The objective of this study was to investigate the concentrations of inorganic elements in the rhizome of
In recent years, numerous studies have found that the efficacy of Traditional Chinese Medicine is not only related to the organic components, but also closely related to the types and concentrations of inorganic elements [
The concentrations of inorganic elements in the rhizomes of PPC with different planting years were determined by using inductively coupled plasma mass spectrometry (ICP-MS), followed by statistical analysis of the measured concentration data, including correlation analysis (CA), principal component analysis (PCA), and cluster analysis (HCA). Meanwhile, the effects of different cultivation conditions (propagation mode, altitude, soil properties, and harvesting time) on the concentrations of inorganic elements were further studied, so as to provide certain theoretical basis for the quality control and rational use of PPC.
The samples of PPC of different planting years and cultivation conditions were all collected from Pengzhou of Chengdu City, Sichuan, China. The rhizome of wild PPC was also collected from Pengzhou. The number of samples of 1-year plants, 2- to 8-year cultivated plants, and wild plants were 60, 30, and 30, respectively, while the number of samples used for different cultivation parameters were 20. All samples were
Nitric acid 67% (Kelong Chemical Reagent, China) and hydrogen peroxide 30% (Kelong Chemical Reagent, China) used for digestion were of analytical purity. Internal standard elements, consisting of 1000 mg·L−1 of 6Li, 45Sc, 73Ge, 115In, and 185Re, were obtained from Thermo Fisher Scientific (USA). Calibration solutions of 1000 mg·L−1 of Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, P, Pb, Se, Sr, Ti, V, and Zn were all purchased from China National Analysis Center for Iron and Steel. The certified reference material used for internal quality control was Citrus leaf reference material (CRM, GBW10020) from the Institute of Geophysical and Geochemical Exploration, China, while, all other chemicals were of analytical grade. Internal standard elements of 6Li, 45Sc, 73Ge, 115In, and 185Re were used as mixed internal standards (200
The samples were processed in microwave digestion apparatus (WX-8000, Preekem Co., China) before digestion analysis; see Table
Microwave operating conditions for the digestion of PPC.
Step |
|
Pressure (atm) | Power (W) | Holding time (min) |
---|---|---|---|---|
1 | 120 | 40 | 1600 | 10 |
2 | 150 | 40 | 1600 | 20 |
3 | 180 | 40 | 1600 | 20 |
Optimum ICP-MS operating conditions for the analysis of PPC.
Instrument parameter | Condition |
---|---|
Radio frequency power | 1.4 kW |
Spray chamber temperature | 2.7°C |
Nebulizer pump | 0.1 rps |
Cool flow | 13 L/min |
Auxiliary flow | 0.8 L/min |
Plasma flow | 13 L/min |
Nebulizer | 0.88/min |
Sampling depth | 5 mm |
No. of replicates per sample | 3 |
Isotopes measured |
6Li |
Powdered samples (200 mg) dried to constant weight were precisely weighed and placed in a polytetrafluoroethylene (PTFE) digestion tank soaked with 10% (V/V) nitric acid. After incubation with 7 mL nitric acid and 1 mL hydrogen peroxide for 1 h, the samples were sealed and digested in microwave. The digestion followed the working procedures of microwave digestion. After the temperature decreased to below 80°C, the smoke was dispersed, and the samples were removed and placed in an electric heating sleeve to expel the acid to about 1 mL. Then, the samples were transferred into a 25 mL volumetric flask after microwave digestion with deionized water. Later, the samples were transferred into another 25 mL volumetric flask after washing three times with deionized water and metered to volume. Finally, the samples were filtered with membrane after mixing evenly to serve as test solution. Three blank solutions were also prepared and digested in the same manner.
