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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 68  |  Page : 64-68  

Rapid identification of the indigenous medicinal crop Cinnamomum osmophloeum from various adulterant Cinnamomum species by DNA polymorphism analysis


1 Department of Chinese Pharmaceutical Science and Chinese Medicine Resources, China Medical University, Taichung, Taiwan
2 Department of Bioresources, Da-Yeh University, Changhua, Taiwan
3 Department of Safety, Health and Environmental Engineering, Mingchi University of Technology, Taipei, Taiwan

Date of Submission24-Jun-2019
Date of Decision16-Sep-2019
Date of Web Publication31-Mar-2020

Correspondence Address:
Meng-Shiunn Lee
91, Hsueh-Shih Road, Taichung
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_267_19

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   Abstract 


Background: Cinnamomum osmophloeum (Co), a member of the Lauraceae, is an indigenous medicinal crop in Taiwan, and it contains higher cinnamaldehyde in essential oil than do other Cinnamomum species. Among these species, Cinnamomum burmannii (Cb) is frequently adulterated as Co because of their similar morphological characteristics or features. Objective: The purpose of this study was to develop a DNA-based molecular method for rapid authentication of the indigenous Co and prevention of its adulteration. Materials and Methods: The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA from various Cinnamomum species were amplified by polymerase chain reaction (PCR), and these obtained sequences were used for sequence analysis. Based on the sequence variants among various Cinnamomum species, restriction fragment length polymorphism (RFLP) was used to differentiate these Cinnamomum plants. Results: Two restriction endonucleases, MylI and EcoRV, were specifically used to digest the PCR-amplified ITS DNA from seven Cinnamomum species. The PCR-RFLP results demonstrated that the different restriction patterns that were produced by these two restriction enzymes clearly distinguished Co from Cb and five other Cinnamomum species simultaneously. Conclusion: The PCR-RFLP analysis developed in this study provides an alternative method for rapidly identifying Cinnamomum plants at the species level using DNA polymorphisms.

Keywords: Cinnamomum burmannii, Cinnamomum osmophloeum, internal transcribed spacers, polymerase chain reaction, restriction fragment length polymorphism


How to cite this article:
Yang BC, Lee MS, Sun FC, Chao HH, Chang WT, Lin MK, Chen HJ, Lee MS. Rapid identification of the indigenous medicinal crop Cinnamomum osmophloeum from various adulterant Cinnamomum species by DNA polymorphism analysis. Phcog Mag 2020;16, Suppl S1:64-8

How to cite this URL:
Yang BC, Lee MS, Sun FC, Chao HH, Chang WT, Lin MK, Chen HJ, Lee MS. Rapid identification of the indigenous medicinal crop Cinnamomum osmophloeum from various adulterant Cinnamomum species by DNA polymorphism analysis. Phcog Mag [serial online] 2020 [cited 2020 May 27];16, Suppl S1:64-8. Available from: http://www.phcog.com/text.asp?2020/16/68/64/281688



SUMMARY

  • Polymerase chain reaction-restriction fragment length polymorphism method described in the present study enabled us to rapidly and conveniently identify Cinnamomum osmophloeum (Co) from various Cinnamomum species. It may be useful and easy to standardize this method, which can be applied practically for Cinnamomum identification. Moreover, in the future, it might be possible to apply this approach for the investigation of the population diversity and structure of Co and other Cinnamomum species that are indigenous to Taiwan.




Abbreviations used: PCR: Polymerase chain reaction; RFLP: Restriction fragment length polymorphism; RE: Restrict enzyme; ITS: Internal transcribed spacer; RAPD: Random amplified polymorphic DNA analysis; AFLP: Amplified fragment length polymorphism.


   Introduction Top


The dried bark of Cinnamomum cassia Presl. (Cc), commonly known as “cinnamon,” is an important medicinal crop that is widely used throughout the world as an ingredient in food and for medical applications. Cinnamon has a sweet taste and is used as a spicy ingredient in the food industry. Cinnamomum osmophloeum Kanehira. (Co) is a tree species that is indigenous to Taiwan and has been frequently planted in forests for landscaping or medicinal applications. Pharmacological studies have reported that Co has antifungal, anti-inflammatory, antitermitic, antibacterial, and antioxidative effects and reduced serum uric acid levels.[1],[2],[3],[4],[5] Research investigating the plant's phytochemical constituents has also shown that cinnamaldehyde makes 76% of essential oil in Co, which is higher than in other Cinnamomum species.[4] Therefore, Co essential oil may have high value added for the production of cosmetics or other related healthy products. Recently, various commercial products using Co as the functional ingredient have been created and marketed in Taiwan, including herbal tea, beverages, soap, and cosmetics.[6]

