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Authentication of Acori Tatarinowii Rhizoma ( Shi Chang Pu ) and its adulterants by morphological distinction, chemical composition and ITS sequencing

View Article: PubMed Central - PubMed

ABSTRACT

Background: Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott) (Shi Chang Pu) is widely used in Chinese medicine (CM) to resuscitate, calm the mind, resolve shi (dampness) and harmonize the wei (stomach). Seven different species have been identified as belonging to the genus Acorus, all of which can be found in China. However, it can be difficult to distinguish the different species of Acorus because of their morphological similarities. The aim of this study was to authenticate Acorus species using macroscopic and microscopic techniques, chemical analysis and DNA authentication and to compare the resolution power and reliability of these different methods.

Methods: Four batches of ATR, Acori Graminei Rhizoma (AGR), Acori Calami Rhizoma (ACR) and Anemones Altaicae Rhizoma (AAR) (totaling 16 samples) were collected from Hong Kong and mainland China. The major characteristic features of these Acorus species were identified by macroscopic and microscopic examination. The identified samples were also analyzed by UHPLC analysis. Principal component analysis (PCA) and hierarchal clustering analysis (HCA) on UHPLC results were used to differentiate between the samples. An internal transcribed spacer (ITS) was selected as a molecular probe and a modified DNA extraction method was developed to obtain trace amounts of DNA from the different Acorus species. All extracted DNA sequences were edited by Bioedit and aligned with the ClustalW. And the sequence distances were calculated using the Maximum Parsimony method.

Results: Macroscopic and microscopic analyses allowed for AAR to be readily distinguished from ATR, AGR and ACR. However, it was difficult to distinguish between ATR, AGR and ACR because of their similar morphological features. Chemical profiling revealed that α- and β-asarone were only found in the ATR, AGR and ACR samples, but not in the AAR samples. Furthermore, PCA and HCA allowed for the differentiation of these three species based on their asarone contents. Morphological authentication and chemical profiling allowed for the partial differentiation of ATR, AGR ACR and AAR. DNA analysis was the only method capable of accurately differentiating between all four species.

Conclusion: DNA authentication exhibited higher resolution power and reliability than conventional morphological identification and UHPLC in differentiating between different Acorus species.

Electronic supplementary material: The online version of this article (doi:10.1186/s13020-016-0113-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


UHPLC-DAD chromatograms of extracts from ATR and its adulterants. a UHPLC-DAD chromatograms of 16 samples (number 1–16 for different samples, shown in Table 1) at 270 nm were shown. Upper panel shows the markers, α-asarone and β-asarone. b Score plots for ATR and its adulterants, using peak areas of α-asarone and β-asarone as input data, were shown. c Hierarchical clustering analysis for the 16 samples. The loading plot was performed with the original peak areas of α-asarone and β-asarone as input data
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Fig4: UHPLC-DAD chromatograms of extracts from ATR and its adulterants. a UHPLC-DAD chromatograms of 16 samples (number 1–16 for different samples, shown in Table 1) at 270 nm were shown. Upper panel shows the markers, α-asarone and β-asarone. b Score plots for ATR and its adulterants, using peak areas of α-asarone and β-asarone as input data, were shown. c Hierarchical clustering analysis for the 16 samples. The loading plot was performed with the original peak areas of α-asarone and β-asarone as input data

Mentions: α-Asarone and β-asarone, which are the most abundant chemicals in these plant species, were used as chemical markers for ATR. These chemicals have also been reported to exhibit biological functions [13–16]. The samples were analyzed using a DAD detector according to the popular criteria. The detection wavelength of the DAD detector was set to 270 nm to acquire the most abundant peaks. Typical chromatograms of ATR, AGR, ACR and AAR from samples 1–16 are shown in Fig. 4a. The results revealed that there were considerable differences in the chromatographic profiles of the ATR, AGR and ACR extracts compared with the AAR extract. The majority of the α-asarone and β-asarone components were eluted in less than 30 min from the ATR, AGR and ACR extracts. In contrast, the chromatogram of the AAR extract did not contain any α-asarone or β-asarone based on a comparison of its retention times and UV spectra with those of the standard compounds (Fig. 4a). The chromatographic fingerprints of all of the extracts were shown to be highly stable and reproducible using the “Similarity evaluation system for the chromatographic fingerprints of TCM” software, which was developed by the Chinese Pharmacopeia Commission. The application of PCA to the chromatographic fingerprints summarized most of the UHPLC-DAD data into the first two principle components, PC1 and PC2, with two-dimensional score plots showing clear differences between the different samples. It is noteworthy that PC1 and PC2 covered more than 90 % of the total variability. Data for α-asarone and β-asarone were found to be distinct from those of the main cluster profiles of the ATR, AGR, ACR and AAR extracts. The scatter points showed that the samples could be readily classified into four different groups, indicating that it was possible to differentiate between the different plants. The α-asarone and β-asarone contents of the different extracts could therefore be used to discriminate ATR from AGR, ACR and AAR (Fig. 4b). Hierarchical clustering analysis (HCA) was also applied to differentiate between the different extracts using Pearson’s correlation as a measurement (Fig. 4c). In a similar manner to the PCA results, HCA allowed for most of the samples to be classified into two groups, including cluster 1 [samples 1–4 (ATR)] and cluster 2 [samples 5–16 (AGR, ACR and AAR)]. This clustering agreed well with the results of ATR and non-ATR samples. Taken together, these results suggest that ATR can be readily differentiated from the other three Acorus species based on differences in their asarone contents. PCA and HCA allowed for the different species to be partially distinguished by visual inspection for conventional quality control purposes.Fig. 4


