Chemical Fingerprint Analysis and Quantitative Analysis of Rosa Rugosa by UPLC-DAD
Chemical Fingerprint Analysis and Quantitative Analysis of Rosa Rugosa by UPLC-DAD
Chemical Fingerprint Analysis and Quantitative Analysis of Rosa Rugosa by UPLC-DAD
v1
Article
Chemical Fingerprint Analysis and Quantitative
Analysis of Rosa Rugosa by UPLC-DAD
Sanawar Mansur 1,2,3, Rahima Abdulla 1,2, Amatjan Ayupbec 1,2 and Haji Akbar Aisa 1,2,*
1 Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute
of Physics and Chemistry, Chinese Academy of Sciences, Xinjiang 830011, China;
sanam0405@163.com (S.M.); rahima@ms.xjb.ac.cn (R.A.)
2 Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical Institute of Physics
Abstract: A method based on ultra performance liquid chromatography with diode array detector
(UPLC-DAD) was developed for quantitative analysis of five active compounds and chemical
fingerprint analysis of Rosa rugosa. Ten batches of Rosa rugosa collected from different plantations in
the Xinjiang region of China were used to establish the fingerprint. The feasibility and advantages
of the used UPLC fingerprint were verified for its similarity evaluation by systematically comparing
chromatograms with professional analytical software recommended by State Food and Drug
Administration (SFDA) of China. In quantitative analysis, the five compounds showed good
regression (R2=0.999 5) within the test ranges and the recovery of the method was in the range of
94.2–103.8%. The similarities of the fingerprints of 10 batches of the samples were more than 0.981.
The developed UPLC fingerprint method is simple, reliable and validated for the quality control
and identification of Rosa rugosa. Additionally, simultaneous quantification of five major bioactive
ingredients in the Rosa rugosa samples was conducted to interpret the consistency of the quality test.
The results indicated that the UPLC fingerprint as a characteristic distinguishing method combining
similarity evaluation and quantification analysis, can be successfully used to assess the quality and
to identify the authenticity of Rosa rugosa.
1. Introduction
Traditional Chinese medicine (TCM) has recently become an attractive subject for many
scientists and drug producers. Many TCM, across history and cultures, have been used for medicinal
purposes as alternative therapies based on plants in order to avoid drug adverse effects, and over the
past years, many articles were reported. When TCM were used, particular attention must be also paid
to standardization process. Additionally, the Food and Drug Administration (FDA) specifies certain
labeling requirements for foods, supplements, and drugs, and the European Union requires that
standardized herbal substances are reported as content of constituents with known therapeutic
activity.
The genus Rosa rugosa Thunb. (Family Rosaceae) is conventionally used as medicinal plants in
TCM. Rosa rugosa is distributed throughout the temperate regions of eastern Asia, including China,
Japan and Korea. In Asia, it is a traditional herbal medicine for treating stomach ache, diarrhea,
menoxenia, pain and chronic inflammatory disease [1]. Phytochemical studies conducted so far
showed the isolation of tannins, flavonoids, terpenoids, triterpenoids, steroids, tocopheral and
carotene [2]. Tannins and flavonoids are of special interest related to their activities, such as
antioxidative, antidiabetes and antinflammatory activity associated with global diseases including
diabetes mellitus, pain and chronic inflammatory [3].
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The qualitative and quantitative analysis of major components, and the analysis of chemical
fingerprint, which has been introduced and accepted by State Food and Drug Administration (SFDA)
of China (2000) (State Food and Drug Administration of China 2000). Different cultivation areas and
climatic conditions may vary the chemical constituents of Rosa rugosa significantly. Since application
is growing steadily, development of a suitable quality control method was urgently required. The
official Chinese pharmacopoeia (China Pharmacopoeia Committee 2010) does not include the quality
evaluation of Rosa rugosa.
Among the chromatographic fingerprinting applied to the authentication and qualitative
evaluation of botanical products over the past decade, high performance liquid chromatography
(HPLC) fingerprinting emerges as the most widely used method because of its convenience and
efficiency [4-6]. However, the acquisition of a fingerprint and quantitative analysis by these methods
was a tedious operation, as it generally needed about one or more hours for a single run. In recent
years, UPLC is emerging as a viable technique for quantitative and chemical fingerprint analysis of
natural product, and some reports have appeared in literature on its applications in the fingerprinting
and quantitative analysis of Chinese herbal medicines [7-8]. The results obtained in these references
demonstrated that UPLC was indeed a very powerful tool in chromatographic fingerprinting
applications and quantitative analysis of the components in these herbal medicines.
To the best of our knowledge, there are no reports regarding the fingerprint analysis of Rosa
rugosa. The objective of this study was to establish an effective UPLC fingerprint method for the
identification and quality evaluation of Rosa rugosa. The chromatograms of the extracted samples
from different Rosa rugosa plantations of Xinjiang were compared visually and analyses by similarity
evaluation. Moreover, twenty-three components in ten batches of Rosa rugosa were simultaneously
quantitated by UPLC method.
