Identification of three Daphne species by DNA barcoding and HPLC fingerprint analysis
Identification of three Daphne species by DNA barcoding and HPLC fingerprint analysis
Yanpeng Li 0 1
Lu Geng 0 1
Yuanyan Liu 0 1
Mingyang Chen 0 1
Qirui Mu 0 1
Xu Zhang 1
Zhongyi Zhang 1
Guangxi Ren 0 1
Chunsheng Liu 0 1
? These authors contributed equally to this work. 1
0 School of Chinese Pharmacy, Beijing University of Chinese Medicine , Beijing, PR China, 2 Pharmaceutical Co. , Ltd., of Qinhuangdao Shanhai Pass , Qinhuangdao, Hebei Province , PR China
1 Editor: Vijai Gupta, Tallinn University of Technology , ESTONIA
In order to well identify the 93 wild Cortex Daphnes samples from different species and habitats in western China and develop a standard operating procedure (SOP) for the authentication and quality of them in the future, a comprehensive and efficient identification system based on DNA barcoding and HPLC fingerprint technologies has been developed. The result showed that only 17 samples (18%) were Daphne giraldii Nitsche (DG), which is recorded in Chinese Pharmacopeia, while the others (82%) might have safety hazards. Additionally, the result of HPLC fingerprint analysis indicated that samples in the same species origins and wild distributions could be clustered together, which was consistent with DNA barcoding analysis. The study can provide a significant system for the authentication and quality of commercial Cortex Daphnes herbs. Undoubtedly, this study undoubtedly confirmed that the chemical compositions of Cortex Daphnes herbs were affected by both species origins and ecological environments, which is required more in-depth research.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This study was supported by the
Pharmaceutical Co., Ltd., of Qinhuangdao Shanhai
Pass (Qinhuangdao 066200, China) which
provided great financial support. The funder did not
have any additional role in the study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: This study was supported by
the Pharmaceutical Co., Ltd., of Qinhuangdao
Cortex Daphnes (Cirald Daphne Bark), a kind of traditional Chinese medicine, is the dried
root bark and stem bark of DG recorded in the Chinese Pharmacopeia. However, many
Daphne Linn plants, such as Daphne gracilis E. Pritz., Daphne limprichtii H. Winkl. and
Edgeworthia Meisn plants, such as Edgeworthia chrysantha Lindl., etc., are also used as Cortex
Daphnes in some parts of China [1?4]. Daphne tangutica Maxim (DT) and Daphne retusa
Hemsl (DR) are just contained in some provincial medicinal material codes [5?6]. The Daphne
Linn comprises approximately 95 species found in the alpine regions of Asia and Europe.
About 44 species are found in China, and mainly distributed in southwest and northwest of
China. The three Daphne species mostly is located in Provinces of Gansu, Shaanxi, Qinghai
and Sichuan [
As a traditional Chinese medicine, Cortex Daphnes is pungent, bitter in taste, warm in
nature, and of a little toxicity. Acting on the liver channel, the medical efficacy of Cortex
Daphnes is dispelling wind and eliminating dampness, promoting blood circulation to remove
blood stasis and scattered stasis pain [
]. Phytochemical studies showed that there are mainly
four active classes of constituents in the dried velamen and bark of Daphnes Cortex, including
coumarins (e.g. daphnetoxin), flavonoids (e.g. luteolin), lignans (e.g. syringaresinol),
triterpenes (e.g. oleanolic acid), it also contains syringin, daucosterol, etc. Daphnetin is not the
characteristic constituents but also the major bioactive constituents of them. Modern
pharmacological studies have shown that Cortex Daphnes extract exhibits various efficacy, such as
anti-inflammation, analgesia, anti-malaria, anti-tumor, antithrombus, anti-fertility. Daphnetin
effectively inhibit tumor cell growth and kill the malaria parasite, and also its extract has
remarkable therapeutic effect on glomerulonephritis [9?12]. In recent pharmaceutical market,
the dosage forms of Cortex Daphnes are mainly injection, tablets and patch, which are used as
the Extra-Strength Pain Reliever and largely used for the treatment of rheumatic arthralgia,
arthralgia, traumatic injury, arthritis, rheumatoid arthritis and promoting blood circulation,
and also can cure some blood vessel diseases, such as cardiovascular and cerebrovascular
diseases, especially for thrombosis angitis obliterans.