Cinnamomum burmannii (Cb) is also a member of the Lauraceae and has morphological features that are highly similar to those of Co. Therefore, Cb is commonly used as an adulterant of Co for planting.[6] In recent years, several studies have reported that various methods have been applied to the authentication of Cinnamomum species. The histological approach was concluded using microscopy, morphological identification, and phytochemical chromatographic analysis.[7],[8],[9] However, identification based on morphological characteristics has relied heavily on the researcher's experience or operation skills, dramatically reducing the efficiency of identification and its precision.[10] Moreover, environmental and other factors, such as growth, climate, and geography, may affect the morphological features and/or the chemical constituents of Cinnamomum; these effects can result in imprecise species identification using the above traditional analysis.[6] Currently, several DNA-based methods for plant identification have been developed, including random amplified polymorphic DNA analysis, amplified fragment length polymorphism, polymerase chain reaction (PCR), PCR restriction fragment length polymorphism (PCR-RFLP), authentication by sequencing of internal transcribed spacers (ITS) regions within the nuclear ribosomal DNA (rDNA), and authentication by sequencing of the chloroplast trnL DNA.[11],[12],[13],[14],[15],[16],[17] However, to date, few genetic identification studies focusing on Cinnamomum species have addressed the authentication of trees or the identification of their misused adulterants.[17]

For the reasons outlined above, the specific aim of this study was to develop a PCR-RFLP method for the rapid identification of Co from its adulterant Cb and related Cinnamomum plants by employing the ITS regions of the nuclear rDNA as a DNA molecular marker. In this study, a DNA restriction endonuclease (RE) pattern was successfully established, providing a more convenient and feasible way to authenticate the Cinnamomum species. To the best of our knowledge, this study is the first to describe the use of RE DNA patterns and the PCR-RFLP system for the genetic identification of Co and its popular adulterants.


   Materials and Methods Top


Plant source

Seven Cinnamomum species, Co, Cb, Cinnamomum kanehirae (Ck), Cinnamomum camphora (Ca), Cinnamomum reticulatum (Cr), Cinnamomum kotoense (Ckk), and Cc, were collected at different localities around Taiwan. The collection points of these specimens are presented in [Table 1]. The plant samples of these Cinnamomum species were identified by Dr. Wen-Te Chang, and voucher specimens were deposited at the Graduate Institute of Chinese Pharmaceutical Science and Chinese Medicine Resources of China Medical University. Voucher specimen numbers are denoted and listed in [Table 1].
Table 1: Collected Cinnamomum species in this study

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DNA extraction

Dried leaves from the Cinnamomum samples were collected and ground with liquid nitrogen into powder to allow DNA extraction. Total DNA was isolated from each sample of homogenized plant tissue using a Geneaid Genomic Extraction Mini Kit (Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. The concentration of purified total genomic DNA was determined using a spectrophotometer (NanoVue™, GE Healthcare, USA), and each sample was then stored at −20°C.

Primers

The specific oligonucleotide primers TCM-5 (5'-CGTAA CAAGGTTTCCGTAGGTGAAC-3') and TCM-12 (5'-GACG CTTCTCCAGACTACAA-3') were designed based on the 18S and 26S rDNA sequences of various Cinnamomum species, respectively.[6] The whole ITS DNA region, including ITS1, 5.8S RNA, and ITS2, was amplified when TCM-5 and TCM-12 were used as primers in the PCR amplification.[6]

DNA amplification and internal transcribed spacer sequence alignment

To align the ITS sequence, the complete ITS regions of the seven Cinnamomum species were amplified using the TCM-5 and TCM-12 primers with genomic DNA from these various Cinnamomum species as template DNA. In brief, 50 ng of total genomic DNA from each Cinnamomum species was individually added to the PCR mixture. PCR was performed in a 25-μl reaction mixture containing 0.4 mM of dNTPs, 5 pmol each of TCM-5 and TCM-12, 1U Pro-Taq™ DNA polymerase (Protech, Taiwan), and 1× Pro-Taq™ buffer (10 mM Tri-HCl, 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl2, 0.1% Triton X-100, pH 9.0). The PCR conditions were 95°C for 5 min followed by 35 cycles of 95°C for 1 min, 57.7°C for 1 min, and 72°C for 1 min and a final extension cycle at 72°C for 10 min.[6] The amplified PCR products were resolved by 2% agarose gel electrophoresis. DNA banding was detected by the observation of the presence of visible DNA bands after staining with ethidium bromide. To sequence the ITS DNAs, each PCR product was individually added to the pGEM™-T vector, and TA cloning was carried out according to the manufacturer's instructions (Promega, USA) using T4 DNA ligase at 4°C with overnight incubation. The ligation mixture was transformed into competent Escherichia coli Top10 cells and recombinant colonies selected followed by plasmid purification and DNA sequencing. The sequences obtained for each ITS DNA region were then subjected to multiple pairwise sequence alignment using Clustal W2 software (http://www.ebi.ac.uk/clustalw/index.html).