Statistical analysis was performed on the citrus leaves reference materials (CRM, GBW10020) using
In the same plot, the seedlings, multiple shoots, and rhizome cuts with buds were cultivated to study the effects of propagation methods. To study the effect of altitude, rhizome cuts with buds were cultivated at altitudes of 500 m, 1000 m, and 1500 m, respectively. Rhizome cuts with buds were cultivated in deep and loose layers of raw soil, mellow soil, loam soil, and humus soil, in order to study the impact of soil properties. Rhizome of the selected PPC, which has been cultivated for eight years using seedlings as propagation materials in the same plot, were collected in June, August, October, and December, respectively, to study the impact of harvesting time.
The determination of inorganic element concentrations in the samples obtained from the cultivation mentioned above was carried out according to the aforementioned method.
The overall parameters of the proposed method for inorganic element analysis were obtained and are listed in Table
Analytical parameters of the ICP-MS method.
Element | Linear range ( |
Regression equation | Correlation coefficient ( |
Limit of detection ( |
Limit of quantification ( |
Recovery (%) |
---|---|---|---|---|---|---|
Al | 0–1000 |
|
0.9994 | 2.646 | 7.937 | 101.5 |
As | 0–500 |
|
0.9990 | 0.006 | 0.021 | 95.7 |
B | 0–500 |
|
0.9999 | 0.011 | 0.033 | 99.7 |
Ba | 0–1000 |
|
0.9988 | 0.005 | 0.016 | 96.8 |
Be | 0–500 |
|
0.9997 | 0.001 | 0.003 | 99.4 |
Bi | 0–1000 |
|
0.9996 | 0.003 | 0.010 | 101.4 |
Ca | 0–1000 |
|
0.9982 | 3.770 | 11.309 | 96.8 |
Cd | 0–500 |
|
0.9992 | 0.008 | 0.027 | 99.1 |
Co | 0–500 |
|
0.9983 | 0.003 | 0.010 | 95.2 |
Cr | 0–500 |
|
0.9992 | 0.116 | 0.354 | 96.8 |
Cu | 0–300 |
|
0.9999 | 0.203 | 0.613 | 97.9 |
Fe | 0–1000 |
|
0.9993 | 1.923 | 5.769 | 100.4 |
K | 0–1000 |
|
0.9988 | 3.618 | 10.867 | 105.1 |
Li | 0–500 |
|
0.9985 | 0.008 | 0.025 | 98.6 |
Mg | 0–1000 |
|
0.9994 | 0.717 | 2.152 | 101.2 |
Mn | 0–1000 |
|
0.9996 | 0.008 | 0.025 | 99.5 |
Na | 0–1000 |
|
0.9987 | 0.049 | 0.148 | 96.7 |
Ni | 0–500 |
|
0.9989 | 0.102 | 0.305 | 97.9 |
P | 0–1000 |
|
0.9998 | 11.957 | 35.870 | 99.4 |
Pb | 0–500 |
|
0.9983 | 0.063 | 0.194 | 105.3 |
Se | 0–500 |
|
0.9993 | 0.450 | 1.358 | 100.2 |
Sr | 0–500 |
|
0.9983 | 0.027 | 0.085 | 103.4 |
Ti | 0–500 |
|
0.9987 | 0.056 | 0.168 | 98.1 |
V | 0–500 |
|
0.9985 | 0.007 | 0.022 | 104.3 |
Zn | 0–500 |
