1. IntroductionChinese herbal medicine (CHM) is popular across the globe as dietary supplements, and traditional and alternative medicine [1,2]. CHM has a clinical effect on vascular dementia [3], cancer immune regulation [4], diabetic nephropathy [5], and hepatocellular carcinoma [6].Recently, the inclusion of CHM in the Chinese protocol for combating the coronavirus disease (COVID-19) has been effective because of its efficacy and comprehensive therapeutic theory [7,8]. CHM can reduce the incidence of severe or critical events, improve the clinical recovery of patients with COVID-19, and helps alleviate symptoms such as cough or fever, likely through its host-directed regulation and certain antiviral effects [9]. CHMs have advantages such as abundant clinical experiences, and their unique diversity of chemical structures and biological activities.Health risks from CHMs present serious concerns. Poisoning by metals and metalloids at concentrations above acceptable regulatory standards has been reported [10]. CHM showed different degrees of trace element contamination, which is affected by different types of CHM, medicament portions, elements, preparations, and CHM sources [11]. In a survey on 247 CHMs, 5–15% of the samples contained higher-than-standard concentrations of arsenic (As), and ~5% of them contained higher-than-standard concentrations of lead (Pb) [12].Earlier studies on the potential risks associated with CHMs mostly focused on the total concentration of trace elements, in which a limited number of studies focus on the bioaccessibility of trace elements in CHMs, which could reflect the actual levels of trace elements exposed to the human body [13]. A better understanding of the bioaccessibility of trace elements is crucial for the assessment of their health risk against humans.Currently, herbal medicines are frequently used in various dosage forms, while consumers do not focus on the particulars of the traditional dosage forms [14]. Herbs are dispensed in the form of decoct, powder, granule, or oral liquid. During CHM production, the use of traditional or modern processing methods results in a risk of trace element contamination throughout the whole procedure [15]. Whether these different processing methods affect the bioaccessibility of trace elements in CHMs is unclear.Astragalus membranaceus, Glycyrrhiza uralensis, and Isatidis radix are three medicine food homologous plants that are the most frequently applied herbs in the world. The root of A. membranaceus is a type of CHM widely used as a health-promoting agent with a long history in China, and it has multiple functions, including the regulation of immune function, antioxidant, anti-aging, antitumor, anti-fibrosis, antibacterial, reducing blood glucose, lowering blood lipid, neuroprotectivity, and hepatoprotectivity [16,17]. The dried roots and rhizome of G. uralensis are often used for the treatment of diseases such as weakness of the spleen and stomach, fatigue, palpitation, shortness of breath, cough and phlegm, epigastrium, limb contracture, acute pain, and carbuncle; it is commonly used to alleviate drug toxicity [18]. I. radix is an important and commonly used drug for clearing heat and detoxification, cooling blood, and regulating the pharynx, and it has been used in China and Asia for thousands of years; it has played an important role in preventing and alleviating the symptoms of SARS (Severe Acute Respiratory Syndrome) [19]. G. uralensis and I. radix are also essential compositions for the Lianhuaqingwen capsule. Lianhuaqingwen capsules can improve the improvement rate of clinical symptoms of COVID-19, such as fever, fatigue, cough, improve lung imaging lesions, shorten the duration of symptoms, and improve the clinical cure rate [20].

To confirm whether different preparation types of CHMs could affect the bioaccessibility of trace elements in CHMs, we prepared three of the most commonly used herbal CHMs, namely, A. membranaceus, G. uralensis, and I. radix, in different ways, and analyzed for the bioaccessibility of trace elements. Results could provide information on better regulation of the safe utilization of CHMs.

