Identification of Campsis Flos and its common adulterants using ITS 2 sequence of DNA molecular barcoding

Internal transcribed spacer 2 (ITS2) is known as a good sequence of DNA molecular barcoding. In this study, the authors authenticated Campsis Flos and its common adulterants based on ITS2 sequence. Campsis Flos has been used in traditional Chinese medicine for many years as a blood activator to promote menstruation. Among its adulterants in the market, the most common are the flowers of Paulownia tomentosa, Hibiscus syriacus and Rhododendron molle. To discriminate Campsis Flos from these adulterants and control its quality, safety and efficacy, the total genomic DNA was extracted from the leaves of Campsis grandiflora, Campsis radicans, and their common adulterants. The internal transcribed spacer 2 (ITS2) of ribosomal DNA was sequenced after PCR amplifying. A neighbor-joining (NJ) tree was constructed using MEGA 6.0. The ITS2 secondary structure was predicted by an ITS2 web server. The results showed that the ITS2 sequence lengths of C. grandiflora, C. radicans, and P. tomentosa were 246–248, 246, and 229–233 bp, respectively. The ITS2 sequence lengths of H. syriacus, H. syriacus L. f. var. syriacus f. amplissimus, and R. molle were all 230 bp. The ITS2 secondary structure of Campsis Flos was then effectively distinguished from its adulterants. In conclusion, barcode ITS2 sequence could be used to rapidly and accurately identify Campsis Flos from its adulterants to promote quality control and standardization.


INTRODUCTION
Campsis Flos is made from the dried flowers of Campsis grandiflora and Campsis radicans and is used in traditional Chinese medicine.Chinese Pharmacopoeia states that Campsis Flos activates blood to promote menstruation (Commission of Chinese Pharmacopoeia 2015).Recently, researchers have found antidepressive (Yu et al., 2015), antioxidative and anti-inflammatory activities (Cui et al. 2006) in Campsis Flos extract.Campsis Flos is not expensive, but there are some adulterants in the market (He et al., 2007).The common adulterants are the flowers of Paulownia tomentosa, Hibiscus syriacus and Rhododendron molle.Misuse will *Corresponding author.E-mail: t_luye@suda.edu.cn.Tel: +86 512 6588 2090.Fax: +86 512 65882089.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License will produce side effects, especially because R. molle flowers are toxic.Therefore, identifying Campsis Flos is very important.Traditional microscopic identification is too specialized and only a few people can know it (He et al., 2007).To solve this problem, there is need to find a simple and rapid molecular identification method to distinguish the Campsis Flos from its common adulterants.
DNA molecular barcoding has become a potential identification tool for plants and animals (Kress et al., 2005(Kress et al., , 2007;;Zhang et al., 2015;Chen et al., 2013).This method can be used to promote quality control and standardization in traditional Chinese medicine, and it has been promulgated by the Chinese Pharmacopoeia.
Among DNA barcoding candidate sequences of the chloroplast genes psbA-trnH, matK, rbcL and nuclear ribosomal DNA ITS (internal transcribed spacer) 2, ITS2 is known as a good sequence of identification (Yao et al., 2010;Pang et al., 2013).Chen et al. (2010) also determined the effectiveness of the ITS2 in identifying medicinal plants using samples from a wide range of taxa.Zhang et al. (2016) also recommended ITS2 for use as a mini-barcode.In this study, total genomic DNA was isolated from C. grandiflora and C. radicans and their adulterants.The ITS2 was amplified by PCR and sequenced.A neighbor-joining (NJ) tree was constructed using MEGA 6.0 software.The maximum genetic distance within species was far less than the minimum genetic distance between species.The Campsis Flos samples were clustered as a class in the NJ tree, and different species were separated.The NJ tree revealed a close genetic relationship between P. tomentosa and Campsis Flos; R. molle has a closer relationship with Campsis Flos, whereas H. syriacus had a farther relationship.The ITS2 secondary structure was predicted by ITS2 web server.ITS2 secondary structures of the different species were significantly different from each other, whereas those of the same species had no significant differences.Thus, ITS2 sequence offers a new alternative for rapid and straightforward identification of Campsis Flos and its common adulterants.

MATERIALS AND METHODS
Leaf samples of Campsis Flos and its adulterants were collected from Suzhou, Hangzhou, and Enshi in Hubei province.Professor Chunyu Liu from Soochow University identified the species.All voucher specimens were deposited in the herbarium of the College of Pharmaceutical Science of Soochow University.Fresh leaves were dried using silica gel until DNA extraction.Table 1 shows the samples used in this study and their origins, time and place of collection.