Cortex Daphnes and its relative products is greatly in the world. Up to the year of 2015 in
the survey areas, the wild resources storage of Cortex Daphnes was only 583.62t, the storage of
DG was 97.64t (16.7%), the storage of DT was 392.66t (67.3%), the storage of DR was 93.32t
(16%), according to the resources survey results from our research group (Beijing University
of Chinese Medicine, Beijing, China). Many Daphne plants are used as Cortex Daphnes in folk
of China and ordinary people is unable to correctly identify them well. Few Daphne plants
have been cultivated and almost all wild materials are obtained from the agricultural markets
in different areas[13?14].
The medicinal plant trade is the primary source of income for herbalists, and economic
constraints may provide incentives for herbalists to substitute cheaper and more readily
available species for rare ingredients and sell them under the same name [
]. The constituents
contained in medicinal materials of Cortex Daphnes herbs collected from different habitats
and different species origins are varied, which may directly influence the quality and efficacy
of corresponding Chinese patent medicine.
Morphological classification is an important method for the identification of medicinal
plants, mostly depending on their leaves, flowers and fruits. The harvest time of Cortex
Daphnes herbs is in March of each year. However, there is no flower or fruit in March when villagers
collect Cortex Daphnes herbs, so bark of the three Daphne species as Cortex Daphnes herbs
circulated in the medicinal markets is more difficult to be identified because of the high
similarity of their external characteristics [
]. Therefore, more scientific and accurate identification
methods are required. Currently, DNA barcoding is recognized as a technology that is able to
accurately and efficiently identify the species of medicinal plants. Internal transcribed spacer
of ribosome gene (ITS) sequence is able to provide sufficient information for species
Generally, the curative effects of traditional Chinese medicine (TCM) are the results of
multiple bioactive components. Chromatographic fingerprinting provide an entire profile of
almost global component of herbal medicines and is considered to be an important method
for evaluating the quality of herbal medicines. It has been internationally accepted by the
World Health Organization (WHO), the Food and Drug Administration of the USA (FDA),
the Chinese State Food and Drug Administration (SFDA) and other authorities. HPLC
fingerprinting is the most widely used method for qualitative evaluation and species identification of
herbal medicines due to its convenience and efficiency [22?25].
In China, 93 wild Cortex Daphnes samples in different species were collected from
different provinces covered the main wild distribution areas. Therefore, the aim of this study was
to develop a valid and accurate system based on DNA barcoding and HPLC fingerprint
methods, to identify and classify the species origins and habitats of the 93 Cortex Daphnes
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samples and control quality. Standard compound daphnetin was used as reference
component for the qualitative of chromatographic peak by an HPLC- UV method. Samples are
compared visually and analyzed using neighbor-joining tree (N-J tree) analysis, similarity
analysis (SA), hierarchical cluster analysis (HCA) and principal component analysis (PCA).
This study will be helpful in development of strategies for conservation, utilization and
quality control of Cortex Daphnes and other herbs. The system could be used by the supervisory
departments for the market supervision of commercial Cortex Daphnes herbs in the future,
and provide a identification model for other commercial Chinese herbal medicines. More
importantly, the classification results can preliminarily lay a foundation for the research that
how species origins and ecological environments effect on the chemical components of
Cortex Daphnes herbs.
Main characteristics on morphology and habitat of the three Daphne
According to the previous resources survey results, the plant morphology (S1 File) of DG is
different from the other two Daphne species (DT and DR). The plant morphology of DT and DR
is quite similar to each other. The flowers of DG are yellow and its leaves are pale green and
membranous. However, the flowers of DT and DR are all fuchsia and the leaves of them are
dark green and leathery. It is easy to separate DG from DT and DR by comparing the
characteristics of their flowers and leaves in the flowering and fruiting stages. The leaves of DT are
long, narrow and lanceolate. The leaves of DR are long ovate, and the front-end of leaves are
concave down, which is able to be selected as a major characteristic to identify DT and DR. It
shows that the genetic relationship between DT and DR is most similar to each other, which is
consistent with records about DR in flora of China [
]. However, the identification of plants
by the external form is restricted by collection time before blooming and growing
environment, and the success of differentiation depending on the difference of the subtle external
morphological features is restricted by professional experience.
