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Quality assessment on Polygoni Multiflori Caulis using HPLC/UV/MS combined with principle component analysis
© Zhao et al.; licensee Chemistry Central Ltd. 2013
- Received: 17 April 2013
- Accepted: 19 June 2013
- Published: 24 June 2013
Polygoni Multiflori Caulis, the dried caulis of Polygonum multiflorum Thunb., is one of the commonly used traditional Chinese medicines having antioxidant, anti-obesity, anti-inflammatory and antibacterial effects. Polygoni Multiflori Caulis used clinically or circulated on market have great differences in their diameters. However, to the best of our knowledge, no study has been reported on the qualities of Polygoni Multiflori Caulis with different diameters.
Systematic HPLC/UV/MS chromatographic fingerprinting and quantitative analytical methods combined with principal component analysis were developed and applied to analyze different Polygoni Multiflori Caulis samples. The contents of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside, the chemical marker for quality control on Polygoni Multiflori Caulis specified in Chinese Pharmacopoeia (2010 edition), were found to have surprising relevance with the samples’ diameters for the first time.
The finding provides a scientific basis for collecting Polygoni Multiflori Caulis in the best time. Moreover, the diameter can be used as the criterion for quality control on Polygoni Multiflori Caulis as a preliminary step in the future. In addition, scores plot obtained from principal component analysis shows the obvious differences between unqualified Polygoni Multiflori Caulis samples and qualified ones visually, which can be used to single out the unqualified ones with qualified ones efficiently and immediately.
- Polygoni Multiflori Caulis
- Quality assessment
- Principle component analysis
Polygoni Multiflori Caulis (PMC), Shou-Wu-Teng in Chinese, is the dried caulis of Polygonum multiflorum Thunb. It is one of the commonly used traditional Chinese medicines (TCMs) listed in Chinese Pharmacopoeia (CP) (2010 edition) . Pharmacological studies indicated that it had antioxidant [2, 3], anti-obesity , anti-inflammatory and antibacterial effects .
Anthraquinones, flavonoids and stilbene glycosides are considered to be the main active constituents in PMC [6, 7]. However, unlike Polygoni Multiflori Radix, He-Shou-Wu in Chinese, there are only a few reports on quality control of PMC. High performance liquid chromatography (HPLC) with ultraviolet detector (UV) were applied to determine the contents of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) and emodin [8–10], but THSG, one of the stilbene glycoside, was not specified as chemical marker for quantitative determination of PMC until CP (2010 edition) was published.
As for original plant morphology, it is regulated in CP (2010 edition) that the diameter of PMC is between 4 and 7 mm. However, it derives from P. multiflorum which is a perennial plant and can be harvested all the year round, so PMC circulated on market have big variations on their diameters. In the PMC samples we collected, the smallest diameter is just 0.5 mm, however, the biggest one reaches to 36 mm. Their differences go so far to 70 times unexpectedly. In that way, are there any differences on their qualities? The issue arouses our great interest.
In the present study, chromatographic fingerprinting and quantitative analytical methods were developed to analyze different PMC samples. Seven peaks, marked as 1 to 7, were designated as characteristic peaks in chromatographic fingerprints. They were identified as THSG, emodin-8-O-β-D-glucoside, emodin-8-O-(6′-O-malonyl)-β-D-glucoside, physcion-8-O-β-D-glucoside, physcion-8-O-(6′-O-acetyl)-β-D-glucoside, emodin and physcion, respectively, based on UV and MS data compared with reference compounds and related literatures [11–17]. THSG, emodin and physcion were quantified at their maximal UV wavelengths. From the results, we found that the contents of THSG had great relevance with the diameters of PMC samples. Principal component analysis (PCA), one of the popular chemometrics, was then used for comprehensive and systematic assessment on PMC samples collected from different regions with different diameters, based on the variables including the contents of the three quantified analytes and the PA/W (peak area divided by sample weight) values of the four unquantified ones. Very useful information were obtained from PCA scores plot, by which unqualified PMC samples could be distinguished from qualified ones visually and immediately. Points of view how variables contributed to samples’ positions in scores plot were also discussed in detail according to PCA loadings plot.