Restriction analysis of the polymerase chain reaction products

The individual PCR products amplified by TCM-5 and TCM-12 were extracted from the agarose gel after DNA electrophoresis and purified using a Gel/PCR DNA fragment extraction kit according to the manufacturer's instructions (Geneaid, Taiwan). Digestion was carried out at 37°C for 1 h in a 50 μl RFLP reaction mixture containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT, 100 ug/ml BSA, 5 U Mly I, and 5 U Eco RV. The resulting restriction digestion product was analyzed by 3% agarose electrophoresis and visualized using ethidium bromide staining and ultraviolet excitation.


   Results Top


Polymorphism of the internal transcribed spacer of the nuclear ribosomal DNA of various Cinnamomum species

To compare the interspecies sequence variation of Cinnamomum spp., the ITS regions of the different Cinnamomum spp. were used as targets for PCR amplification. Using the primers TCM-5 and TCM-12, a major single PCR product of approximately 800 bp was observed after DNA amplification from each Cinnamomum species [Figure 1]a. Sequencing of these ITS fragments, as illustrated in [Figure 2], showed that these obtained ITS DNA regions of five Co from different localities with 790 bp in size demonstrated these sequences, including partial 18S and 26S rDNA, a complete 5.8S rDNA, ITS1, and ITS2. All ITS DNA of five Co sequences was identical. The other six Cinnamomum species, Cb, Ck, Ca, Cr, Ckk, and Cc, have ITS regions of 770 bp, 811 bp, 792 bp, 808 bp, 805 bp, and 800 bp, respectively. All obtained sequences of different Cinnamomum species were aligned to determine their similarities and to recognize their restriction maps using the software NEBcutter V2.0 (http://tools.neb.com/NEBcutter2/). As illustrated in [Figure 2], the results showed that a number of Mly I restriction sites were present in the abovementioned six Cinnamomum species, except in the sequence of Cc. Moreover, a unique Eco RV restriction site was only present in the sequence of Ca [Figure 2]. No Eco RV restriction sites were present in the other six Cinnamomum species. Thus, two restriction enzymes, Mly I and Eco RV, spanned in these ITS sequences may have the potential to be molecular markers for use in specific species identification of these seven Cinnamomum plants by PCR-RFLP analysis.
Figure 1: (a) Amplification of internal transcribed spacer DNA from various Cinnamomum plants by polymerase chain reaction. Polymerase chain reaction was performed using universal primers, TCM-5 and TCM-12, designed based on the sequence of nuclear ribosomal DNA. Polymerase chain reaction conditions were described in the methods and materials. (b) Polymerase chain reaction-restriction fragment length polymorphism analysis. Myl I and Eco RV restriction pattern of a polymerase chain reaction product of internal transcribed spacer DNA from seven Cinnamomum plants were resolved using 3% agarose gel. Lanes 1–5 indicate Cinnamomum osmophloeum; lanes 6–8 indicate C. burmannii; lanes 9–13 indicate Cinnamomum kanehirae, Cinnamomum camphora, Cinnamomum reticulatum, Cinnamomum kotoense, and Cinnamomum cassia, respectively. Lane M: DNA marker

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Figure 2: Sequence comparison of a region of internal transcribed spacer of nuclear ribosomal DNA for seven Cinnamomum plants. An asterisk (*) represents the aligned nucleotide that is identical to the upper sequence. A hyphen (-) represents a gap in the aligned sequence. The restriction enzyme sites of Myl I and Eco RV are indicated by underline and bold, respectively

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Polymerase chain reaction-restriction fragment length polymorphism DNA polymorphism of the internal transcribed spacer regions from the seven Cinnamomum species

To rapidly identify these seven Cinnamomum plants using PCR-RFLP analysis, the restriction enzyme patterns of the seven species are predicted and listed in [Table 2]. The predicted restriction profiles suggested that if only one restriction enzyme, Mly I, is used, it appears not to be possible to clearly differentiate all the Cinnamomum species simultaneously in this study. Using Mly I, it was enabled to theoretically verify Co between Cb, Ck, Ca, Cr, Ckk, and Cc in terms of digestion pattern of restriction enzyme. However, the predicted restriction profiles with Mly I between Ca and Ckk have quite similar DNA banding patterns after electrophoresis. For the Ca sample, a 348-bp DNA fragment and a 460-bp DNA fragment were produced; similarly, a 346-bp DNA fragment and a 459-bp DNA fragment were produced in the Ckk. Based on this prediction, another restriction enzyme, Eco RV, was selected by the prediction software for use to improve the discrimination. As illustrated in [Figure 1]b, the restriction patterns created by Mly I and Eco RV showed that these DNA fragments were varied and clearly verified above seven Cinnamomum plants. The obtained RFLP patterns were notably different from those of the other Cinnamomum species, especially regarding the identification of Co and Cb. Taken together, RFLP analysis with Mly I and Eco RV established in this work is powerful and has the potential to be applied for molecular identification among popular adulterated Cinnamomum plants, especially for discriminating Co and Cb.
Table 2: Predicted restriction fragments in sizes (in bp) among Cinnamomum species when restriction enzymes were used for digestion

