Multi-elements characteristic and fingerprint
The concentrations of 54 elements in the studied MC samples were expressed as mean (c) ± standard deviation (SD) of the sampling sites, the means and RSDs% of all the studied elements were calculated for further analysis whether there were differences in the samples of MC from different regions. Data of K, Na, P, Ca, Mg, Al, S, Si, B, Fe, Mn, Zn, Ba, Sr and Cu were obtained by ICP-AES, and the rest elements were determined by ICP-MS. Internal standard solution (Rh) was used during the ICP-MS determination. According to the average content of 54 elements from different producing areas, we established the multi-elements distribution fingerprint of MC herbs using Origin 9.0, as shown in Fig. 2. These elements are divided into 4 groups: (a) K, Ca, Mg, Na, Al, Fe, S and P are at the above 100 mg.kg−1; (b) Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu are rare earth elements (REEs); (c) As, Cd, Hg, Pb and Cu are heavy metals and toxic elements that must be monitored according to “Green Trade Standards of Importing & Exporting Medicinal Plants & Preparations” (WM2-2001); (d) Ba, V, Mo, Sr, Li, I, Zn, Mn, B, Ti, Si, Co, Sb, Sn, Rb, Ni, Cs, U, Be, Bi, Tl, Th, Ge, Br and Cr are the remaining elements to be studied.
The content of K, Ca, Mg, Na, Al, Fe, S and P is above 100 mg.kg−1 in Table 2 and Fig. 2, accounting for 98.79% of the total amount (27,306 mg.kg−1, S5). The RSDs% values were all higher than 48, indicating significant differences in the levels of each element from different regions. The herbs of Chizhou got the highest K (11,812 mg.kg−1) and Ca (25,369 mg.kg−1) while the lowest content of K (3364 mg.kg−1) and Ca (8370 mg.kg−1) was in Tongling and in Xuancheng, respectively. The Na content in Bozhou sample (890 mg.kg−1) is extremely high, which is 9.08 times of that in Tongling sample (98 mg.kg−1). The herbs of Bozhou got the highest Mg (2832 mg.kg−1), but was only 824 mg.kg−1 in herbs of Wuhu. The Fe content is the highest in the herb produced in Bozhou (744 mg.kg−1), which is 4.4 times the lowest in Wuhu (170 mg.kg−1). Al is an element that accumulates in the body and causes chronic toxicity. The highest Al content in Chizhou was 1254 mg.kg−1, while the Al content in Wuhu was only 286 mg.kg−1. P and S are non-metallic elements, and their contents are in the range of 805 mg.kg−1 (Wuhu) ~ 2214 mg.kg−1 (Chizhou) and 412 mg.kg−1 (Wuhu) ~ 1325 mg.kg−1 (Bozhou), respectively. The contents of K, Ca, Mg, Na, Al, Fe and P in MC from different producing areas were close to those of other reports (Liu et al. 2017; Zhu et al. 2020). In short, the content of K, Mg, Al, Fe, Na, S and P of the MC herbs from Tongling and Wuhu is much lower than those from other regions in Group (a). Except Bozhou, the contents of metal elements in other origins are arranged in the following order: Ca > K > Mg > Al > Fe > Na, and Ca is the most abundant element of all MC herbs.
The concentration of elements in Group (b), (c) and (d) are significantly lower than the group (a). Therefore, RSDs% of the studied elements below 50 are considered to have little difference in origin. In group (b), 16 REEs (Sc, Y and Lanthanide, Pm was excluded) are determined in Table 3 and Fig. 2, and all REEs are found in all MC herbs. The content of REEs (i.e., Eu, Tb, Y, Pr, La, Nd, Th, Sm, Dy, Ho and Gd) displayed similar levels as the RSDs% is 25–52. The highest and lowest contents of REEs were Wuhu and Bozhou, which got 5.094 mg.kg−1 and 2.000 mg.kg−1, respectively. The abundance of even atomic number elements was higher than that of their odd atomic number neighbors (Fig. 2), which conforms to the law of Oddo–Harkins (Markert 1987). The ratio of light to heavy rare earth elements (LREEs/HREEs = ∑([La]-[Eu])/∑([Gd]-[Lu] + Y + Sc) is between 3.807 and 5.504, indicating the enrichment of LREEs in all MC samples. Except for Bozhou, the contents of REEs in the other four production areas of MC are as follows: Ce > La > Nd > Y > Pr > Er > Yb > Eu > Ho > Tb > Tm > Lu. These results can not only see the differences between MC growing in the plain and hilly areas, but also can be used for the identification of MC.
