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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.


Micrographs show powders of ATR and its adulterants. AATR Acori Tatarinowii Rhizoma; BAGR Acori Graminei Rhizoma; CACR Acori Calami Rhizoma; DAAR Anemones Altaicae Rhizoma. Notations are: 1 crystal fibers; 2 starch granules; 3 secretory cells; 4 vessels; 5 epidermal cells of leaf sheath; 6 fibers; 7 stone cell; 8 parenchymatous cell. a Indicated features under the light microscope; b Indicate features under the polarized microscope
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Fig3: Micrographs show powders of ATR and its adulterants. AATR Acori Tatarinowii Rhizoma; BAGR Acori Graminei Rhizoma; CACR Acori Calami Rhizoma; DAAR Anemones Altaicae Rhizoma. Notations are: 1 crystal fibers; 2 starch granules; 3 secretory cells; 4 vessels; 5 epidermal cells of leaf sheath; 6 fibers; 7 stone cell; 8 parenchymatous cell. a Indicated features under the light microscope; b Indicate features under the polarized microscope

Mentions: There were no significant differences in the microscopic features of the ATR, AGR and ACR powders. The fibers were mostly bundled, colorless or pale yellow in color, tapered towards the ends and lignified with distinct pit canals. Fibrous bundles surrounded by cells containing calcium oxalate prisms, which formed crystalline fibers. Polyhedral, sub-polygonal or polycone-like calcium oxalate prisms, which appeared yellowish-white or polychromatic under a polarized microscope. Simple starch granules, which were ellipsoid, spheroidal, long-ovoid, hilum pointed, V-shaped or short slit-shaped with indistinct striation marks; black and cruciate-shaped under a polarized microscope; compound starch granules composed of 2–20 units. Secretory cells abundant, sub-rounded or elongated-rounded, filled with orange-yellow secretions. The epidermal cells of the leaf sheath were greyish-green or pale yellowish-brown and rectangular in shape. The vessels were mainly reticulate (Fig. 3A–C).Fig. 3


Authentication of Acori Tatarinowii Rhizoma ( Shi Chang Pu ) and its adulterants by morphological distinction, chemical composition and ITS sequencing
Micrographs show powders of ATR and its adulterants. AATR Acori Tatarinowii Rhizoma; BAGR Acori Graminei Rhizoma; CACR Acori Calami Rhizoma; DAAR Anemones Altaicae Rhizoma. Notations are: 1 crystal fibers; 2 starch granules; 3 secretory cells; 4 vessels; 5 epidermal cells of leaf sheath; 6 fibers; 7 stone cell; 8 parenchymatous cell. a Indicated features under the light microscope; b Indicate features under the polarized microscope
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Micrographs show powders of ATR and its adulterants. AATR Acori Tatarinowii Rhizoma; BAGR Acori Graminei Rhizoma; CACR Acori Calami Rhizoma; DAAR Anemones Altaicae Rhizoma. Notations are: 1 crystal fibers; 2 starch granules; 3 secretory cells; 4 vessels; 5 epidermal cells of leaf sheath; 6 fibers; 7 stone cell; 8 parenchymatous cell. a Indicated features under the light microscope; b Indicate features under the polarized microscope
Mentions: There were no significant differences in the microscopic features of the ATR, AGR and ACR powders. The fibers were mostly bundled, colorless or pale yellow in color, tapered towards the ends and lignified with distinct pit canals. Fibrous bundles surrounded by cells containing calcium oxalate prisms, which formed crystalline fibers. Polyhedral, sub-polygonal or polycone-like calcium oxalate prisms, which appeared yellowish-white or polychromatic under a polarized microscope. Simple starch granules, which were ellipsoid, spheroidal, long-ovoid, hilum pointed, V-shaped or short slit-shaped with indistinct striation marks; black and cruciate-shaped under a polarized microscope; compound starch granules composed of 2–20 units. Secretory cells abundant, sub-rounded or elongated-rounded, filled with orange-yellow secretions. The epidermal cells of the leaf sheath were greyish-green or pale yellowish-brown and rectangular in shape. The vessels were mainly reticulate (Fig. 3A–C).Fig. 3

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.