There are obvious differences in habitats of the three Daphne species. DG often grows
under the bushes, where the community mostly consists of shrubs and small trees. The soil
types are mostly humus with good permeability. The air humidity is relatively modest.
Theoretically, the habitat altitude range of DG is 1600*2600 m, however, we only found them
where the altitude range is 2186*2554 m in the resources survey. DT often grows in the sparse
forest, alpine grassland and forest edge, especially on the sunny slope, with the altitude range
1000*3800 m. The coenotype is relatively diverse. According to Meteorological Data Center of
China Meteorological Administration [
], there are large differences in the climate type of
each DT habitat, which is able to be divided into four groups. Group 1: the climate types of
habitats including Menyuan, Ledu, Hualong, Datong and Zhuoni counties are all plateau
continental climate; the climate types of Huzhu and Tianzhu counties are continental cold
temperate climate and plateau continental monsoon climate. Characteristics of these climate types in
group 1 are more similar. Group 2: the climate types of habitats including Heishui and Mao
counties are all plateau monsoon climate. Group 3: the climate types of habitats including
Foping, Liuba and Ningqiang counties are all warm temperate humid monsoon climate, the
climate type of habitat Zhenan is semi-humid climate. Characteristics of these climate types are
relatively mild and more similar. Group 4: the climate types of habitats including Tianshui and
Pingwu are temperate monsoon climate and subtropical mountain humid monsoon climate.
Kang counties is the excessive area that subtropical transition to warm temperate zone.
Characteristics of climate types in group 4 are mild, abundant rainfall and more similar. DR often
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Fig 1. Distributions of the 93 collected Cortex Daphnes samples. This map was generated by the software ESRI ArcGIS Desktop, version: 10.3.0.4322,
URL http://www.esri.com/. It was created by author L. G. (The border line is vectorized China map data.).
grows on alpine grass slope, with the altitude range 3000*3900 m. The climate types of
habitats including Jinchuan, Kangding and Maerkang counties are continental plateau climate,
alpine plateau climate and low latitude, high altitude special geography and alpine canyon
three-dimensional climate. These characteristics of climate types are a bit similar to the plateau
monsoon climate. For the same Daphne species, habitat may be the major factor, which result
in their chemical composition and gene segment exist differences. The specific habitat
distributions of the 93 Cortex Daphnes samples are shown in Fig 1 (generated by the software ESRI
ArcGIS Desktop, version: 10.3.0.4322, URL http://www.esri.com/).
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Genetic taxonomy of Cortex Daphnes samples
All of the collected samples were identified according to their ITS sequences. To identify the
species of the 93 Cortex Daphnes samples more accurately and visually, a phylogenetic tree
was constructed based on the ITS sequences (S2 File) obtained from GenBank and the
samples. Likewise, the modified ITS sequences were submitted to DNAMAN software to compare
similarities of the samples. Among them, 76 Cortex Daphnes samples of DT and DR were
identified with the similarities higher than 99%, and the other 17 Cortex Daphnes samples of DG
were also identified. The similarity of ITS sequences between DG and DT was 93% and the
similarity between DG and DR was 92%. However, the similarity between DT and DR was
99%. The genotype of ITS sequences in DT at 60bp and 156bp were C and G. But, the genotype
of ITS sequences in DR at the same locus were all A, based on this, it also able to identify them
HCA based on ITS sequences was used to analyze the genetic data to characterize the
population genetics of the Cortex Daphnes samples and determine their genetic diversity and
population differentiation. Thymelaea aucheri (AJ549445.1), Daphne cneorum (AJ549490.1),
Thymelaea dioica (AJ549468.1) and Daphne blagayana isolate DB45_SL (GQ167491.1)
downloaded from GenBank-NCBI were selected as out-group gene sequences to obtain
more accurate branching of the phylogenetic tree. The wild Cortex Daphnes populations
distributed in western China were grouped separately through their ITS sequences (as shown in
All ITS sequences of Cortex Daphnes samples were merged with Daphne blagayana isolate
DB45_SL (GQ167491.1), which indicated that these samples belonged to Daphne Linn. All
samples were divided into two main characteristic clusters. The first cluster consisted of DG.