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   Discussion Top


Generally, most Cinnamomum plants are highly economically valuable tree species. However, Cinnamomum species share similar morphological features in the taxonomy. Thus, developing a rapid and feasible method for the identification of Cinnamomum plants is needed to prevent their adulteration of trees. In Taiwan, due to the applications of this plant to food, cosmetic, and medical usage, Co is widely planted by farmers in the field. However, in Taiwan, tree adulteration of Cb during planting results in significant economic losses in agriculture.

In this study, we have successfully developed a PCR-RFLP method for the rapid and specific discrimination of Co trees from other Cinnamomum species samples. Previous studies have reported that many genetic markers, such as ITS of the rDNA region and specific genes in the chloroplast DNA, could be used for molecular identification.[17],[18] Among these markers, ITS has strong potential for use as a barcode for plant authentication.[18] However, which DNA regions or specific genes are best or suitable for molecular identification depends on the species in the family. In this study, DNA from the ITS region was chosen for amplification using PCR primers. Sequence alignment analysis of the ITS regions from seven Cinnamomum species revealed that the ITS1 and ITS2 regions showed higher interspecies variation than intraspecies. In contrast, the sequence divergence across the Cinnamomum species was not great. Therefore, based on the sequence variation of ITS, it is not easy to design species-specific PCR primers for use in direct identity identification via a diagnostic PCR method. Normally, sequencing of an ITS DNA fragment during medicinal herb identification is a useful approach, and it directly overcomes the problem of species identification. However, a lack of truly universal primers for the amplification of ITS regions from a wide variety of plants has limited the usefulness of this approach as a general method of species identification.[18] Moreover, the direct sequencing of a PCR product is a relatively costly procedure and is time-consuming. To improve such problems, other molecular techniques, such as PCR-RFLP, have been developed and used for species identification; such an approach requires a good universal primer pair for the amplification of the ITS region of Cinnamomum species for further organization of genetic information.[10],[19],[20] In most plants, the length of the ITS1-5.8S rDNA-ITS2 region ranges from 550 to 850 bp.[18],[21],[22] The present studies showed that the length of the ITS regions of the various Cinnamomum species varied [Figure 2]. Nevertheless, the Cinnamomum universal primers, TCM-5 and TCM-12, were able to conveniently amplify the ITS DNA regions of the seven Cinnamomum species and thus seem to be both compatible with and effective for ITS amplification from Cinnamomum samples in general.

As to the analysis on the investigation of the restriction maps of the ITS regions of seven Cinnamomum plants, Mly I was selected as a candidate restriction enzyme that might be useful when discriminating between Co and other Cinnamomum species [Table 2]. However, the predicted restriction profiles of Ca and Ckk with Mly I were highly similar and difficult to distinguish their differences from each other in terms of conventional DNA banding by agarose gel electrophoresis (data not shown). As a result, digestion of the PCR product of ITS DNA with Mly I and Eco RV was performed for the experiment. As expected, this enzyme combination is capable of distinguishing between Ca and Ckk. It is worth noting that in this study, plant leaves were used to extract genomic DNA for tree identification in the farm field. In fact, the barks of Cinnamomum are being used in traditional medicine or in the food industry after agricultural harvesting. However, different regions of extracted DNA used in this study did not influence the molecular identification theoretically. Thus, this developed PCR-RFLP may not only be employed on tree authentication but may also be useful as an alternative way to identify cinnamon products.


   Conclusion Top


The PCR-RFLP method described in the present study enabled us to rapidly and conveniently identify Co from various Cinnamomum species. It may be useful and easy to standardize this method, which can be applied practically for Cinnamomum identification. Moreover, in the future, it might be possible to apply this approach for the investigation of the population diversity and structure of C. osmopholeum and other Cinnamomum species that are indigenous to Taiwan.

Acknowledgements

Bo-Cheng Yang and Meng-Shiunn Lee contributed equally to this work.

Financial support and sponsorship

This work was supported by a grant from the China Medical University of Taiwan, ROC (CMU 108-MF-91).

Conflicts of interest

There are no conflicts of interest.



 
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