|
0.9989 | 0.031 | 0.096 | 101.7 |
The linear ranges were beyond at least 5 calibration points, which were separated into three intervals, including 0–300
The accuracy and precision of this method were validated using certified reference materials of citrus leaves (CRM, GBW10020), and the certified value versus the obtained values by using ICP-MS is given in Table
Accuracy assessment by analysis of the citrus leaves certified reference material (CRM, GBW10020;
Element | Certified value ( |
Obtained value ( |
---|---|---|
Al | 1150 (8.7%)a | 1090 (7.3%)a |
As | 1.1 (18.2%)a | 1.0 (20%)a |
B | 32 (9.4%)a | 31 (16.1%)a |
Ba | 98 (6.1%)a | 96 (9.4%)a |
Be | 0.031 (22.6%)a | 0.033 (18.2%)a |
Bi | 0.23 (10.9%)a | 0.228 (11.1%)a |
Ca | 42000 (9.5%)a | 41800 (9.7%)a |
Cd | 0.17 (11.8%)a | 0.16 (18.8%)a |
Co | 0.23 (26.1%)a | 0.24 (33.3%)a |
Cr | 1.25 (8.8%)a | 1.27 (8.7%)a |
Cu | 6.6 (7.6%)a | 6.4 (10.9%)a |
Fe | 480 (6.3%)a | 492 (11.4%)a |
K | 7700 (5.2%)a | 7920 (8.2%)a |
Li | 1.0 (10%)a | 0.9 (11.1%)a |
Mg | 2340 (3%)a | 2280 (7.1%)a |
Mn | 30.5 (4.9%)a | 30.1 (14%)a |
Na | 130 (15.4%)a | 124 (25%)a |
Ni |
|
1.0 (10%)a |
P | 1250 (7.2%)a | 1220 (9%)a |
Pb | 9.7 (9.3%)a | 9.5 (8.4%)a |
Se | 0.17 (17.6%)a | 0.18 (27.8%)a |
Sr | 170 (5.9%)a | 175 (13.7%)a |
Ti | 38 (26.3%)a | 40 (22.5%)a |
V | 1.16 (11.2%)a | 1.12 (25%)a |
Zn | 18 (11.1%)a | 19 (15.7%)a |
% differences between the theoretical and experimental values are given in brackets. aValues within each line followed by the same character is not statistically different (
The rhizome of PPC was not only abundant in Ca, K, Mg, Mn, Na, and P, but also rich in B, Ba, Cr, Cu, Fe, Mn, Ni, Sr, and Zn, regardless of the planting years. In addition, it contained not only essential trace elements such as Se and V, but also toxic elements such as As, Cd, and Pb, even though the concentrations of these toxic elements were very low (Table
Element concentrations in the rhizomes of different planting years of PPC (
Element | 1st year ( |
2nd year ( |
3rd year ( |
4th year ( |
5th year ( |
6th year ( |
7th year ( |
8th year ( |
---|---|---|---|---|---|---|---|---|
Al | 3561 ± 22 | 1131 ± 31 | 419 ± 12 | 197 ± 8 | 213 ± 11 | 123 ± 10 | 303 ± 9 | 621 ± 20 |
As | 1.32 ± 0.13 | 0.81 ± 0.08 | 0.23 ± 0.02 | 0.31 ± 0.01 | 0.11 ± 0.02 | 0.09 ± 0.01 | 0.08 ± 0.01 | 0.25 ± 0.02 |
B | 5.75 ± 0.41 | 5.37 ± 0.25 | 4.94 ± 0.34 | 5.38 ± 0.19 | 3.23 ± 0.11 | 3.85 ± 0.15 | 3.48 ± 0.24 | 5.36 ± 0.27 |