The other cluster was made up with DT and DR which was divided into five main branches.
The first branch was consisted of DT and contained 33 samples (S25?S48, collected from
Qinghai Province; S49?S57 collected from Tianzhu and Zhuoni counties in Gansu Province,
the altitude of habitats higher than 2900 m). The second branch including DR contained 13
samples (S66?S78, all gathered from Sichuan Province). The first branch merged with the
second branch (DR) to form a larger branch that merged with the other branches. The third
branch was consisted of DT and included 8 samples (S58?S65, collected from Tianshui and
Kang counties in Gansu Province). The fourth branch was consisted of DT and included 22
samples (S84?S93, all gathered from Shaanxi Province; S79?S83, S18?S24, gathered from
Pingwu, Mao and Heishui counties in Sichuan Province). According to plant morphological,
the fifth branch contained 4 samples (S14?S17, gathered from Taibai county in Shaanxi
Province) was identified to be DG. However, it was merged with DT and DR cluster. Likewise, it
was also close to the DG cluster. That may be the species variation of DG caused by specific
ecological environment or the coenospecies of DG and DT. The NJ tree obtained from ITS
sequences is able to easily distinguish and identify the 93 Daphnes Cortex samples gathered
from different species origins and habitats. The results of HCA were in good agreement with
the results of the morphological taxonomy. HCA placed all DT samples into one of two main
branches. DT samples with the altitude of habitats more than 2900 m were grouped in one
branch, and the others with the altitude of habitats below 2900 m merged gradually became
one branch. It likely showed that altitude of habitats may have important influence on the
ITS sequences of Cortex Daphnes. Accordingly, it revealed that samples from the same
species origins, similar geographical environments and nearby regions had similar ITS
sequences and were in the same or close clusters. DNA barcoding could successfully identify
the Cortex Daphnes samples.
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Fig 2. Results of NJ-tree analysis based on ITS sequences of the 93 Cortex Daphnes samples.
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Method validation of HPLC fingerprint
Optimization of extraction and chromatographic conditions. Four different concentra
tions of methanol and ethanol (60, 70, 80, and 100%) extractions were compared. The best
extracting way was ultrasonic and extracted for 45 min in 70% methanol. Under such
condition, more chromatographic peaks and more chemical contents were detected. As a result, the
best extracting condition was established as follows: the samples were extracted by ultrasonic
extraction using 70% methanol as the extracted solvent and the duration was 45min.
To obtain more chromatographic peaks, the optimized gradient elution program was used
in this study. To make the chromatograms with better separation and sharper peaks, the
mobile phase, column temperature and detection wavelength were all optimized. The elution
effect of the mobile phase constitution (methanol / water, methanol / 0.5% FA (formic acid)
water, acetonitrile / water, acetonitrile / 0.5% FA water) on the chromatographic separation
was compared. It showed that acetonitrile / 0.5% FA water was the best mobile phase
composition with higher elution efficiency. In this study, three column temperatures (20, 25, 30?C)
were selected to assess their efficiency on gaining higher resolution of the chromatographic
peaks. As a result, the best column temperature was 25?C. The wavelength for the detection of
compounds was selected by UV detector. Most of the chromatographic peaks could be
detected at approximately 327 nm, at which the chromatograms could provide maximum
absorption of daphnetin. So 327nm were chosen as detection wavelength for the HPLC
Precision, repeatability and stability tests. The test of precision, repeatability and
stability were performed by calculating the relative standard deviations (RSDs) of relative retention
times (RRTs) and relative peak areas (RPAs) based on common peaks respectively. The results
were showed in Table 1. All RSDs including RRTs and RPAs were < 3%, which indicated that
the method was sufficiently accurate, stabilized and sensitive for the fingerprint analysis of the