Authentication of Acori Tatarinowii Rhizoma ( Shi Chang Pu ) and its adulterants by morphological distinction, chemical composition and ITS sequencing
UHPLC-DAD chromatograms of extracts from ATR and its adulterants. a UHPLC-DAD chromatograms of 16 samples (number 1–16 for different samples, shown in Table 1) at 270 nm were shown. Upper panel shows the markers, α-asarone and β-asarone. b Score plots for ATR and its adulterants, using peak areas of α-asarone and β-asarone as input data, were shown. c Hierarchical clustering analysis for the 16 samples. The loading plot was performed with the original peak areas of α-asarone and β-asarone as input data
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Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5037583&req=5

Fig4: UHPLC-DAD chromatograms of extracts from ATR and its adulterants. a UHPLC-DAD chromatograms of 16 samples (number 1–16 for different samples, shown in Table 1) at 270 nm were shown. Upper panel shows the markers, α-asarone and β-asarone. b Score plots for ATR and its adulterants, using peak areas of α-asarone and β-asarone as input data, were shown. c Hierarchical clustering analysis for the 16 samples. The loading plot was performed with the original peak areas of α-asarone and β-asarone as input data
Mentions: α-Asarone and β-asarone, which are the most abundant chemicals in these plant species, were used as chemical markers for ATR. These chemicals have also been reported to exhibit biological functions [13–16]. The samples were analyzed using a DAD detector according to the popular criteria. The detection wavelength of the DAD detector was set to 270 nm to acquire the most abundant peaks. Typical chromatograms of ATR, AGR, ACR and AAR from samples 1–16 are shown in Fig. 4a. The results revealed that there were considerable differences in the chromatographic profiles of the ATR, AGR and ACR extracts compared with the AAR extract. The majority of the α-asarone and β-asarone components were eluted in less than 30 min from the ATR, AGR and ACR extracts. In contrast, the chromatogram of the AAR extract did not contain any α-asarone or β-asarone based on a comparison of its retention times and UV spectra with those of the standard compounds (Fig. 4a). The chromatographic fingerprints of all of the extracts were shown to be highly stable and reproducible using the “Similarity evaluation system for the chromatographic fingerprints of TCM” software, which was developed by the Chinese Pharmacopeia Commission. The application of PCA to the chromatographic fingerprints summarized most of the UHPLC-DAD data into the first two principle components, PC1 and PC2, with two-dimensional score plots showing clear differences between the different samples. It is noteworthy that PC1 and PC2 covered more than 90 % of the total variability. Data for α-asarone and β-asarone were found to be distinct from those of the main cluster profiles of the ATR, AGR, ACR and AAR extracts. The scatter points showed that the samples could be readily classified into four different groups, indicating that it was possible to differentiate between the different plants. The α-asarone and β-asarone contents of the different extracts could therefore be used to discriminate ATR from AGR, ACR and AAR (Fig. 4b). Hierarchical clustering analysis (HCA) was also applied to differentiate between the different extracts using Pearson’s correlation as a measurement (Fig. 4c). In a similar manner to the PCA results, HCA allowed for most of the samples to be classified into two groups, including cluster 1 [samples 1–4 (ATR)] and cluster 2 [samples 5–16 (AGR, ACR and AAR)]. This clustering agreed well with the results of ATR and non-ATR samples. Taken together, these results suggest that ATR can be readily differentiated from the other three Acorus species based on differences in their asarone contents. PCA and HCA allowed for the different species to be partially distinguished by visual inspection for conventional quality control purposes.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Acori Tatarinowii Rhizoma (ATR; rhizome of Acorus tatarinowii Schott) (Shi Chang Pu) is widely used in Chinese medicine (CM) to resuscitate, calm the mind, resolve shi (dampness) and harmonize the wei (stomach). Seven different species have been identified as belonging to the genus Acorus, all of which can be found in China. However, it can be difficult to distinguish the different species of Acorus because of their morphological similarities. The aim of this study was to authenticate Acorus species using macroscopic and microscopic techniques, chemical analysis and DNA authentication and to compare the resolution power and reliability of these different methods.

Methods: Four batches of ATR, Acori Graminei Rhizoma (AGR), Acori Calami Rhizoma (ACR) and Anemones Altaicae Rhizoma (AAR) (totaling 16 samples) were collected from Hong Kong and mainland China. The major characteristic features of these Acorus species were identified by macroscopic and microscopic examination. The identified samples were also analyzed by UHPLC analysis. Principal component analysis (PCA) and hierarchal clustering analysis (HCA) on UHPLC results were used to differentiate between the samples. An internal transcribed spacer (ITS) was selected as a molecular probe and a modified DNA extraction method was developed to obtain trace amounts of DNA from the different Acorus species. All extracted DNA sequences were edited by Bioedit and aligned with the ClustalW. And the sequence distances were calculated using the Maximum Parsimony method.

Results: Macroscopic and microscopic analyses allowed for AAR to be readily distinguished from ATR, AGR and ACR. However, it was difficult to distinguish between ATR, AGR and ACR because of their similar morphological features. Chemical profiling revealed that α- and β-asarone were only found in the ATR, AGR and ACR samples, but not in the AAR samples. Furthermore, PCA and HCA allowed for the differentiation of these three species based on their asarone contents. Morphological authentication and chemical profiling allowed for the partial differentiation of ATR, AGR ACR and AAR. DNA analysis was the only method capable of accurately differentiating between all four species.

Conclusion: DNA authentication exhibited higher resolution power and reliability than conventional morphological identification and UHPLC in differentiating between different Acorus species.

Electronic supplementary material: The online version of this article (doi:10.1186/s13020-016-0113-x) contains supplementary material, which is available to authorized users.

No MeSH data available.