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formic acid (pH 2.98, v/v) and 0.1% formic acid solution (pH 2.67, v/v) was chosen for the
determination of Rosa rugosa with a large number of peaks on the chromatogram achieved within 71
min. More detectable peaks could be obtained and the baseline was well improved around 260 nm,
and therefore, better results for 5 target compounds in Rosa rugosa and reference standards could be
obtained. Hence characteristic chromatographic patterns were obtained by using 260 nm as the
detection wavelength. Optimal UPLC condition used in this study was shown in UPLC condition
Section.
Figure.2 The typical UPLC chromatographic profile of five standards peaks 1, GA (3.8 3min); 2, EA
(30.7 min); 3, Kaempferol-3-O-sophoroside (38.9 min) 4, Hyperoside (48.9 min); 5, Astragalin (57.2
min).
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The recovery test was determined by standard addition method. The samples (Sample 1) were
spiked with the high, intermediate and low levels of mixed five standards solution in triplicates, and
then extracted, processed and quantified in accordance with the established procedures. The results
of the recovery rates are summarized in Table 1. The recovery rates were performed using a Waters
Acquity BEH Shield C18 column (100 mm 2.1 mm i.d., 1.8 µm), The recovery rates of the five
compounds were in the range of 94.2–103.8% and their RSD values were less than 2.1%. Therefore,
the UPLC-DAD methods were precise, accurate and sensitive enough for simultaneously
quantitative evaluation of five compounds in Rosa rugosa.
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Table 2: Analytical results of precision, stability and repeatability tests of 23 characteristic common
peaks in Rosa rugosa samples (Sample 8) (n = 5).
8(S) 0 0 0 0 0 0
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Figure.3: The reference fingerprint of Rosa rugosa: (1) GA (3.783 min); (5) 2-Phenylethyl-O-β-D-
glucopyranoside (26.9 min); (6) Quercetin -3-O-(2′′-O-β-D-glucopyranosyl)- β-D-glucopyranoside)
(28.0min); (7) Juglanin (28.9 min) (8) EC (30.7 min); (9) Avicularin (36.0 min); (10) Quercetin (38.3 min);
(11) Kaempferol-3-O-sophoroside (38.9 min) (16) Hyperoside (48.9 min); (18) Astragalin (57.3 min).
To standardize the fingerprint, 10 samples were analyzed. The software named Similarity
Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version
2004A) (Fig. 4) was used to evaluate chromatograms. These samples had similar UPLC profiles. Peaks
that existed in all 10 samples with relatively high intensity and good resolutions were assigned as
“characteristic common peaks” for identification of the plant. There were 23 characteristic peaks
(from peak 1 to 23) found in the chromatogram, which covered more than 65% of the total area (Fig.
3). 10 components were identified as peak1 GA, peak5 2-Phenylethyl-O-β-D-glucopyranoside, peak6)
Quercetin -3-O-(2′′-O-β-D-glucopyranosyl)- β-D-glucopyranoside), peak7 Juglanin (28.9 min) , peak8
EC (30.7 min), peak9 Avicularin (36.0 min), peak10 Quercetin (38.3 min) , peak11 Kaempferol-3-O-
sophoroside (38.9 min) , peak16 Hyperoside (48.9 min), peak18 Astragalin (57.3 min).by comparing
their retention time and UV spectrum with those of standard compounds. The other 13 common
fingerprint peaks were unknown. To calculate the RRT and RPA of each characteristic peak, a
reference peak should be chosen. Ellagic acid (peak 8) had a considerably high content of more than
0.46% of the total area, and it also had moderate retention time, stable peak area and good shape in
the Rosa rugosa chromatograms. Therefore, it was chosen as the reference peak. Then the retention
time and peak area of the 23 common peaks were measured; RRT and RPA of all characteristic
common peaks with respect to this reference peak were calculated (Table 3).
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Table 3: The retention time (tR), relative retention time (RRT), peak area (PA) and relative peak area
(RPA) of 23 common peaks in Rosa rugosa (n = 10).
tR RRT PA RPA
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our literature review regarding the plant species under study, it appears that these five compounds
have not been quantified before and are reported for the first time in this paper.
S1-5 of Rosa rugosa were collected from north of Xinjiang while the last parts were collected from
south of Xinjiang. The Average contents of Hyperoside, Astragalin, Kaempferol-3-O-sophoroside
were and GA in Rosa rugosa collected from south of Xinjiang higher than that in north of Xinjiang.
The Average contents of EA higher than that in south of Xinjian (Table 4).
Ten batches of raw material samples of Rosa rugosa were collected from Xinjiang Uyghur
Autonomous Region, China. S1 was collected from Qitai of Xinjiang. S2 – S5 were collected from
Jimsar o f Xinjiang. S6 was collected from ShaChe of Xinjiang. S7 was collected from YuTan of
Xinjiang. S8 was collected from HuTan of Xinjiang. S9 was collected from Hutan country of Xinjiang.
S10 was collected from MoYu of Xinjiang.All the voucher specimens identified by research fellow
Guanmian Shen, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences.