Ba | 68.8 ± 0.7 | 41.8 ± 0.5 | 26.1 ± 0.2 | 22.7 ± 0.3 | 15.1 ± 0.2 | 13.6 ± 0.2 | 18.5 ± 0.1 | 39.9 ± 0.2 |
Be | 0.107 ± 0.002 | 0.052 ± 0.002 | 0.012 ± 0.001 | 0.008 ± 0.001 | 0.007 ± 0.001 | 0.007 ± 0.001 | 0.008 ± 0.001 | 0.024 ± 0.003 |
Bi | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ |
Ca | 8339 ± 60 | 7924 ± 22 | 6717 ± 35 | 7291 ± 50 | 3419 ± 40 | 4026 ± 40 | 3094 ± 35 | 4782 ± 50 |
Cd | 0.50 ± 0.02 | 0.14 ± 0.01 | 0.08 ± 0.01 | 0.12 ± 0.01 | 0.04 ± 0.00 | 0.03 ± 0.00 | 0.08 ± 0.01 | 0.15 ± 0.02 |
Co | 2.49 ± 0.07 | 1.39 ± 0.05 | 0.34 ± 0.03 | 0.22 ± 0.02 | 0.15 ± 0.01 | 0.14 ± 0.01 | 0.17 ± 0.01 | 0.51 ± 0.03 |
Cr | 12.72 ± 0.44 | 10.34 ± 0.23 | 2.92 ± 0.21 | 1.42 ± 0.14 | 1.45 ± 0.12 | 1.33 ± 0.09 | 1.42 ± 0.08 | 4.84 ± 0.16 |
Cu | 15.2 ± 0.21 | 7.74 ± 0.34 | 4.41 ± 0.28 | 4.51 ± 0.29 | 1.62 ± 0.03 | 1.33 ± 0.11 | 1.93 ± 0.16 | 3.03 ± 0.14 |
Fe | 3149 ± 42 | 277 ± 30 | 122 ± 8 | 100 ± 5 | 40 ± 3 | 112 ± 5 | 214 ± 7 | 666 ± 15 |
K | 12234 ± 80 | 7621 ± 68 | 6476 ± 45 | 11291 ± 52 | 6111 ± 50 | 5375 ± 47 | 5890 ± 37 | 8929 ± 45 |
Li | 4.22 ± 0.20 | 1.81 ± 0.13 | 0.43 ± 0.03 | 0.25 ± 0.01 | 0.28 ± 0.02 | 0.23 ± 0.02 | 0.22 ± 0.03 | 0.85 ± 0.07 |
Mg | 4489 ± 50 | 1396 ± 32 | 1451 ± 44 | 1790 ± 38 | 1120 ± 42 | 1042 ± 51 | 944 ± 36 | 1789 ± 44 |
Mn | 193 ± 4 | 85.3 ± 2.3 | 40.5 ± 1.7 | 45.4 ± 1.5 | 28.9 ± 1.1 | 25.3 ± 1.4 | 31.5 ± 0.9 | 78.8 ± 1.4 |
Na | 1683 ± 54 | 374 ± 36 | 1658 ± 26 | 481 ± 50 | 886 ± 13 | 1094 ± 31 | 263 ± 11 | 656 ± 10 |
Ni | 41.9 ± 0.6 | 35.7 ± 0.7 | 22.6 ± 0.4 | 34.0 ± 0.3 | 1.15 ± 0.13 | 1.65 ± 0.16 | 1.30 ± 0.14 | 4.36 ± 0.24 |
P | 2387 ± 33 | 2937 ± 27 | 2983 ± 50 | 4422 ± 41 | 1631 ± 29 | 1157 ± 18 | 1341 ± 34 | 1727 ± 24 |
Pb | 8.02 ± 0.25 | 3.17 ± 0.04 | 0.79 ± 0.02 | 0.58 ± 0.02 | 0.36 ± 0.01 | 0.35 ± 0.01 | 0.42 ± 0.01 | 1.12 ± 0.03 |
Se | 0.33 ± 0.02 | 0.19 ± 0.01 | 0.10 ± 0.01 | 0.12 ± 0.01 | 0.07 ± 0.01 | 0.08 ± 0.01 | 0.07 ± 0.01 | 0.13 ± 0.01 |
Sr | 28.5 ± 0.4 | 20.9 ± 0.5 | 21.4 ± 0.5 | 19.7 ± 0.3 | 9.2 ± 0.4 | 8.21 ± 0.08 | 6.96 ± 0.19 | 16.1 ± 0.8 |
Ti | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ | <LOQ |
V | 7.90 ± 0.07 | 3.18 ± 0.02 | 0.90 ± 0.01 | 0.47 ± 0.02 | 0.50 ± 0.02 | 0.40 ± 0.01 | 0.30 ± 0.02 | 1.39 ± 0.03 |
Zn | 190 ± 4 | 102 ± 3 | 64.4 ± 2.9 | 33.2 ± 3.1 | 75.7 ± 4.6 | 62.8 ± 3.1 | 59.4 ± 1.1 | 166 ± 5 |
Bi and Ti concentrations were below the limit of quantification (LOQ).