Chemicals, solvents and herbal materials
Collected information of the nineteen PMC samples and the content (%) of THSG, emodin and physcion in the samples
Content (mg/g dry weight) (Mean ± SD)
Moisture content (%)
19.547 ± 0.0016
0.791 ± 0.0003
0.785 ± 0.0001
Raw Material a
3 ~ 9
23.766 ± 0.0116
1.295 ± 0.0007
1.394 ± 0.0005
3 ~ 9
0.742 ± 0.0007
0.023 ± 0.0001
Medicinal Slices b
7 ~ 12
1.275 ± 0.0007
0.054 ± 0.0001
0.043 ± 0.0000
0.5 ~ 2
0.351 ± 0.0004
0.015 ± 0.0000
3 ~ 9
0.268 ± 0.0001
0.074 ± 0.0000
0.086 ± 0.0000
3 ~ 11
5.955 ± 0.0022
0.452 ± 0.0002
0.592 ± 0.0005
4 ~ 8
11.184 ± 0.0070
0.424 ± 0.0004
0.836 ± 0.0008
2 ~ 5
1.195 ± 0.0032
0.031 ± 0.0000
0.102 ± 0.0002
11 ~ 26
0.427 ± 0.0003
0.010 ± 0.0000
9 ~ 27
0.501 ± 0.0002
0.025 ± 0.0001
0.015 ± 0.0001
10 ~ 26
1.482 ± 0.0011
0.039 ± 0.0000
0.042 ± 0.0000
4 ~ 14
0.880 ± 0.0005
0.027 ± 0.0000
0.013 ± 0.0000
10 ~ 28
1.683 ± 0.0014
0.014 ± 0.0000
12 ~ 26
7 ~ 36
0.840 ± 0.0005
4 ~ 14
8 ~ 23
7 ~ 15
41.361 ± 0.0009
0.864 ± 0.0009
1.054 ± 0.0006
2 ~ 6
Dried PMC samples were sliced into small pieces and were ground into fine powders (20 mesh) using a grinder with a knife blade. Half gram of each PMC powder was carefully weighed into a 50 mL centrifuge tube. Twenty microliters of 75% methanol was then added into the tube and shaken briefly to mix. Each sample was then sonicated in an ultrasonic cleaner (Delta DC400H) at a frequency of 40 kHz at 25°C for 30 min. The extract was centrifuged for 10 min at 3000 rpm and the supernatant was then transferred into a 50 mL volumetric flask. The procedure was repeated for one more time and the supernatants were combined. The final volume was made up to 50 mL with 75% methanol. The final combined extract was filtered through a 0.45 μm PVDF syringer filter (VWR Scientific, Seattle, WA) before analysis. An aliquot of 10 μL solution of each sample was used for HPLC and HPLC-ESI-MS analyses.
Stock standard solutions of the three accurately weighed reference compounds were prepared in 75% methanol. A standard mixture was obtained by mixing the individual stock standard solution to give THSG at a concentration of 252.5 mg/L, emodin at 138.325 mg/L and physcion at 15.4 mg/L. The standard mixture was diluted with 75% methanol to appropriate concentrations for calibration curves. The solutions were brought to room temperature and filtered through 0.45 μm PVDF syringer filter and an aliquot of 10 μL of each solution was used for HPLC analysis.
HPLC analyses were performed on a Waters 2695 HPLC system equipped with Waters 2998 photodiode array detector (PDA), Waters e2695 separations module and column heater module. A Grace Alltima C18 column (250 mm × 4.6 mm i.d., 5 μm) was used. The mobile phase consisted of 0.5% v/v formic acid aqueous solution (A) and acetonitrile (B). The optimized elution conditions were as follow: 0–22 min, 16% B; 22–45 min, 16-34% B; 45–60 min, 34-38% B; 60–70 min, 38-95%; 70–80 min, 95% B. The flow rate was 1 mL/min and the injection volume was 10 μL. UV spectra were acquired from 190 nm to 400 nm. The autosampler and column compartment were maintained at 25°C and 35°C, respectively.