DNA extraction, PCR amplification and sequencing
Samples of approximately 50 mg were pulverized using liquid nitrogen and 2% polyvinylpyrrolidone (PVP) with a pestle and mortar.Total genomic DNA was subsequently extracted using the Plant Genomic DNA Kit (CWBiotech Co., Ltd., Beijing, China) following the recommended protocol.The extracted DNA concentrations of all samples were determined using a Q3000-type micro ultraviolet spectrophotometer (Bio-Rad Company, Hercules, CA, USA), and optical density values at 230, 260, and 280 nm of each DNA sample was determined.One pair of universal primers, up (5'-AACCATCGAGTCTTTGAACGC-3') and down (5'-CCTTGTAAGTTTCTTTTCCTCC-3'), was designed for PCR amplification of the ITS2 sequence of Campsis Flos and its adulterants.PCR amplification was carried out in My Cycler (BioRad Lab Inc., USA).PCR was performed using 50 µl reaction mixtures containing 25 µl of 2 × Taq Master Mix (Sangon Biotech Co., Ltd., Shanghai, China), 1 µl of each PCR primer (10 µM), and 0.5 to 1 µl of DNA extract, and the total volume was adjusted to 50 µl with sterile deionized water.PCR amplification was conducted according to the following procedure: 94°C for 3 min, 30 cycles of 94°C for 50 s, 54°C for 50 s, and 72°C for 1 min, and a final extension at 72°C for 10 min.PCR products were analyzed by electrophoresis in a 1% agarose gel in 0.5 × TAE buffer with 0.5 µg/ml Gel Red at a constant voltage of 100 V for 40 min.To estimate the size of separated fragments, DNA ladder was loaded into the first lane of each gel.The PCR products were sequenced bidirectionally using an ABI 3730XL sequencer (Sangon Biotech Co., Ltd., Shanghai, China).

Sequence alignment and phylogenetic analysis
The ITS was located in the middle of 18S and 28S of nuclear ribosomal RNA (nrRNA), and the middle part was divided into ITS1 and ITS2 by 5.8S.The 5.8S and 28S sections of the obtained and GenBank sequences were removed using a Hidden Markov model (HMM) (Keller et al., 2009), leaving the ITS2 sequence.All ITS2 sequences were analyzed using MEGA 6.0 (Tamura et al., 2013).An NJ tree (Saitou et al., 1987) was constructed via bootstrap method that was conducted with 1000 replicates; the substitution model was Kimura-2-parameter (K2P) (Kimura, 1980); and gaps were treated as missing data (complete deletion).Intra-and interspecies sequence divergences were also calculated using the K2P model, and gaps were treated as missing data.The ITS2 secondary structure was predicted via the ITS2 database (http://www.its2.bioapps.biozentrum.uni-wuerzburg.de/)(Koetschan et al., 2010).

DNA quality
Table 2 shows the qualities of the extracted DNA.The quality of DNA extracted from the flowers is low.Therefore, DNA was extracted from leaves instead.The results indicated that the leaf DNA was of sufficient quality for subsequent experiments.

ITS2 sequence analysis and intra-and inter-species variations
The average G-C content and length of ITS2 of all samples are listed and aligned, respectively, using the MUSCLE method in Table 3.The C. grandiflora from Enshi, when compared with Genbank, had 17 interspecies variable sites (Table 4).The evolution speed of ITS is fast.Nucleotide analysis of the ITS2 sequence could provide more information on inter-and intra-species divergences.The average intraspecific genetic distances calculated by the Kimura-2-parameter model (Kimura, 1980) for Campsis Flos were 0.01375.The interspecific diversities ranged from 0 to 0.055 between C. grandiflora from Genbank, Garden Hotel Suzhou, and from Enshi; from 0.005 to 0.010 Hibiscus syriacus Linn.f. var.syriacus f. amplissimus (Suzhou Jinji Lake Avenue) 59.5 230 between H. syriacus and H. syriacus L. f. var.syriacus f. amplissimus from Baitang Ecological Botanic Garden, Yangcheng Lake Campus, and Jinji Lake Avenue in Suzhou; and from 0.040 to 0.82 between P. tomentosa from Main Campus of Soochow University and Suzhou International Foreign Language School (Table 5).
In this study, the minimum genetic distances of intraspecies of samples (0.220) were greater than the maximum genetic distances of inter-species of samples (0.055).Therefore, it would be easier to distinguish Campsis Flos from the other samples using the ITS2 sequence.

Prediction and analysis of secondary structure of ITS2
The secondary structure of ITS2 provided the most accurate phylogenetic analysis (Keller et al., 2010), and its information correlated with the biological species concept (Müller et al. 2007).Studies on the secondary structures of nrRNAs have been considered important for inferring phylogenetic relationships at some taxonomic levels.The authors predicted the secondary structure of all samples using the ITS2 web site (http://its2.bioapps.biozentrum.uni-wuerzburg.de/).
Generally, secondary structures of samples contain a central loop and four helical regions (arms): I, II, III, and IV (Figure 1).There were no significant differences in the number, size and angle of the arm loops within the same species.However, there were significant differences among different species.The six species, C. grandiflora, C. radicans, P. tomentosa, H. syriacus, H. syriacus L. f. var.syriacus f. amplissimus, and R. molle, could be directly identified based on the nrRNA secondary structure of their ITS2 regions.