The planting years affected the content of inorganic elements in the rhizome of PPC. The variation curves of inorganic elements in samples with different planting years are shown in Figure
Variation curves of inorganic elements of different planting years of PPC.
Among samples of all planting years, the elements Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, and Ni in Year 1 samples were the highest. The contents of majority inorganic elements were lower in Years 2–6 samples than in Year 1 samples. However, the contents of inorganic elements were slightly higher in Year 8 samples than in Year 7 samples. The reasons for above changes may lie in the accumulation of starch and other substances in the rhizome of PPC. In Year 1, the contents of starch and other substances were the lowest, resulting in the highest contents of inorganic elements. With planting years increasing, the accumulation of starch and other substances increased significantly, resulting in a gradual decline in the percentage of inorganic elements. Year 8 is another accelerating growth period for the rhizome of PPC. The folds of rhizome dry weight gain decreased [
Correlation analysis (CA) was used to analyze the correlation of inorganic elements in PPC of different planting years. See Table
Principal component analysis (PCA) was conducted to determine the inorganic elements in PPC of different planting years. See Table
The first two principal component loadings and scores of inorganic elements in PPC of different planting years are presented in Figure
Varimax rotated principal component (a) loadings and (b) scores of different planting years of PPC.
Hierarchical cluster analysis (HCA) was used to further analyze PPC of different planting years (Figure
Dendrogram of hierarchical cluster analysis for PPC samples collected in different planting years.
With the spreading of cultivated PPC in Southwestern China, researches on the cultivation should be deepened. Therefore, this study also determined the concentrations of inorganic elements in the rhizomes of PPC under different conditions (propagation mode, altitude, soil properties, and harvesting time). See Table
Influence of (a) propagation mode, (b) altitude, (c) soil property, and (d) harvesting time on element concentrations in the rhizomes of PPC.
As shown in Figure
For the impact of altitude (Figure
As shown in Figure
In the present study, on the impact of harvesting time (Figure
In this study, ICP-MS was used to determine the concentrations of inorganic elements in the rhizomes of PPC with different planting years or under different cultivating conditions. The results show that the rhizome of PPC contains numerous elements, including Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, P, Pb, Se, Sr, V, and Zn. Planting years have significant effects on the concentrations of inorganic elements. The elemental patterns of element concentrations in samples collected in different planting years are different. The elemental patterns are similar in samples collected in Years 5–7, but significantly different from samples collected in Year 8. In addition, propagation mode, altitude, soil properties, and harvesting time also have certain effects on the concentrations of these elements in the rhizome of PPC. When cultivated PPC is compared with wild ones, the concentrations of inorganic elements are lower in most cultivated samples than those in wild samples, which can explain to some extent why a better medicinal effect is observed with wild PPC. In future studies, it is expected to unravel the relationship between inorganic elements and pharmacological actions of PPC.
The data used to support the findings of this study are included within the article and the supplementary information file.
The author declares that there are no conflicts of interest.
The authors are grateful for National Key R&D Program of the Ministry of Science and Technology of China (2017YFC1700705); the Sixth Training Session for Academic Experience Inheritance of National Senior Chinese Medicine Experts, and National Talent Training Program for Inheritance of Traditional Chinese Medicine, sponsored by the State Administration of Traditional Chinese Medicine; Sichuan Science and Technology Programs (2019YFS0190, 2019YFN0096, 2018NZZJ011, 2017RZ0051, and 2016JY0081); Sichuan Science and Technology Research Projects of Traditional Chinese Medicine (2018PC011 and 2017PC002); Sichuan Science and Technology Innovation Seedling Project (2018114); and Sichuan Basic Scientific Research Project of Public Research Institutes (A-2018N-25).
(1) Table S1 shows the correlation matrix for the inorganic elements concentrations in the rhizomes of different planting years of PPC. (2) Table S2 shows the varimax rotated factor loadings of the first two principal components obtained by principal component analysis of inorganic elements in the rhizomes of different planting years of