Safety Limit Test of Heavy Metals and Pesticide Residues in the Zhuang Ethnic Medcine Herba Polygoni Chinensis from 13 Producing Areas

Fengfeng XIE Anda WEI Liba XU Hua ZHU Guodong HUANG Shiyan HE Linmiao LI

Abstract ［Objectives］ This study was conducted to determine the contents of such five heavy metals as lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg) and copper (Cu) and the contents of nine kinds of organochlorine pesticide residues including total BHC (α-BHC, β-BHC, γ-BHC, δ-BHC) and total DDT (pp -DDE, pp-DDD, op-DDT, pp-DDT) and pentachloronitrobenzene in the Zhuang medicine Herba Polygoni Chinensis from 13 producing areas.

［Methods］The five heavy metals were determined in accordance with the second method of general rule 2341, the inductively coupled plasma mass spectrometry method, in part 4 of the 2020 edition of Chinese Pharmacopoeia; and the nine organochlorines were determined in accordance with the first method of general rule 2341, organochlorine pesticide residue determination method (chromatography), in part 4 of the 2020 edition of Chinese Pharmacopoeia.

［Results］ In the Herba Polygoni Chinensis from 13 origins, the lead contents were in the range of 0.50-3.28 mg/kg; the cadmium contents were in the range of 0.06-4.21 mg/kg; the arsenic contents were in the range of 0.16-6.9 mg/kg; the copper contents were in the range of 10.5-45.1 mg/kg; the mercury contents were in the range of 0.00-0.003 mg/kg; and the nine organochlorines including the total benzene hexachloride (α-BHC, β-BHC, γ-BHC, δ-BHC), total DDT (pp-DDE, pp-DDD, op-DDT, pp-DDT) and pentachloronitrobenzene were all not detected. Referring to part 4 of the 2020 edition of Chinese Pharmacopoeia, the Cu and As of the Herba Polygoni Chinensis produced in Hezhou exceeded the standards, the Cu of the Herba Polygoni Chinensis produced in Wuzhou exceeded the standard, and the As of the Herba Polygoni Chinensis produced in Yulin City and Guilin City exceeded the standard.

［Conclusions］This study provides safety limit test data for the development and utilization of Herba Polygoni Chinensis of the Zhuang medicine.

Key words Different producing areas; Zhuang medicine Herba Polygoni Chinensis; Heavy metals; Pesticide residues

Received: January 28, 2021  Accepted: March 30, 2021

Supported by Development of Huotanmu Capsules, a New Zhuang Ethnic Medicine for the Treatment of Hepatitis B (20183046-1); Collaborative Innovation Center  of  Zhuang and Yao Ethnic Medicine (GJKY ［2013］ 20); Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicines (GKJZ ［2014］ 32); Guangxi Key Discipline: Zhuang Pharmacology (GJKY ［2013］ 16); First-class Discipline in Guangxi of Traditional Chinese Pharmacology (Direction of Ethnic Medicine) (GJKY ［2018］ 12).

Fengfeng XIE (1989-), female, P. R. China, research asistant, PhD, devoted to research about pharmacy and industry of fine chemicals.

*Corresponding author.

In order to improve the safety and effective control of drugs, the 2020 edition of Chinese Pharmacopoeia further strengthens the control of safety evaluation indexes such as heavy metals and harmful elements and banned pesticide residues in medicinal pieces. The "0212 General Rules for the Verification of Medicinal Materials and Decoction Pieces" (Part 4) was revised, stipulating that banned agrochemicals (33 prohibited agrochemicals) shall not be detected in botanical medicinal materials and decoction pieces, and a fifth method "Method for the determination of banned agrochemical residues in medicinal materials and decoction pieces (plants)" was added to the "2341 Methods for Determination of Agrochemical Residues". The "9302 Guidelines for the Establishment of Limits for Hazardous Residues in Traditional Chinese Medicines" was revised, adding a fifth item "Guidelines for the Consistency Limits of Heavy Metals and Hazardous Elements in Traditional Chinese Medicines (Plants)"［1］. And compared with the 2015 edition of Chinese Pharmacopoeia, the limits for heavy metals and harmful elements in the new version of the pharmacopoeia are revised from "Lead shall not exceed 5 mg/kg; cadmium shall not exceed 0.3 mg/kg; arsenic shall not exceed 2 mg/kg; mercury shall not exceed 0.2 mg/kg; copper must not exceed 20 mg/kg" to "Lead must not exceed 5 mg/kg; cadmium must not exceed 1 mg/kg; arsenic must not exceed 2 mg/kg; mercury must not exceed 0.2 mg/kg; copper must not exceed 20 mg/kg", and the number of heavy metals and harmful elements tested has been increased from 17 to 28. The 11 newly added varieties are Angelicae Dahuricae Radix, Angelicae Sinensis Radix, Radix Puerariae, Polygonati Rhizoma, Radix Ginseng, Notoginseng Radix Et Rhizoma, Fructus Gardeniae, Persicae Semen, Semen Ziziphi Spinosae, Corni Fructus and Cordyceps sinensis, all of which are Chinas bulk medicinal materials. It can be seen that China has been continuously strengthening the safety control requirements of Chinese medicinal materials and improving the controllability of drug quality.