HPLC-ESI-MS analyses were performed on a TSQ Quantum Access Max Triple Stage Quadrupole Mass Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with an Accela 1250 UHPLC system equipped with an Accela 1250 photo diode array (PDA) detector, an Accela HTC PAL autosampler, and an Accela 1250 binary pump. The column and elution conditions used were the same as those used in “HPLC analysis” except that the flow rate was set at 0.25 mL/min with a split ratio. Ultrahigh pure helium (He) and high purity nitrogen (N2) were used as collision gas and for nebulizer, respectively. The optimized parameters in negative/positive ion modes were as follows: ion spray voltage, -2.5 kV/3.0 kV; auxiliary gas, 40 arbitrary units; sheath gas, 15 arbitrary units; capillary temperature, 350°C; vaporizer temperature, 350°C; capillary offset, -35 V/18 V; tube lens offset, -33 V/102 V. Spectra were recorded in the range of m/z 100–1000 for full scan data, meanwhile, the normalized collision energy was set at 45% for MS2 data with dependant scan.
Quantitative analytical method validation
The limits of detection (LOD) and quantitation (LOQ) were defined as the lowest concentrations of analytes in the sample that can be detected and quantified, which were determined on the basis of signal-to-noise ratios (S/N) at 3:1 and 10:1, respectively. Intra- and inter-day variations were chosen to evaluate the precision of the developed method. The intra-day variation was determined by analyzing one of the mixed stock solutions (THSG at 50.5 mg/L, emodin at 27.665 mg/L and physcion at 3.08 mg/L) five times within one day. While for inter-day variability test, the same solution was examined in triplicate for three consecutive days. Repeatability was confirmed with five different working solutions prepared from sample PMC-01. Stability was tested with the same sample solution at 0, 2, 4, 8, 12, 24 h.
Fingerprinting and principal component analyses
The data obtained from chromatographic fingerprints were analyzed with Solo (Eigenvector Research, Inc.,Wenatchee, WA) for chemometric analysis. Normalize (2-Norm, length = 1) and mean center were used for data reprocessing before principal component analysis (PCA) was performed.
Optimization of extraction method
The extraction solvents were optimized based on the extraction efficiency of THSG. Four solvents, ethanol, 50% methanol, 75% methanol and methanol were investigated with sonication at room temperature for 30 min. As a result, 75% methanol was proved to be superior to other solvents (Additional file 1: Figure S1 A and Figure S1 B), which was selected as the extraction solvent. The optimal extraction times for 75% methanol was further investigated. The powder of Polygoni Multiflori Caulis (0.5 g) was extracted with 20 mL of 75% methanol for three times (30 min for each time). It showed that most THSG was extracted (> 99%) after the second extraction. (Additional file 1: Figure S1 C). Finally, the optimal extraction method was finalized, as described in “Sample preparation”.
Optimization of chromatographic conditions
Assignments of the seven characteristic peaks
Assignments of the seven characteristic peaks by HPLC/UV/MS
MS in Neg. mode
MS2in Neg. mode
MS in Pos. mode
MS2in Pos. mode
407 [M + H]+
245 [M + H-glucosyl]+
451 [M-H + HCOOH]-
429 [M + Na]+
811 [2 M-H]-
245 [M + H-glucosyl]+
433 [M + H]+
455 [M + Na]+
887 [2 M + Na]+
271 [M + H-glucosyl]+
863 [2 M-H]-
541 [M + Na]+
271 [M + H-malonylglucosyl]+
469 [M + Na]+
285 [M + H-glucosyl]+
491 [M-H + HCOOH]-
511 [M + Na]+
533 [M-H + HCOOH]-
285 [M + H-acetylglucosyl]+
271 [M + H]+
285 [M + H]+
307 [M + Na]+
Peak 1 occurs at retention time of 19.6 min with maximal UV absorption at 319 nm. In negative ion mode, the deprotonated molecular ion at m/z 405 [M-H]-, formic acid adduct ion at m/z 451 [M-H + HCOOH]- and 811 [2 M-H]- were found in its MS spectrum. Fragmentation of the ion at m/z 405 [M-H]- yielded a product ion at m/z 243 arising from the loss of a glucosyl (−C6H10O5) unit. In positive ion mode, the protonated molecular ion at m/z 407 [M + H]+ and a sodium adduct ion at m/z 429 [M + Na]+ were found in its MS spectrum. The MS2 fragmentation of the ion at m/z 407 was further investigated and a dominant product ion at m/z 245 [M + H-glucosyl]+ was observed, corresponding to the loss of the glucosyl unit (162 amu). This peak was unequivocally identified as THSG by comparison with MS data of the standard as well as literatures [11–14].