2. Materials and Methods 2.1. CHM Materials and Analysis of Total Concentration of Trace elementsDry roots of A. membranaceus, G. uralensis, and I. radix originating from Gansu Province, China was selected as the CHM materials (Figure 1). They were identified according to the Pharmacopoeia of China, and the voucher specimens were preserved in a specimen cabinet at the Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences (Beijing, China).The total concentrations of trace elements in CHMs were analyzed by grinding the plant roots and digesting them with a mixture of HNO3–HClO4 (4:1, v/v) [21,22]. There were six replicates for each CHM material. 2.2. Micro-XRF Analysis for the Distribution of Elements and the Inorganic CompositionsThe high-resolution distribution of different mineral elements was obtained using a high-performance micro-X-ray fluorescence spectrometer (Bruker, M4 TORNADO PLUS, Berlin, Germany) [23,24,25]. Roots were fixed to the sample stage by using an X-ray sample film (Prolene* Thin-film, gauge: 0.00016,” 4 µm; 40,640). The analysis parameters were set in accordance with the manufacturer’s instructions as follows: X-ray beam spot size, ≤20 μm for Mo–K; step size, 15 μm; scanning time for each step, 20 ms; excitation, high-brilliance X-ray tube with polycapillary X-ray optics, target material, Rh; 50 kV, 600 μA; vacuum path; and silicon drift detector, detector energy resolution 2.3. Preparation Types of CHMs

Three different preparation types were designed for the CHMs.

(1)

Decoct: the raw CHM material was placed in a bottle with 678.8 mL of ultrapure water, soaked for 40 min, and then boiled for 3 min once every 40 min. This process was repeated thrice. The volume of the solution was controlled within 200 mL and centrifuged at 5000× g for 5 min.

(2)

Powder: the raw CHM material was first ground to powder (<150 μm), and the same procedure for decoction was followed.

(3)

Granule: the raw CHM material was soaked and boiled twice using the same procedure as a decoction, and then 95% ethanol was added to the decoction until the ethanol content reached 70%. The supernatant was collected and condensed into a 10 mL extract. The extract was mixed with dextrin, sucrose, and 95% ethanol, dried at 50 °C for 15 min, and granulated into particles with the size of <1.18 mm.

(4)

Liquid: the raw CHM was soaked and boiled twice by using the same procedure as decoction and condensed to an extract with a density of 1.08–1.12 g/cm3. The supernatant was collected, added with 95% ethanol until the ethanol content reached 60%, and condensed again to an extract with a density of 1.30–1.33 g/cm3. The supernatant was mixed with sucrose syrup (60%) and diluted to a volume of 1000 mL.

To analyze the total concentrations of trace elements in different preparation types, the solution obtained above was digested with a mixture of HNO3–HClO4 (4:1, v/v) [21,22]. 2.4. Extraction and Analysis for the Bioaccessible Fraction of Trace Elements in CHMsBioaccessible fraction extraction included two steps [13]. During the first step, 0.5 g material was mixed with 30 mL of simulated gastric fluid, shaken and extracted in an incubator-rotary at 30 rpm for 1 h at 37 °C, and then centrifuged at 3500× g for 5 min. The supernatant was collected and condensed to approximately 3 mL at a low temperature by using an electro-thermal plate.

Then, the residue from step 1 was extracted using simulated intestinal fluid via the same procedure. The supernatant was collected and condensed using the same procedure as step 1. Two parts of the supernatant were measured for the concentration of trace elements.

The simulated gastric fluid was 1.25 g pepsin, 0.50 g sodium citrate, 0.50 g sodium malate, 500 μL of acetic acid, and 420 μL of lactic acid made up to 1 L by deionized water. The pH was adjusted to 2.0 by 30% HCl.

The simulated intestinal fluid consisted of 1.75 g bile salts and 0.5 g pancreatin made up to 1 L by deionized water. The pH was adjusted to 7.0 by NaHCO3.

To analyze the total concentrations of trace elements in different extracts, the solution obtained above was digested with a mixture of HNO3–HClO4.

2.5. Chemical Analysis and Quality Control

The As concentrations were measured via atomic fluorescence spectrometry (Haiguang AFS-2202, Beijing Kechuang Haiguang Instrumental Co., Ltd., Beijing, China). The concentrations of cadmium (Cd), chromium (Cr), nickel (Ni), Pb, and zinc (Zn) were measured via inductively coupled plasma–mass spectrometry (ICP–MS; ELAN DRCe; PerkinElmer, Shelton, CT, USA). For quality control, the samples of certified standard reference materials for plants (GBW07603) from the China National Standard Materials Center were digested with the experimental samples. The recovery rates of trace elements were 95~110%.