The medicinal materials conventionally used by various nationalities recorded in the 2020 edition of Chinese Pharmacopoeia are mainly derived from Tibetan, Mongolian, Uygur, and Dai［2］, with a total of 16 species, namely Fructus Podophylli, Fructus Terminaliae Billericae, Fructus Phyllanthi, Herba Lamiophlomis, Herba Lagotis, Acori Calami Rhizoma, Pterocephali Herba, Herba Cissampelotis, Fructus Choerospondiatis, Malvae Fructus, Aconiti Kusnezoffii Folium, Fructus Hippophae, Saussureae Involucratae Herba, Herba Cichorii, Semen Nigellae, and Entadae Semen among which eight are traditional Tibetan medicinal materials, four are traditional Mongolian medicinal materials, three are Uyghur traditional medicinal materials, one is Dai traditional medicinal material, and one is unspecified ethnic customary medicinal material (Entadae Semen). Furthermore, the Fructus Hippophae is called "the traditional medicinal materials of Tibetan and Mongolian nationality". No medicinal materials commonly used by Zhuang people are included. Therefore, striving for Zhuang medicine to enter the national standard is a huge challenge for the future development of Zhuang medicine.

The Zhuang nationality is the most populous ethnic minority in China (more than 18 million). More than 90% of the Zhuang population live in the Guangxi Zhuang Autonomous Region. The Zhuang medicine formed and developed in the long production and life and the practice of fighting against diseases is an important part of traditional medicine in China, and it is still one of the main health resources for the masses of people to prevent and treat diseases and ensure their health. According to research, Zhuang medicine has a long history［3］, and especially in recent years, China has attached great importance to Zhuang medicine, and has issued related policies and measures that promote the development of Zhuang medicine. All three volumes of Quality Standards for Zhuang Medicine in Guangxi Zhuang Autonomous Region have been officially published and implemented［4-6］, and a total of 489 herbal medicines have been collected. Herba Polygoni Chinensis refers to the dried whole plants of Polygonum chinense L. and Polygonum chinense L. var. hispidum Hook.f. in Polygonum of Polygonaceae and is recorded in Quality Standards for Zhuang Medicine in Guangxi Zhuang Autonomous Region (Volume 1) to have the effects of clearing away heat and damp toxin and cooling blood to relieve pain. It is used for treating Ayimi (dysentery), Baidong (diarrhea), Nengbang (jaundice), Huoyanma (sore throat), Xiehan (colpitis mycotica), Beixi (acute mastitis), Nenghannenglei (eczema), Eha (venomous snake bite). In modern research, Herba Polygoni Chinensis has liver injury-protecting［7-8］, feverand jaundice-relieving［9］, anti-inflammatory, analgesic［10-11］, and antibacterial effects［12-13］. According to the new regulations on heavy metals, harmful elements, and banned pesticide residues in the 2020 edition of Chinese Pharmacopoeia, Guangxi pays more and more attention to the safety evaluation of Zhuang medicinal materials. In the technical requirements for the drafting of the Guangxi Zhuang Medicine Quality Standard (Volume 3), relevant regulations are made for "heavy metals and harmful elements" and "pesticide residues", which need to be inspected in accordance with the national pharmacopoeia. According to reports, there are no relevant literatures on the research on the safety evaluation indexes of the Zhuang medicine Herba Polygoni Chinensis including its heavy metals, harmful elements, pesticide residues, etc. In this study, in accordance with the method 2321 in the fourth part of the 2020 edition of Chinese Pharmacopoeia, five heavy metals, i.e., lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg) and copper (Cu) were determined by inductively coupled plasma mass spectrometry; and in accordance with the method 2341 in the fourth part of the 2020 edition of Chinese Pharmacopoeia, gas chromatography was used to determine the residues of 9 kinds of organochlorine pesticides. This study provides a basis for the resource development and utilization and safety evaluation of the Herba Polygoni Chinensis medicinal materials, and provides experimental data for the Zhuang medicine to "enter the national standard, enter the pharmacopoeia, and identify itself", so as to promote the high-quality development of Guangxi Zhuang medicine.