Peak 3 occurs at retention time of 48.2 min with maximal UV absorption at 281 nm. Ions at m/z 517 and 473 were observed in its MS spectrum in negative ion mode, which were speculated as [M-H]- and [M-H-CO2]- ions, respectively. The ions at m/z 269 [M-H-malonylglucosyl]- and 225 [M-H-malonylglucosyl-CO2]- were found in its MS2 spectrum. Protonated molecular ion was not found in its MS spectrum in positive mode, but sodium adduct ion at m/z 541 [M + Na]+ and the one which lost a malonylglucosyl unit at m/z 271 [M + H-malonylglucosyl]+ were predominant. This peak was tentatively identified as emodin-8-O-(6′-O-malonyl)-β-D-glucoside based on its MS data and the literature .
Peak 5 shows the retention time of 55.8 min with maximal UV absorption at 270 nm. Characteristic ions at m/z 487 [M-H]-, 533 [M-H + HCOOH]- and 283 [M-H-acetylglucosyl]- were produced from this peak in MS spectrum in negative ion mode. The deprotonated ion at m/z 487 [M-H]- gave a predominant ion at m/z 240 in MS2 spectrum resulting from the losses of a acetylglucosyl unit, a neutral molecular of CO and a methyl group. In positive ion mode, we did not find the protonated molecular ion, however it yielded a predominant sodium adduct ion at m/z 511 [M + Na]+ and the ion at m/z 285 by losing a acetylglucosyl unit. By comparison with the reported paper [11, 16], the peak was identified as physcion-8-O-(6′-O-acetyl)-β-D-glucoside.
Peak 6 was eluted at retention time of 70.1 min with maximal UV absorption at 222 and 288 nm, which produced the [M-H]- ion at 269 in the MS spectrum in negative ion mode. It further gave fragment ions at m/z 241 [M-H-CO]-, 225 [M-H-CO2]- and 182 [M-H-CO-CO2-CH3]- in the MS2 spectrum. In positive ion mode, the peak yielded weak protonated molecular ion at m/z 271 [M + H]+ in MS spectrum, and no useful information was obtained in its MS2 spectrum. By comparison with MS behaviors of the standard and the literatures [11–17], the peak was unequivocally identified as emodin.
Peak 7 was eluted at retention time of 73.8 min with maximal UV absorption at 223 and 286 nm. Deprotonated molecular ion at m/z 283 [M-H]- was observed in its MS spectrum in the negative ion mode, which further generated a predominant ion at m/z 255 in MS2 spectrum owing to the loss of a neutral CO molecule. Other fragment ions at m/z 240 and 212 were also observed owing to the successive losses of a methyl unit and a CO molecule from 255. In positive ion mode, protonated molecular ion at m/z 285 [M + H]+ and sodium adduct ion at m/z 307 [M + Na]+ were observed in MS spectrum of the peak. Based on the MS data reported in publications [12–17] and the comparison with the standard, it was unequivocally identified as physcion.
Calibration curves, LODs and LOQs
Regression data, LODs and LOQs for the three analytes tested in HPLC-UV chromatograms
Linear range (mg/L)
y = 29885 x - 210960
y = 38332 x + 23549
y = 22565 x - 1352.2
Precision, repeatability and stability
Results of precision, repeatability and stability of the three analytes, expressed as RSD (%)
Repeatability (n = 5)
Stability (n = 6)
Intra-day RSD (%) (n = 5)
Inter-day RSD (%) (n = 9)
Quantification of THSG, emodin and physcion in PMC samples
The established HPLC-UV quantitative analytical method was successfully applied for simultaneous quantification on the three compounds in nineteen PMC samples (eight were from mainland China, and eleven were from local pharmacies of Hong Kong). The contents (mg/g dry weight) were calculated and summarized (n = 2) in Table 1.
Firstly, the results showed that the contents of each compound in different PMC samples varied markedly. To our surprise, the contents of THSG, emodin and physcion ranged from 0.268 to 41.361 mg/g, 0.010 to 1.295 mg/g and 0.013 to 1.394 mg/g, respectively. In addition, THSG was not detected (below LOD) in L-PMC-07, L-PMC-09 and L-PMC-10, emodin was not detected in L-PMC-07, L-PMC-08, L-PMC-09 and L-PMC-10, and physcion was not found in PMC-03, PMC-05, L-PMC-02, L-PMC-06, L-PMC-07, L-PMC-08, L-PMC-09 and L-PMC-10. The results indicated that significant differences of the concentrations of each compound in different PMC samples were found.