For heavy metals and toxic elements (Table 4 and Fig. 3), the content of Pb, Cu and Cd had remarkable different as the RSDs% are 65, 56 and 67, while As and Hg are slightly less affected by origin. The highest content of As and Hg is 0.223 mg.kg−1 and 0.004 mg.kg−1, respectively. The content of Pb was the highest in Wuhu (2.897 mg.kg−1) and the lowest in Bozhou (0.457 mg.kg−1). The content of Cu varied in the range of 3.395–11.029 mg.kg−1, and the herbs from Bozhou is the highest. Tongling, commonly known as Tongdu, has many copper mines. It is believed that MC has grown here with a high content of Cu, but in fact it is only 4.432 mg.kg−1. The herbs of Wuhu got the highest Cd (0.778 mg.kg−1) whereas other regions content was in the range of 0.070–0.448 mg.kg−1. According to the regulations in the “Green Trade Standards of Importing & Exporting Medicinal Plants & Preparations” (WM2-2001), the maximum limits of Cu, Cd, Pb, Hg and As in CHMs are 20, 0.3, 5, 0.2 and 2 mg.kg−1, respectively. Among all the herbs, Cu, Pb, Hg and As are below the limiting value. Some of herbs from Wuhu, Xuancheng and Chizhou were found to have excessive Cd levels, while the sample from Bozhou contains low level of Cd. This result should be related to the soil environment of producing area. The soil types in Wuhu, Xuancheng and Chizhou are mainly red (brown) soil, limestone soil and paddy soil in some areas. The soil is mostly acidic (Tao et al. 2011). The soil in Bozhou is developed from the parent material of yellow flood alluvium and mainly consists of yellow tide soil, which is alkaline (Ye et al. 2007). Studies have shown that the proportion of ion-exchangeable Cd in acidic soil is high, and that Cd bound with carbonate is released, increasing the fluidity and bioavailability of Cd. The pH of Bozhou soil is higher than 8, which reduces the solubility of Cd in soil, increases the negative charge of soil particles, reduces the exchangeable Cd in soil and reduces the mobility of Cd (Song et al. 2015; Li et al. 2013a, b). The exceedance ratios of Cd in the 66 samples were 59.09%, which pointed out that the Cd pollution is serious in MC herbs. Li et al. (2018) also reported that Cd content in MC from Daodi-producing areas exceeded the standard, which is consistent with the results of this study.
In the group (Table 5), due to the origin, the concentration of Si, U, Li, Be, Cr and Sr had remarkable difference, and the RSDs% varies from 54 to 77. There are a wide variety of Si in herb, ranging from 162 (Tongling) to 23.29 mg.kg−1 (Bozhou). Sr in Xuancheng was the lowest at 21.034 mg.kg−1 while it was the highest at 135.277 mg.kg−1 in Bozhou. Cr content of the herbs was in the range of 2.302–8.280 mg.kg−1 with the highest value from Xuancheng. The content of Si and Sr in MC in this study was higher than that in previous reports (Guo et al. 2008; Liu et al. 2017). Nevertheless, the concentrations of other elements in this group of samples from different origins display similar levels as the RSDs% are 8 and 43. Average content of Ti, Rb, B, Mo, Ni and Ba is 47.334 mg.kg−1, 5.680 mg.kg−1, 23.984 mg.kg−1, 0.260 mg.kg−1, 1.360 mg.kg−1 and 35.208 mg.kg−1, respectively. Br and I are typical non-metallic elements, Br level of all herbs is higher than I. They were determined the mean of 1.674 mg.kg−1 and 0.154 mg.kg−1, respectively. Zn in the herbs was ranged from 12.996 mg.kg−1 (Xuancheng) to 25.041 mg.kg−1 (Bozhou), which was consistent with Zn content in 32 batches of MC reported by Zhu et al. (2020).
Chemical forms of Cd
Due to the phenomenon of Cd exceeding the standard detected in MC herbs, Cd chemical forms were further analyzed. The percentage of chemical forms of Cd in MC is shown in Table 6 and Fig. 4. The MC from Bozhou has the highest residue state Cd (PR) in 6 chemical forms, accounting for 30.6%, while the other 4 producing areas have the highest integration with pectate and protein Cd (PNaCl). Owning to higher migration ability and higher biological activity, PEtOH (inorganic Cd) and PH2O (water-soluble Cd) are more harmful to plant cells than other Cd forms (Li et al 2014; Wang et al. 2008; Qiu et al.2011). The lowest sum of inorganic Cd and water-soluble Cd in MC from Wuhu and Tongling is 18.4% and 19.7%, followed by that from Xuancheng and Chizhou, which is 29.8% and 27.7%, the highest is from Bozhou as 31.2%. The average percentage of PHAc (insoluble CdHPO4, Cd3(PO4)2), PHCl (Cd oxalate) and PR (residues) in all forms were 14.6% (8.0–21.3%), 5.4% (2.8–8.2%) and 19.4% (13.1–30.6%), respectively. The percentage of PNaCl (Cd integrated with pectate and protein) in herbs from Wuhu, Tongling, Xuancheng and Chizhou was the highest, accounting for 41.2% (25.2–56.4%) of all chemical forms. However, PNaCl percentage in MC from Bozhou is much lower than other regions, which only contained 9.1%. In terms of chemical form distribution, the herbs from Bozhou are quite different from those from other origins, and the proportion of PR, PH2O and PHAc is the highest top 3 among all samples. This is probably due to the geological environment where herbs are grown. Wuhu, Tongling, Xuancheng and Chizhou are hilly but Bozhou is plain.