Materials and Methods

Experimental materials

Herba Polygoni Chinensis was identified as the dried whole plants of P. chinense L. and P. chinense L. var. hispidum Hook.f. in Polygonum of Polygonaceae collected from various regions of Guangxi, and the specific producing areas are shown in Table 1.

Experimental equipment and reagents

Inductively coupled plasma mass spectrometry (Agilent Technology Co., Ltd., 7800); gas chromatograph (Agilent Technology Co., Ltd., Agilent 7890B ECD detector); gas chromatography column (Agilent Technology Co., Ltd., DB-5 122-5032 30 m×0.250 m×0.25 μm ); microwave digestion instrument (CEM, Mars6, USA).

Mixed reference solution of organochlorine pesticides (including: α-BHC standard, β-BHC standard, γ-BHC standard, δ-BHC standard, P-P-DDT standard , P-P-DiDiDi standard, P-P-DDD standard, O-P-DDT standard, pentachloronitrobenzene), lot number: 610005-201802, 1.5 ml/bottle, National Institutes for Food and Drug Control.

Mixed control solution of Pb, Cd, As, Hg and Cu, lot number: 610014-201701, 2 ml/bottle (in which Pb was calculated at 198.7 μg/ml, Cd was calculated at 10.0 μg/ml, As was calculated at 103.2 μg/ml, Hg was calculated at 10.0 μg/ml, Cu was calculated at 1 007.5 μg/ml), National Institutes for Food and Drug Control.

The remaining reagents were all high-quality pure or analytically pure, and the water was first-grade water, second-grade pure water and third-grade pure water.

Experimental methods

The five heavy metals, Pb, Cd, As, Hg and Cu, were determined in accordance with the second method of general rule 2341, the inductively coupled plasma mass spectrometry method, in part 4 of the 2020 edition of Chinese Pharmacopoeia; and the nine organochlorines were determined in accordance with the first method of general rule 2341, organochlorine pesticide residue determination method (chromatography), in part 4 of the 2020 edition of Chinese Pharmacopoeia.

Agricultural Biotechnology2021

Results and Analysis

The test results of heavy metals and pesticide residues in Herba Polygoni Chinensis from different origins are shown in Table 1. From the test results in Table 1, it can be seen that in the Herba Polygoni Chinensis from 13 origins, the Pb contents were in the range of 0.50-3.28 mg/kg; the Cd contents were in the range of 0.06-4.21 mg/kg; the As contents were in the range of 0.16-6.9 mg/kg; the Cu contents were in the range of 10.5-45.1 mg/kg; the Hg contents were in the range of 0.00-0.003 mg/kg; and the nine organochlorines including the total benzene hexachloride (α-BHC, β-BHC, γ-BHC, δ-BHC), total DDT (pp-DDE, pp-DDD, op-DDT, pp-DDT) and pentachloronitrobenzene were all not detected. Referring to part 4 of the 2020 edition of Chinese Pharmacopoeia, "In Chinese medicinal materials other than minerals, animals, and marine materials, Pb must not exceed 5 mg/kg; Cd must not exceed 1 mg/kg; As must not exceed 2 mg/kg; Hg must not exceed 0.2 mg/kg; and Cu must not exceed 20 mg/kg", the As of the Herba Polygoni Chinensis produced in Hezhou, Yulin and Guilin exceeded the standard, the Cu of the Herba Polygoni Chinensis produced in Hezhou exceeded the standard, and the Cd of the Herba Polygoni Chinensis produced in Wuzhou exceeded the standard, while the rest of the indexes were in compliance with the pharmacopoeia standards. According to literature reports, due to the differences in the growth environment of the original plants, the contents of heavy metals and harmful elements contained in wild and artificially planted Chinese medicinal materials of the same species are very different［14］. For example, Yang et al.［15］ discussed the pesticide residues and heavy metals in wild and cultivated Sarcandra glabra in different regions of Fujian Province, and found that the pesticide residues in wild and cultivated S. glabra in different regions were all less than 0.01 mg/kg, but the Cd of the wild samples exceeded the standard, while the heavy metal indexes of the cultivated products met the requirements of the national pharmacopoeia, that is to say, the cultivated products were safer than the wild products. Studies on the safety of wild products and cultivated products of Atractylodes lanceolata, Polyporus umbellatus and other medicinal materials have also shown that the wild products in some production areas have excessive Cd［16-17］, indicating that the growth environments of wild products, including soil, water and air, are in their natural states and are not restricted, and it is thus difficult to control the heavy metal contents of wild products. Therefore, the heavy metals of wild products are often higher than cultivated products, while the pesticide residues are higher in cultivated products than wild products, which is closely related to whether the cultivation environment and methods and field management of the cultivated products are standardized.

In this study, 13 batches of Herba Polygoni Chinensis were collected from various regions in Guangxi, and were wild products, the heavy metal contents of which could not be monitored and exceeded the standard. In addition, the original plant of Herba Polygoni Chinensis is a perennial herb with a strong rhizome, and whether the heavy metal contents exceeding the limits were due to its high heavy metalenrichment capacities requires further research and verification.

Conclusions and Discussion

In this study, inductively coupled plasma mass spectrometry (ICP-MS) was used to simultaneously determine five heavy metals in Herba Polygoni Chinensis powder. This method has the widest dynamic linear range, high analytical precision, high accuracy and high speed, and the linear dynamic range of concentration is up to 9 orders of magnitude, realizing direct measurement from 10-12 to 10-6, and it has been widely used in various fields such as biomedicine, food, chemical industry, environment, agriculture, life science, material science and so on. Especially for the determination of trace and ultra-trace elements, the ICP-MS method has the advantages that other traditional analysis cannot meet. In the field of traditional Chinese medicine and ethnic medicine, the ICP-MS method has more advantages than atomic absorption spectrophotometry (AAS). Wei et al.［18］ used ICP-MS and AAS methods to determine such five Chinese medicinal materials as Schisandrae Chinensis Fructus, Radix Ginseng, Panacis Quinquefolii Radix, Platycodonis Radix and Gentianae Radix Et Rhizoma grown in five producing areas in Antu, Wangqing, Fusong, Jingyu and Jian, Jilin Province and their cultivated soils. Specifically, As and Hg were measured by ICP-MS method, and Pb, Cd and Cr were measured by the AAS method. The ICP-MS method could be operated under the same conditions, with fast speed and easy operation, while the AAS method needed to set the detection parameters of each element, and had more complicated steps. Yue［19］ established ICP-MS and AAS methods for determining the contents of inorganic nutrient elements and heavy metal elements in Chuanmingshinis Radix samples. The ICP-MS method could determine the low contents of inorganic elements in Chuanmingshinis Radix, while AAS was suitable for the determination of high contents of inorganic elements. The characteristics and advantages of ICP-MS make it very suitable for the needs of trace and ultra-trace element analysis and the rapid analysis of certain isotope ratios, and it thus has been rapidly developed.

The heavy metal limits and pesticide residue limits for Chinas traditional Chinese medicine and traditional medicine of ethnic minorities have been continuously improved. With the publication of the new edition of the pharmacopoeia, the control requirements for the safety indexes of traditional Chinese medicine and traditional medicine of ethnic minorities continue to increase. In recent years, national policies and policies of the Guangxi Zhuang Autonomous Region have played a huge role in promoting Guangxi ethnic medicine, especially Zhuang medicine. To give full play to the unique advantages of Zhuang Medicine and support the Guangxi Zhuang Medicine brand, safety control is the first condition. In this study, through the determination of heavy metal limits and pesticide residues of the Zhuang medicine Herba Polygoni Chinensis from 13 production areas in Guangxi, it is proposed to put forward some prospects for the safety index control of Guangxi Zhuang medicine: ① In order to ensure the control standards for harmful substances in Zhuang medicinal materials, it is necessary to strengthen the risk assessment of heavy metals, harmful elements and pesticide residues in medicinal materials, establish a tracking system for controlling the generation of harmful substances from sources, and evaluate the planting environments includes soil, water and air. ②   Fast, simple and economical analysis methods not only reduce the cost of investment, but also facilitate market monitoring. For example, rapid pesticide residue detectors and ion immunoassay method have been further developed to effectively improve the detection efficiency and meet the needs of grassroots or on-site detection. ③ Local standards for the limits of harmful substances in Zhuang medicine can be formualted. On the basis of Chinese Pharmacopoeia, combining Guangxis unique Karst landform and the growth environment of various herbal medicines and their own enrichment capacities, local standards suitable for Zhuang medicine can be comprehensively formulated to clarify the limits of harmful substances.

In this study, only the medicinal material samples from 13 cities in Guangxi were measured. Whether it can be used as a basis for the formulation of the quality standardd of the Zhuang medicine Herba Polygoni Chinensis still needs further study. The number of samples needs to be further expanded, and at least one batch of samples should be collected from each city, so as to ensure that the sample quantity is representative. Meanwhile, the soil and water sources of each production area should be monitored to eliminate the capacity of medicinal materials to accumulate heavy metals, so as to formulate standardized and reliable limits.

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