Secondly, according to the regulation of China pharmacopoeia (2010 edition) that the content of THSG in dried PMC sample should not be less than 0.20% (2.0 mg/g), only five samples in our study, including PMC-01, PMC-02, PMC-07, PMC-08 and L-PMC-11, were definitely qualified raw medicinal materials for clinic use. It was worth mentioning that one of the eleven local PMC samples from Hong Kong, L-PMC-11, had the highest content of THSG in all the tested samples, which was also the only one qualified sample from local pharmacy of Hong Kong. Emodin and physcion are not the specified chemical markers in China pharmacopoeia, but they usually exist in the plants from family Polygonaceae, which were also quantified in PMC or its related commercial product [18, 19]. The data obtained in the present study showed that except the samples in which emodin were not detected, the content of emodin was the highest in PMC-02, however, the lowest, in L-PMC-02. In the same way, the content of physcion in PMC-02 was the highest and the one in L-PMC-05 was the lowest. The results also indicated that emodin and physcion were not the dominant chemical compounds in PMC compared with THSG.
Thirdly, the PMC samples tested in the present study were mainly from southlands of China. L-PMC-11 was found to have the highest content of THSG at 41.361 mg/g and relative higher contents of emodin (0.864 mg/g) and physcion (1.054 mg/g) in all the tested samples. But its origin was unknown. The sample from Yunnan, numbered PMC-02, had the highest contents of emodin (1.295 mg/g) and physcion (1.394 mg/g) as well as the second highest content of THSG (23.766 mg/g) in all the samples. However, the samples, in which the three analytes were not detected, were from different origins. Seeing from the results, we find that the qualities of PMC samples collected for the present study do not have necessary relations with their origins.
Fingerprinting and principal component analyses
Although the quantification results can confirm the contents of THSG, emodin and physcion in a PMC sample, there is no way to know intuitively how similar a PMC sample to another one on the whole just by quantification a few compounds. Fingerprinting and chemometrics analyses, however, can show the chemical similarities between one and another one holistically and visually. Principal component analysis, one of the chemometrics, is an unsupervised mathematical procedure that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables called principal components. Its operation can be thought of as revealing the internal structure of the data in a way which best explains the variance.
According to PC2 loadings plot, peak 3 and peak 5 mainly contribute to high PC2 scores, however, peak 1, peak 6 and peak 7 contribute to low PC2 scores (P < 0.05). PMC-07 has the highest PA/W value of peak 3 and the second highest PA/W value of peak 5. PMC-01 has the highest PA/W value of peak 5 and the second highest PA/W value of peak 3. So, they are positioned on the top in scores plot, having higher PC2 scores. The highest content of peak 1 was found in L-PMC-11, and then in PMC-02. The highest PA/W value of peak 6 and the highest content of peak 7 were both obtained in PMC-02, and then in L-PMC-11. The above two reasons lead to the positions of the two samples at the bottom.
Other samples are clustered tightly around the corner due to their similar and low contents or PA/W values of the seven peaks.
All in all, scores plot shows the distributions of the tested samples intuitively and clearly, meanwhile, loadings plots indicate the influences of the variables on the positions of PMC samples. From the scores obtained in the present study, qualified and unqualified PMC samples can be distinguished easily and efficiently.
For the first time, systematic HPLC/UV/MS chromatographic fingerprinting and quantitative analytical methods combined with principal component analysis were developed to analyze different PMC samples. The contents of THSG were found to have surprising relevance with the samples’ diameters. Diameters of the five qualified PMC samples basically fell in the specified range, which also had higher contents of emodin and physcion than others. However, diameters of the unqualified PMC samples generally exceeded the specified range. Seven characteristic peaks in chromatographic fingerprints marked 1 to 7 were identified, and based on the contents or PA/W values of the seven variables, PCA scores plot was generated. The finding in the present study provides a scientific basis for collecting PMC in the best time, and with the aid of PCA, unqualified PMC samples can be singled out from qualified ones easily and efficiently.
We like to thank Taiwan Department of Health Clinical Trial and Research Center of Excellence, China Medical University Hospital (DOH99-TD-B-111-004) for providing UPLC/MS facility for this study. The authors also like to acknowledge the grant support from Chinese Materia Medica Standard Office, Department of Health, Hong Kong for this study.
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