DNA measurement also confirmed that DNA isolation was not successful in all cases. Aconite species contain toxic diterpene alkaloids, like aconitine, hypaconitine and mesaconitine (Chan, 2009; Niitsu et al., 2013). For reducing these toxic compounds, crude aconite roots have to be decocted for one to two hours (Chan, 2011; Singhuber et al., 2009). This kind of detoxification may lead to a degradation of DNA and be the reason for the failure of the proximate PCR-reaction, as all Aconite species investigated here were processed. Similar processing procedures were applied to many other herbal substances investigated here, and in many cases, DNA isolation with standard methodology was not successful (e.g. Anemarrhenae rhizoma, Aucklandiae radix, Gastrodiae rhizoma, Magnoliae officinalis cortex, Rehmanniae radix, Saposhnikoviae radix, Scutellariae radix).

In summary, all adulterations found are native to China (Duke and Ayensu, 1985; Tian et al., 2013; Turner and Fearing, 1964), hence a contamination of the medicinal plants, e.g. during harvesting of the plants seems plausible.

Table 26: List of NCBI nucleotide blast alignment results for DNA sequences of herbal substances belonging to TCM. (Unexpected results shown in blue. Maximum identity [Max. id.]:

the extent to which two nucleotide sequences have the same residues at the same positions in an alignment, expressed as a percentage. (http://www.ncbi.nlm.nih.gov/books/NBK62051/ 2014)

No. EDQM Sample (EDQM code)

Pao Zhi (Common processing procedures of the herbal substances (Körfers and Sun, 2008)

Result of NCBI nucleotide blast alignment

Max.

Id (%)

Highest homology to accession no.

(NCBI database)

1 Acanthopanacis cortex (32961)

Not common Eleutherococcus

gracilistylus (syn.

Acanthopanax gracilistylus)

98 FJ980422

2 Acanthopanax giraldii harms cortex (32987)

Not common Eleutherococcus

gracilistylus (syn.

Acanthopanax gracilistylus)

99 FJ980422

3 Acanthopanacis cortex (32527)

Not common No result 4 Aconiti kusneoffii

radix preparata (33022)

Zhi Chuan Wu / Sheng Chuang Wu: