Elemental Analysis of Nine Herbal Drugs for Nourishing Blood by Inductively Coupled Plasma Atomic Emission Spectroscopy with Microwave Digestion

CHEN Lin-wei, CAI Hao, WANG Qin, QIAO Feng-xian, QIN Kun-ming, CAI Bao-chang

School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

Elemental Analysis of Nine Herbal Drugs for Nourishing Blood by Inductively Coupled Plasma Atomic Emission Spectroscopy with Microwave Digestion

CHEN Lin-wei, CAI Hao*, WANG Qin, QIAO Feng-xian, QIN Kun-ming, CAI Bao-chang

School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

An analytical method has been developed for the determination of traces of Zn, Mn, Na, K, Ca, Mg, Ni, Pb, Se, Fe, Cu, Al, B, Ti, Sn, Hg, and Li in nine herbal drugs for nourishing blood by ICP-AES. The data showed that nine herbal drugs for nourishing blood were abundant in the contents of major and trace elements which are healthy for human body, including the trace elements with higher contents, such as Fe, Se, Ni, Mn, and Zn, which are well known for nourishing blood. Elements (macro-elements, essential elements, non-essential elements, and toxic elements) classified in nine herbal drugs for nourishing blood were illustrated in cylindrical histograms. Principal component analysis and hierarchical cluster analysis were applied to the data matrix to evaluate analytical results. The results of hierarchical cluster analysis showed that dendrogram obtained by Ward’s method of hierarchical cluster analysis for seventeen variables and nine samples mainly displayed three clusters. After optimizing the microwave digestion technology, the recoveries of seventeen elements were in the ranges from 97.89% to 103.25% and the RSDs were all lower than 3.0%. ICP-AES combined with microwave digestion is accurate and precise in determining major and trace elements in nine herbal drugs for nourishing blood.

Herbal drugs for nourishing blood; Elements; Microwave digestion; ICP-AES

Introduction

In drugs for nourishing blood, some Chinese herbal medicines includingGinsengRadixetRhizoma,AstragaliRadix,AngelicaeSinensisRadix,PaeoniaeRadixAlba,CitriReticulataePericarpium,PolygoniMultifloriRadix,PolygonatiOdoratiRhizoma,PolygonatiRhizoma, andLyciiFructushave been used for more than thousand years to nourish the ‘Blood’ (body circulation) and raise the ‘Qi’ (vital energy)[1-2]. The medicinal value of Chinese herbal medicine is very important and has a direct relationship with major and trace elements[3-4]. However, inorganic components classified in above nine herbal drugs for nourishing blood are still remained unclear and no investigations have been reported referring to the element compositions of above nine herbal drugs for nourishing blood. Therefore, it is essential to develop a reasonable method for profiling the major and trace elements of Chinese herbal medicine.

The methods of atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS) are often used for determining major and trace elements[5-6]. Among them, ICP-AES has been widely used with the advantages of low cost, rapid analysis, wide linear range, low detection limit, and simultaneous determination of multi-element. In recent years, microwave digestion has become an internationally recognized digestion method with the advantages of fast and effective dissolving effect, low evaporation loss, easy to control, multiple sample digestion, and good reproducibility[7-8]. In this study, the concentrations of seventeen elements were determined in nine herbal drugs for nourishing blood by inductively coupled plasma-atomic emission spectroscopy with microwave digestion. The developed method might provide an approach to study the relationship between the elements and the actions of nine herbal drugs for nourishing blood.

1 Materials and Methods

1.1 Analytical instrumentation and test method

Optima 2100 DV inductively coupled plasma atomic emission spectrometer (ICP-AES, PerkinElmer, USA), dual array solid-state CCD detector projection optical system, RYTON material atomizer, 40.68 MHz solid-state high-frequency self-excited generator, polyscience circulating water cooling system and MAS microwave digestion system (Preekem, China). ICP-AES operating parameters were shown in Table 1.

The digestion solution is HNO3and H2O2, different types of herbs (0.5 g) were taken in triplicate, which were set in the high-pressure digestive tract, and 5 mL of HNO3were mixed, and then 1 mL of H2O2was added，the microwave digestion procedure was conducted in three steps [(1) -120 ℃/3 min, (2) -150 ℃/5 min, and (3) -190 ℃/15 min]. The resulting solutions were transferred into 25 mL volumetric flasks and diluted with deionized water. A reagent blank was prepared with the same acid solution and subjected to the same digestion procedure as the sample. All sample solutions were prepared in triplicate and stored at 4 ℃ and finally analyzed at room temperature.

Table 1 ICP-AES operating parameters

1.2 Samples

Nine herbal drugs for nourishing blood ofGinsengRadixetRhizoma,AstragaliRadix,AngelicaeSinensisRadix,PaeoniaeRadixAlba,CitriReticulataePericarpium,PolygoniMultifloriRadix,PolygonatiOdoratiRhizoma,PolygonatiRhizoma, andLyciiFructuswere purchased from Nanjing Haichang Chinese Medicine Group Corporation (Nanjing, China), which were identified by Prof. CAI Hao.

1.3 Reagents and standard solutions

HNO3(AR grade) and H2O2(UP grade) were purchased from Nanjing Chemical Reagent Co., Ltd. (China), deionized water was obtained with a Milli-Q purification system (Millipore, Milford, MA, USA). The metal standards were provided by the National Institute of Metrology (China) and the metal standard solutions were prepared by appropriate dilutions of 1 000 μg·mL-1stock solutions immediately before use. The standard stock solution was diluted with 2% HNO3step by step. The mixed standard solutions of Zn, Mn, Na, K, Ca, Mg, Se, Fe, Cu, Al, Ti, Sn, and Li were prepared by the gradients of 0.00, 0.50, 1.00, 2.00, 4.00, and 8.00 μg·mL-1, and the mixed standard solutions of Pb, Ni, Hg, and Cd were prepared by the gradients of 0.00, 0.25, 0.50, 1.00, 2.00, and 4.00 μg·mL-1.

2 Results and Discussion

2.1 Concentration

The relative standard deviations (RSDs), the recoveries, and the detection limits of seventeen elements in nine herbal drugs for nourishing blood were shown in Table 2, and the concentrations of seventeen elements in nine herbal drugs for nourishing blood were shown in Table 3. The results showed that nine herbal drugs for nourishing blood were abundant in the contents of K, Ca, Mg, Na, Fe, Mn, Zn, Se, and Al, and the contents of elements in descending order are K>Na>Mg>Ca>Al>Fe>Mn>Se>Zn>B>Ti>Cu>Li>Hg>Sn>Pb>Ni. Seventeen elements in nine herbal drugs for nourishing blood play irreplaceable roles in their efficacies for nourishing blood, improving immune function, and preventing disease, especially the trace elements of Fe, Se, Ni, Mn, and Zn, which are well known for nourishing blood. The results also showed that the concentrations of heavy metals were very low in nine herbal drugs for nourishing blood. Elements (macro-elements, essential elements, non-essential elements, and toxic elements) classified in nine herbal drugs for nourishing blood were illustrated in cylindrical histograms (Fig.1).

2.2 Principal component analysis

Principal component analysis (PCA) was subsequently carried out on the correlation matrix using the PCA subroutine of the SPSS V10.0 package[9-10]. Six principal components were selecued with eigen values were more than 1. The first six principal components represented 94.01% of all the variable information on the contents of elements in nine herbal drugs for nourishing blood. The first factor has high loadings for K, explains about 34% of the total variance. In this factor, the second factor accounts for 19% of the total variance. It is highly positively correlated with Na. The third factor is loaded by Ca, explains 18% of the total variance. The fourth factor has high loadings for Fe, explains 9% of the total variance. The fifth factor is loaded by Mg, explains 7% of the total variance. The sixth factor accounts for 6% of the total variance. It is highly positively correlated with Al. Pb and Hg does not load as highly as other elements of the factor. A 3-D plot of the PCA loadings was illustrated in Fig.2.