93 Cortex Daphnes samples.
Establishment of chromatographic fingerprint of Cortex Daphnes samples. To
establish the chromatographic fingerprint, 93 Cortex Daphnes samples from different species and
habitats were analyzed under the optimized chromatographic analysis conditions. Sample 1
was the reference sample, and all chromatograms (as shown in Fig 3a) through multipoint
correction and free matching were matched and reference chromatogram (as shown in Fig 3b)
was generated by the computer-aided Similarity Evaluation System for Chromatographic
Fingerprint of TCM (Version 2004A). Peaks in the fingerprint with quite large area and good
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Fig 3. (a) HPLC fingerprints of the 93 Cortex Daphnes samples (S1-S93) and (b) reference chromatogram. 4: Syringoside 5: Daphnetin 9:
8 / 17
resolution shared by all the chromatograms of the tested samples were selected as ?common
characteristic peaks? to represent the characteristics of all the samples. A total of 10 common
peaks shared by all samples (black peak No.) which covered more than 90% of the total area
and 28 common peaks detected in portion samples (green peak No.) were determined in the
reference chromatogram. One component was identified as daphnetin (peak 5) by comparing
its retention time and UV spectrum with the standard compound. The other common
fingerprint peaks were not identified.
SA of HPLC fingerprint of Cortex Daphnes samples
The similarity values between 93 Cortex Daphnes samples and the reference chromatogram
were calculated using the Similarity Evaluation System for Chromatographic Fingerprint of
TCM (Version 2004A), and the results were presented in Table 2.
According to the results, the similarity values were all >0.9. It was indicated that the main
chemical compositions among the 93 Cortex Daphnes samples were relatively consistent.
However, there were still many differences of chemical compositions among the Cortex
Daphnes samples gathered from different habitats and species origins. According to the fingerprint
chromatograms, some characteristic peaks especially in the range of 12?55 min only appeared
in part of the samples collected from similar ecological environment or the same species
origins. These special common characteristic peaks were also marked with green peak No. in the
reference chromatogram. Obviously, the types and quantities of chemical components among
Cortex Daphnes samples were not completely consistent. The varied chemical profiles of
Cortex Daphnes samples may be attributed to the different species origins and habitats, which
were the results of plants adapting to the environment.
HCA of HPLC fingerprint of Cortex Daphnes samples
HCA, one of the chemical pattern recognition and classification evaluation methods, is used to
set the level of bottom-up decomposition for a given data set until certain conditions are
fulfilled. HCA has been commonly applied for fingerprint analysis with standard normal variant
transformation of the data, which led to meaningful classification of herbal samples collected
from different regions [28?29]. In order to show the degree of similarity and differences
among the 93 Cortex Daphnes samples more clearly, the HCA in this study was performed
based on the RPAs (S3 File) of all common characteristic peaks (peaks 1?38, as shown in Fig
3b) by the professional analysis software SIMCA 13.0 Demo. The results of HCA were shown
in Fig 4a.
According to the results, all samples were classified into two main clusters: S1?S17 in cluster
1, S18?93 in cluster 2 (as shown in Fig 4a). Cluster 1 including 17 samples belonged to DG.
Additionally, cluster 2 was divided into six groups: S25?S57 in group1and group 2, S18?S24 in
group 3, S66?S78 in group 4, S84?S93 in group 5, S58?S65 and S79?S83 in group 6. These five
groups including 63 samples belonged to DT. Group 4 including 13 samples belonged to DR
(all gathered from Sichuan Province). Group 1 and group 2 including 33 samples were
collected from Qinghai and Gansu Province with the altitude of habitats more than 2900 m.
Group 3 including 7 samples were gathered from two adjacent counties (Maoxian and Heishui
counties in Sichuan Province). Group 5 including 10 samples were all gathered from Shaanxi
province. Group 6 including 13 samples were gathered from Sichuan and Gansu Province.