Based on the content of 54 elements in MC samples from 22 sampling sites in 5 producing areas, the Fisher discriminant function general discriminant method was used to perform multivariate discriminant analysis through Statistical software, SPSS (Version23). Five elements (K, Na, Sc, Cr and Cd) with obvious regional distribution differences were taken as indicators as independent variables for judgment analysis to establish a discriminant model of MC origin, which is listed in S6. The discriminant function 1 and 2 cumulative explain ability of discriminant model is 94.8%, and the correlation coefficient was greater than 0.95, which shows that discriminant function 1 and 2 to five MC origins classification of main contribution, using the discriminant function score value of the discriminant function 1 and 2 as a scatter plot. It can be seen from Fig. 5. The group centroid of MC from Tongling and Wuhu is close to each other and overlap, which is also related to the special climate and soil conditions of Phoenix Mountain. The group centroid of MC from Xuancheng and Chizhou is closer to that of Wuhu and Tongling, and this may be due to their hilly terrain and location along the Yangtze River. It should be noted that the distance between Bozhou and other producing areas is far, so the distribution of inorganic elements is easy to distinguish the producing areas.
Health risk evaluation
The average Cd content of MC from Wuhu, Xuancheng and Chizhou was 0.778 mg.kg−1, 0.448 mg.kg−1 and 0.358 mg.kg−1, respectively. According to the maximum dose of 12 g for Chinese medicinal materials prescribed by the Chinese Pharmacopoeia 2020, the average weekly intake of Cd in MC (PTWI = Ci × 12 × 7)/62.5) from these three regions was 1.046 μg.kg−1, 0.602 μg.kg−1 and 0.481 μg.kg−1, respectively (Table 7). But in fact, oral Chinese medicine is mostly taken in the form of water decoction. Wang et al. (2019) reported that the dissolution rate of Cd in traditional Chinese medicine was 20.5%, so the actual weekly average intake of Cd in MC (PTWIfact = PTWI × 20.5%) from Wuhu, Xuancheng and Chizhou was 0.215 μg.kg−1, 0.166 μg.kg−1 and 0.133 μg.kg−1, respectively. That's less than 0.6 μg.kg−1 for Cd prescribed by WHO. Moreover, the highest migration and toxicity are PEtOH (inorganic Cd) and PH2O (water-soluble Cd) in 6 chemical forms of Cd, and the sum of the two percentage of Wuhu, Xuancheng and Chizhou was 18.4%, 29.8% and 27.7%, respectively. Corresponding to PIWI was 0.192 μg.kg−1, 0.180 μg.kg−1 and 0.133 μg.kg−1, which were also below limits (0.6 μg.kg−1) as well. Therefore, in Wuhu, Xuancheng, Chizhou the residual amount of Cd in MC ingested by water decoction had little harm to human health.
Low doses of REEs are beneficial to human body, such as inhibiting tumors and protecting brain nerves (Xia et al. 2012; Jin et al. 2014), while high doses are opposite. The REEs that people consume accumulate in their internal organs, bones, brain, hair and blood and have significant effects on human digestive, respiratory, reproductive, nervous, blood and immune systems (Chen et al. 2005; Chen et al. 2008). The total amount of REEs in MC is in the range of 2.000–5.094 mg.kg−1. Based on the maximum daily consumption of 12 g.person−1 (Chinese Pharmacopeia Commission 2020), the lifetime average daily pollutant intake (ADI = Ci × 12 × 90 × 70 /62.5 × 365 × 70) is only 0.0947–0.241 ug.kg−1.d−1 (Table 7), and the REEs intake is 70 ug.kg−1.d−1, as the safe dose (Li et al.2013a, b; Zhu et al.1997). Judging from the amount of REEs in MC herbs, it is not harmful to human health.