Table 2 Analytical wavelengths, detection limits, relative standard deviations, recoveries, and

Table 3 Concentrations of seventeen elements in nine herbal drugs for nourishing blood (μg·mL-1)

Fig.1 Cylindrical histograms of elements classified in nine herbal drugs for nourishing blood

(a): Macro-elements; (b): Essential elements; (c): Non-essential elements; (d): Toxic elements. SW representsPolygoniMultifloriRadix; CP representsCitriReticulataePericarpium; DG representsAngelicaeSinensisRadix; HQ representsAstragaliRadix; BS representsPaeoniaeRadixAlba; GQ representsLyciiFructus; RS representsGinsengRadixetRhizoma; YZ representsPolygonatiOdoratiRhizoma; HJ representsPolygonatiRhizoma

Fig.2 PCA results in the three dimensional space: plot of loadings of the first three principal components

2.3 Hierarchical cluster analysis

A hierarchical clustering by applying Ward’s method, which uses the squared Euclidean distance as a similarity measure, was performed with the standardized data set[11]. The results obtained in the cluster analysis were presented as a dendrogram in Fig.3. Fig.3 mainly displayed three clusters: (1)AstragaliRadix,AngelicaeSinensisRadix,PaeoniaeRadixAlba,CitriReticulataePericarpium,PolygoniMultifloriRadix,andLyciiFructus; (2)GinsengRadixetRhizomaandPolygonatiOdoratiRhizoma; (3)PolygonatiRhizoma. These clusters represented the similarity on the origins of elements for medicinal herb samples. Similar results to cluster analysis were also obtained after the application of PCA.

Fig.3 Dendrogram obtained by Ward’s method of hierarchical cluster analysis for seventeen variables and nine samples (the distances reflect the degree of correlation between different elements)

3 Conclusions

A simple, quick, and sensitive method, based on microwave digestion and ICP-AES, has been developed for the determination of Zn, Mn, Na, K, Ca, Mg, Ni, Pb, Se, Fe, Cu, Al, B, Ti, Sn, Hg, and Li in nine herbal drugs for nourishing blood. The simultaneous determination of multi-element in the radial observation by ICP-AES with microwave digestion reveals its advantage in application of herbal drugs.

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陳林偉，蔡 皓： 并列第一作者

*通訊聯(lián)系人

O657.3

A

微波消解ICP-AES法分析九種補血類中藥元素

陳林偉，蔡 皓*，王 琴，喬鳳仙，秦昆明，蔡寶昌

南京中醫(yī)藥大學藥學院，江蘇 南京 210023

采用微波消解法處理樣品，以電感耦合等離子體發(fā)射光譜法(ICP-AES)測定并分析九種補血類中藥中Zn，Mn，Na，K，Ca，Mg，Ni，Pb，Se，F(xiàn)e，Cu，Al，B，Ti，Sn，Hg和Li元素的含量。結(jié)果顯示，九種補血類中藥富含有益于人體健康的主要及微量元素，其中眾所周知補血類元素Fe，Se，Ni，Mn，Zn含量較高。九種補血中藥按各大元素分類(大量元素，必需元素，非必需元素，有害元素)分布于柱形直方圖。應用主成分分析和聚類分析于數(shù)據(jù)矩陣去評價元素分析結(jié)果。聚類分析的結(jié)果顯示九種補血類中藥中十七種元素的含量所形成的樹狀系統(tǒng)圖主要分為三個集群。通過優(yōu)化微波消解條件，各元素的回收率均在97.89%～103.25%之間, 相對標準偏差均小于3.0%。ICP-AES結(jié)合微波消解法可以精確和準確地測定九種補血類中藥中的元素。

補血類中藥； 元素； 微波消解； 電感耦合等離子體發(fā)射光譜

2015-06-01，

2015-12-23)

Foundation item： the National Natural Science Foundation of China (81173546, 30940093)

10.3964/j.issn.1000-0593(2016)07-2358-05

Received： 2015-06-01; accepted： 2015-12-23

Biography： CHEN Lin-wei, (1989—), a graduate student in School of Pharmacy, Nanjing University of Chinese Medicine e-mail: simply0518@126.com CHEN Lin-wei and CAI Hao: joint first authors *Corresponding author e-mail: haocai_98@126.com