The Group 3 (DT) merged with the Group 4 (DR) to form a larger branch1. Maybe it's because
they were gathered from nearby areas. According to the HCA of chemical components and
N-J tree of ITS sequences, the DR samples merged with the DT samples consistently which
indicated that the chemical compositions and the ITS sequences between the two species were
much of a muchness. The results were consistent with the recordation about DR and DT in
Flora of China [
]. The Group 5 (DT) merged with the Group 6 (DT) to form a larger branch
2. All samples in the branch 2 were gathered from Qinling and Dabashan mountain areas
where the ecological environments were more similar. The branch 1 and branch 2 merged to
form a larger branch in which the altitude of the sample collection sites were below 2900 m
then merged with group 1and group 2. The classification results of HCA were consistent with
the classification of climate types of habitats, and agreed well with the categorized results of the
genetic taxonomy and the visual comparisons of their representative chromatograms, which
may provide more references for further quality control and evaluation of the commercial
Cortex Daphnes herbs.
PCA of HPLC fingerprint of Cortex Daphnes samples
PCA is a multivariate method and widely used in data analysis to summarize variation, which
is implemented as a data-reduction technique to generate a visual scatter plot for the
qualitative evaluation of resemblances and differences between the studied samples [
]. In order to
differentiate all the Cortex Daphnes samples clearly, the PCA was carried out based on the
RPAs (S3 File) of all common characteristic peaks (peaks 1?38, as shown in Fig 3b) by the
professional analysis software SIMCA 13.0 Demo. The score plot was structured based on the first
three principal components which accounted for more than 89.31% of the total variability, and
the other principal components which had little effect on the model were discarded.
According to the results of PCA, all samples were divided into seven groups according to
their different sources (as shown in Fig 4b). Group 1 contained 17 samples (S1-S17) belonging
to DG. Group 2 and group 3 contained 33 samples (S25-S57) belonging to DT. Group 4
contained 13 samples (S66-S78) belonging to DR. Group 5 contained 7 samples (S17-S24)
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Fig 4. (a) Results of HCA based on HPLC fingerprint of the 93 Cortex Daphnes samples and (b) score plot of PCA of the
93 Cortex Daphnes samples.
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belonging to DT. Group 6 contained 10 samples (S84-S93) belonging to DT. Group 7
contained 13 samples (S58-S65, S79-S83) belonging to DT. Groups excepting group 1, group 2
and group 3 were quite close to each other. The results of PCA were in good agreement with
the results of HCA based on the fingerprint. The classification results of the scatter plot
adequately showed the noticeable provenance and geographical differences among the samples.
In this study, samples (S14-S17) were divided into DG groups according to the results of
phytochemical and morphological taxonomy which was not completely consistent with results
of the genetic taxonomic. This conclusion illustrated that the accurate identification of
medicinal materials requires comprehensive applications of multifarious identification techniques.
In this study, a system based on DNA barcoding and HPLC fingerprint analysis to identify
and classify the Cortex Daphnes herbs has been established. Two universal classification
techniques include four different analysis methods: SA, HCA and PCA of the chemical
components and the NJ-tree analysis of ITS sequences. 93 Cortex Daphnes samples, gathered from
different species origins and wild areas in Western China, were identified and classified
successfully. The results of genetic taxonomic were greatly consistent with the phytochemical
taxonomy results. The morphological taxonomy also played an important role in the
identification of Cortex Daphnes samples, such as the distinction of species DR. The conclusions
drawn from the classification system were more objective and scientific. The classification
system could efficiently identify and control the quality of the three Daphne species. Furthermore,
these methods are convenient and suitable for practical use, further pharmacological research
and development of the three species. The system is also able to provide a identification model
for other commercial Chinese herbal medicines to guarantee their clinical safety.
The results of genetic and phytochemical taxonomic showed that samples belonging to the
same species origins and nearby habitats could be clustered together, which demonstrated that
the type and composition of the chemical components of the medicinal plants were the
combined effect of both genetic materials and habitats. For the same Daphne species, climate types
of their habitats may be the decisive factor that make their chemical compositions exist larger
differences. The altitude may play an important role, which may be as a classification
foundation (as shown in Fig 1). In addition to the altitude, there are many other ecological factors
which can influence the type and composition of the chemical components of the medicinal
plants, such as temperature, soil, light, moisture and so on. In order to guarantee the quality
stability of the Cortex Daphnes herbs, to realize the artificial cultivation and protect wild
plants, the effects exerted by the genetic materials and ecological factors to the active
ingredients of the three Daphne species is required deep study in future.