3.2. Reagents
Acetonitrile (Fisher, optima®, LC-MS grade, Fair Lawn, NJ 07410, U.S.A.) and formic acid
(Merck, EMSURE®, analytical grade, Darmstadt 64271, Germany) were used. Water used in the
experiment was deionized and further purified by the Milli-Q Plus water purification system
(Millipore Ltd., Bedford, MA, USA). Other reagents and chemicals were of analytical grade.
Standard preparation: The five chemical standards (hyperoside, gallic acid, ellagic acid;
astragalin, Kaempferol-3-O-sophoroside were confirmed by UV, ESI-MS, and the chromatogram of
mixture standards was shown in Figure 1. The purity of each compound was determined to be higher
than 98% by normalization of the peak area detected by HPLC. The reference compounds were
accurately weighed and dissolved in methanol and diluted to appropriate concentration ranges for
the establishment of calibration curve. All stock and working standard solutions were stored at 4 ℃
until used for analysis.
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Sample preparation: Dried and finely powdered flowers of Rosa rugosa were extracted with 60%
aqueous ethanol (100 mL) for 1 hour under reflux. The solution was filtered through a 0.22 µm filter
before UPLC analysis.
UPLC analysis was performed on a Waters Acquity UPLCTM system (Waters, Milford, MA, USA)
equipped with binary solvent delivery pump, an auto sampler and photodiode array detector (PAD).
The instrument was controlled by Waters Empower 2 software. The chromatographic separation was
performed using a Waters Acquity BEH Shield C18 column (100 mm 2.1 mm i.d., 1.8 µm, Waters,
Massachusetts, U.S.A.), operated at 35 ℃ . The mobile phase consisted of 0.1 % formic acid–
acetonitrile (A) and 0.1 % formic acid–water (B) with a gradient elution of 2 % A (0–1 min), 2–5 % A
(1–2 min), 5-10 % A (2–7 min), 10-11 % A (7–10 min), 11-18 % A (10–56 min), 18-28 % A (56-69 min),
28-43 % A(69–71min). Chromatograms were recorded at an absorbance of 260 nm. The mobile phase
was eluted at a flow rate of 250 µL min-1, and injection volume was 2.00 μL.
Standard curves, limits of detection and recovery rates of quantitative analysis: The standard
curves were obtained by plotting the peak area against nominal concentration of each compound and
were fitted to a linear function of type y = ax + b. In this equation, y and x represent peak area and
nominal concentration in mg/L, respectively. The limit of detection (LOD) was estimated as the
minimum concentration of the compounds needed to produce signals that were at least three times
stronger than the noise signal (S/N). The accuracy tests were carried out by spiking the known
contents of mixed standard solution into the known concentration of Rosa rugosa samples, and the
assessment was done by analyses the three different spiking concentrations of analyses in triplicates.
The percent recovery rates for the analyses were presented as mean of the three results.
The precision of the UPLC fingerprint method was determined by analyses the replicated
extraction solution of the same sample five times within a day. The sample stability test was
determined with one sample on two consecutive days. The repeatability was assessed by analyses
five independently prepared extraction solutions of Rosa rugosa samples. During this period, the
solution was stored at room temperature.
Establishment of UPLC fingerprint and similarity analysis: To establish the representative
chromatographic fingerprint, ten batches of Rosa rugosa samples were analyzed under the established
UPLC method. The obtained UPLC data from ten batches of Rosa rugosa samples were exported from
Waters Empower 2 software in AIA format and imported to the professional software named
Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine
(Version 2004A). This system could reflect the similarity of the distribution ratio of the chemical
composition accurately, as recommended by the SFDA.
5. Conclusions
In this study, UPLC-DAD method proved to be simple, accurate, and reliable for developed
UPLC fingerprint and the determination of five bioactive compounds in Rosa rugosa. For the
fingerprint analysis, 23 characteristic fingerprint peaks were applied to evaluate the similarities
among ten batches of Rosa rugosa and they showed good similarities. For the quantitative
determination, five components of ten batches of Rosa rugosa were successfully separated and
determined. The UPLC fingerprint method was well validated by systematically comparing
chromatograms of all samples from different regions. The method developed in this study will
provide an important reference to establish the quality control method for other related traditional
Chinese medicinal preparations.
Supplementary Materials: The following are available online at www.mdpi.com/link, Figure S1: title, Table S1:
title, Video S1: title.
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Acknowledgments: This study was financially supported by Key Deployment Projects of the Chinese Academy
of Sciences (No. KSZD-EW-Z-004-04).
Author Contributions: Sanawar Mansura, Rahima Abdulla, Amatjan Ayupbec and Haji Akbar Aisa conceived
and designed the experiments; Sanawar Mansura, performed the experiments; Sanawar Mansura and Rahima
Abdullab analyzed the data; Haji Akbar Aisacontributed reagents/materials/analysis tools; Sanawar Mansura
wrote the paper. All authors read and approved the final manuscript.
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Sample Availability: Samples of the compounds GA, EA hyperoside, astragalin and Kaempferol-3-O-
sophoroside are available from the authors.
© 2016 by the authors; licensee Preprints, Basel, Switzerland. This article is an open access
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Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).