Phylogenetic analysis
The phylogenetic tree was constructed using MEGA6.0 with 1000 bootstrap replicates for the ITS2 sequences, as shown in Figure 1.The 12 samples can be clustered into classes I, II, III and IV (Figure 2).Class I included C. radicans and C. grandiflora from Genbank, Garden Hotel Suzhou, and Enshi.The samples of Campsis Flos and P. tomentosa were clustered into class II.Classes I, II and R. molle were clustered into Class III.Class III and H. syriacus and H. syriacus L. f. var.syriacus f. amplissimus were clustered into class IV.The phylogenetic tree and conventional taxonomy of the plants were consistent.

DISCUSSION
Campsis Flos has been used to encourage blood circulation and mediate blood stasis to treat diseases caused by blood stagnation.It is effective in promoting blood circulation.The flowers of P. tomentosa, H. syriacus, and R. molle are its common adulterants.P. tomentosa flowers can treat respiratory infections.H. syriacus and H. syriacus L. f. var.syriacus f. amplissimus flowers can alleviate fever and promote diuresis.Rhododendri Mollis Flos are dried R. molle flowers, which can expel wind and remove dampness.The effects of these four common adulterants and Campsis Flos are vastly different and will produce serious consequences if misused.Although, microscopic identification can identify them, this traditional method is time-consuming and takes intensive training and experience to fully grasp (He et al.,  2007; Zhang et al., 2005).Therefore, molecular identification might be a good alternative.In recent years, DNA barcoding identification has emerged useful in species identification; in particular, ITS2 sequence is an efficient marker for authenticating traditional Chinese medicines.ITS2 can effectively distinguish Campsis Flos from its adulterants.
In addition, successful DNA extraction is a prerequisite for identifying herbal materials (Moyo et al., 2008).The majority of the plant materials used in herbal medicine is procured from markets in dried or powdered forms; thus, the qualities of the DNA extracted in the experiment were poor.The medicinal portions of herbs are the leaves, which provided better DNA quality.
DNA barcoding ITS2 sequence was obtained by PCR amplification and sequencing.The ITS2 secondary structures of all species are composed of a central loop (the main loop) and four helical regions (arms), and each has a different size and number of arm loops (Schultz and Wolf, 2009;Tippery and Les, 2008).The ITS2 secondary structures of C. grandiflora and C. radicans and H. syriacus and H. syriacus L. f. var.syriacus f. amplissimus have little differences.However, the ITS2 secondary structures of Campsis Flos, P. tomentosa, R. molle and H. syriacus were significantly different.Their sizes, positions, numbers and angles of helix arm loops, and center ring shapes are also different.The central loops of P. tomentosa, R. molle and H. syriacus have variable regions, and that of P. tomentosa is irregular.Thus, the prediction of ITS2 secondary structure can be used to distinguish Campsis Flos from its adulterants.
The phylogenetic trees of Campsis Flos and its adulterants that were constructed using the ITS2 sequence could easily distinguish the six species.Moreover, the clustering observed was consistent with traditional classifications.C. grandiflora, C. radicans, P. tomentosa and R. molle belong to sympetalae, and H. syriacus belongs to archichlamydeae.Thus, the genetic relationships among C. grandiflora, C. radicans, P. tomentosa  Adulteration is frequent in traditional Chinese medicine.If we could standardize the cultivation of Campsis Flos and establish a Good Agriculture Practice Area, the adulteration problem would be solved to a certain extent.Furthermore, ITS2 barcodes could help control the quality of planting Campsis Flos.

Figure 1 .
Figure1.The ITS2 secondary structure of Campsis Flos and its adulterants.Their differences were mainly present in Helixes I, III and IV, and, therefore, were distinguishable at the molecular level.

Figure 2 .
Figure 2. The NJ tree of Campsis Flos and its adulterants with the ITS2 sequences.The bootstrap scores (1000 replicates) are shown (≧75%) for each branch.

Table 1 .
Plant samples used in present study.

Table 2 .
DNA quality and concentration of Campsis Flos and its adulterants.

Table 3 .
The average G-C content and length of the ITS2 sequence of Campsis Flos and its adulterants.

Table 4 .
Sequence alignment of C. grandiflora from Enshi in Hubei province and from Genbank.

Table 5 .
genetic distance of Campsis Flos and its adulterants.