Materials and methods
Chemicals and materials
Methanol (analytical grade) and acetonitrile (HPLC grade) were purchased from Fisher
Scientific International (Fair Lawn, New Jersey, USA). Ultra-pure water was generated by an
Ultrapure Water System (Shanghai Ultrapure Technology, Shanghai, China). Standard daphnetin
was purchased from the National Institutes for Food and Drug Control (Beijing, China). The
93 wild Cortex Daphnes samples including 93 stem bark coupled with 93 leaves were collected
from four local provinces of China: Gansu, Shaanxi, Qinghai and Sichuan. They were
identified as genuine samples of DG, DT and DR by Professor Chunsheng Liu (Beijing University of
Chinese Medicine, Beijing, China) through their leaves (as listed in Table 3 [
]). All samples
were dried at 22?25?C. The 93 stem bark were comminuted to powder separately, and sieved
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through a 74?m (or 200 mesh) screen for HPLC analysis and the leaves were used for DNA
DNA barcoding analysis
DNA extraction. Genomic DNA from dried leaves of 93 Cortex Daphnes samples which
were collected in Beijing University of Chinese Medicine were extracted according to the
instructions of the plant DNA extraction kit (Tiangen, Beijing, China). All the DNA were
stored at -20?C before analysis [
PCR and sequencing. A total of 30?L PCR system contains: template DNA 3?L, Mix-Taq
enzyme 15?L, ITS sense primer 1.2?L, ITS antisense primer 1.2?L, ddH2O 9.6?L. PCR
amplification was performed: 95?C for 4 min, followed by 35 cycles of 94?C for 30s, 55?C for 1 min,
72?C for 1 min, and final extension 72?C for 10min (Bio-Rad T100? Thermal Cycler). 5?L of
PCR products were examined via electrophoresis in a 1.0% agarose gel, and were sequenced by
Shanghai sangong company. To ensure the accuracy, samples were all sequenced in two-way.
All the ITS sequences obtained were cut and spliced using DNAMAN and ContigExpress
software, then were submitted to GenBank-NCBI for comparison with the deposited sequences of
near-source species using the tool BLAST. The modified ITS sequences were aligned and a N-J
tree was constructed based on standard parameters with bootstrap testing of 1000 replicates
using ClustalX and MEGA version 5.0 software. The N-J tree was used to observe the natural
interrelationships and differences for each of the Cortex Daphnes samples by ITS sequences
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Samples and standard solution preparation. The dried powdered sample (0.500g) was
precisely weighed and extracted with 50 mL of 70% v/v methanol by ultrasonic extraction for
45 min. The extraction solution was supplemented to the weight of pre-extracted before
analysis when it cooled down to room temperature. The solution was filtered through a 0.45 ?m
membrane filter and stored at 4?C out of light before HPLC analysis.
The standard daphnetin was prepared for the qualitative of chromatographic peak. The
concentration of standard solution was 60?g/mL in methanol and 10?L solution was injected
into the HPLC for analysis according to the standard [
]. The standard solution was filtered
through 0.45 ?m membrane filters and stored at 4?C out of light and brought to room
temperature before HPLC analysis.
Instrumentation and analytical conditions. HPLC analyses were performed using a
Waters system (Waters e2695/2489/Emp2) with a UV detector. Chromatographic separations
were achieved using gradient elution on a C18 reserve-phase column (4.6 mm? 150 mm, 5 ?m;
Agilent Technologies). The column temperature was maintained at 25?C. The absorption
wavelength was 327 nm which was selected by UV detector according to max UV absorption
of the reference. The mobile phase consisted of acetonitrile (A) and 0.5% FA water (B) with a
linear gradient elution at a flow rate of 1.0 mL/min. The gradient elution program was as
follows: 5?27.4% (A) in 0?40 min, 27.4?42%(A) in 40?45 min, 42?53% (A) in 45?58 min, 53?
90% (A) in 58?68 min and 90?5% (A) in 68?78 min. The sample injection volume was 10?L.
The daphnetin as the common components of the three species which chromatographic
peak was selected to be the referential chromatographic peak, the RRTs and RPAs were
analyzed. Method precision was determined by injecting one Cortex Daphnes sample solution
(S1) six times continuously. The repeatability was assessed through six independently prepared
sample solutions (S1). The stability of the injection solution was determined periodically by
injecting samples (S1) stored at 4?C ranging from 0 to 24 h (0, 3, 6, 9, 12, and 24h). The results
were all expressed by RSDs of RRTs and RPAs, respectively, of all common peaks (1, 2, 3, 4, 5,
7, 9, 11, 23 and 29) from six chromatographic profiles of Cortex Daphnes sample (S1).
SA analysis. The HPLC analyses of all the samples were carried out under the established
experimental condition. The data of all chromatographic profiles was converted into ?AIA?
form from the software of Empower. SA was performed on the basis of the RRTs and RPAs
using the professional software named Similarity Evaluation System for Chromatographic
Fingerprint of TCM (2004A), which was recommended by the State Food and Drug
Administration of China (SFDA) for calculating the similarity coefficient (SC) of the chromatographic
profiles of TCM. The similarities among different chromatograms were quantified by
calculating the correlation coefficient or the cosine value of the vectorial angle [32?33].
HCA and PCA analysis. The statistical analysis was performed using the professional
analysis software SIMCA 13.0 Demo for HCA and PCA. HCA and PCA were used to show the
unsupervised clustering pattern of the Daphne Linn species and discover the differences in
samples caused by complex factors. HCA and PCA were used to discover the natural
interrelationships among the chemical components for each of the Cortex Daphnes samples [
The RPAs of main characteristic peaks were selected as the clustering variable and the critical
P value for all analyses in this study was set to 0.05.
All the data were pretreated including background deduction and the chromatograms
alignment before SA, HCA, and PCA. The datasets generated and analyzed during the current
study are available from the corresponding author on reasonable request.
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S1 File. Photos of three Daphne species.
S2 File. ITS sequences obtained from samples and GenBank.
S3 File. Results of HCA and PCA of Cortex Daphnes samples.
We gratefully thank students Xuezhong Wang, Tianda Zhou, Heng Wang, Wencang Tian,
Kunpeng Wang, Sitong Li and Jiwen Li for the collection of wild Cortex Daphnes samples.
This study was supported by the Pharmaceutical Co., Ltd., of Qinhuangdao Shanhai Pass
(Qinhuangdao 066200, China) which provided great financial support. The funder did not have
any additional role in the study design, data collection and analysis, decision to publish, or
preparation of the manuscript. We would also thank agriculture and forestry sectors in Gansu,
Shaanxi, Sichuan and Qinghai provinces for providing great support and help in the research
Conceptualization: Yanpeng Li.
Data curation: Yanpeng Li, Lu Geng, Yuanyan Liu.
Formal analysis: Yanpeng Li, Mingyang Chen.
Funding acquisition: Xu Zhang, Chunsheng Liu.
Investigation: Mingyang Chen, Qirui Mu, Zhongyi Zhang.
Methodology: Lu Geng, Mingyang Chen.
Project administration: Yanpeng Li, Xu Zhang, Chunsheng Liu.
Resources: Lu Geng, Mingyang Chen, Qirui Mu, Xu Zhang, Zhongyi Zhang.
Software: Yanpeng Li, Lu Geng, Mingyang Chen, Qirui Mu, Zhongyi Zhang.
Supervision: Yuanyan Liu, Guangxi Ren, Chunsheng Liu.
Validation: Yuanyan Liu, Xu Zhang, Guangxi Ren, Chunsheng Liu.
Writing ? original draft: Yanpeng Li, Lu Geng.
Writing ? review & editing: Yanpeng Li, Lu Geng, Yuanyan Liu, Qirui Mu, Guangxi Ren.
15 / 17
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