Influence of the rhizosphere soils on essential elements of Ephedra sinica herbaceous stems

*Corresponding author: Yunsheng ZHAO, Ningxia Research Center of Modern Hui Medicine Engineering and Technology, Ningxia Medical University, Pharmacy College, No.692 Shengli South Street, Yinchuan 750004, Ningxia, China. Tel: +86-13619501878, Fax: +86-09516980193. E-mail: zhaoyunsheng1886@163.com, zwhjzs@126.com. Received: 24 January 2017; Accepted: 23 January 2018


INTRODUCTION
Ephedra herb (also known as Ma Huang), is one of the wellknown traditional Chinese medicines, and it has been widely used in crude form for over 3000 years.This herb is used as a diaphoretic, antiasthmatic, or diuretic to relieve colds, bronchial asthma, and edema (Abourashed et al., 2003).Ma Huang contributes to weight loss in obesity and enhances performance in endurance training, and is used as dietary supplement and weight loss product in the Western world (Khasbagan and Soyolt, 2007;Xin et al., 2015).Chinese Pharmacopoeia defines Ephedra sinica Stapf, Ephedra intermedia Schrenk C.A Mey., or Ephedra equisetina Bge as the official source of Ma Huang and indicates that the sum contents of ephedrine and pseudoephedrine measure not less than 0.8% (Pharmacopoeia., 2015).E. sinica, as the the primary medicinal species, is widely distributed in China, except in the lower reaches of Yangtze River and Pearl River Basin, and is especially common in northwest Chinese territories, such as Ningxia, Inner Mongolia, Xinjiang, and Gansu regions (Shen, 1995).E. sinica is an important component of desert grassland ecological systems, and its market demand is strong.Wild E. sinica resources have been severely reduced by excessive harvesting, and the Chinese government has enacted related legislations to strictly control the collection of wild E. sinica (Hong et al., 2011).
Mineral elements may not only influence the production of active ingredients by involving plant secondary metabolism (Nasim and Dhir, 2010;Singh and Garg, 1997;Suchacz and Wesolowski, 2013).but also play important parts as the curative materials (Han et al., 2006;Tuo et al., 2010).Soil provides the main mineral elements for plants.Some studies have measured the correlations between mineral elements in soils and the herbs grown on them.Chen et al. (2009) observed that the growth of Paeonia lactiflora improved at the moderate levels of Fe, Mn, Cu, and Zn in soil, and the paeoniflorin content increased at the same mineral levels, but the opposite effect appeared at the higher levels of such elements.
Mineral elements are important for medicinal plant growth, they exert a direct influence on yields and organic compounds of herbs and also act as important curative materials.However, few studies had been conducted on essential element levels in E. sinica and its rhizosphere soil.Influences of soil elements and element composition on those of E. sinica remain unclear.Therefore, the present study (1) investigated the essential element characteristics of E. sinica and soil samples; (2) explored the relationship between soil and herb elements; and (3) revealed major controlling factors and established prediction models for essential element transfer from soil to E. sinica.

Materials
Stems of wild E. sinica and their rhizosphere soils were collected in September and October 2012 from Ningxia, Inner Mongolia, Xinjiang, and Shanxi (Table 1).Plants were identified as authentic stems of E. sinica by associate Professor Minsheng Yan (Northwest Normal University, China).

Sample preparation
All plant samples were gently washed with deionized water, dried at 105°C, and ground into fine powders (100 mesh).Afterward, fine powders of each plant sample were homogenized in a metal-free mortar and stored in paper bags at room temperature before analysis.Soil samples were treated identically to plant samples, except for washing, and stored in polyethylene bags before use.
For microwave-assisted digestion of plant samples (in triplicate) a Mars-6 Microwave System (CEM Co., Ltd, USA) was used to implement the following procedure: 1.0000 g of homogenized sample was weighed into a Teflon reaction vessel.The samples were digested with 5.0 mL HNO 3 + 3.0 mL H 2 O 2 in a three-step program (1-120°C/20 min, 2 -160 °C/20 min, and 3 -180°C/45 min).Microwaveassisted digestion of soil samples (in triplicate) followed the procedure: 0.5000 g of homogenized sample was weighed into a Teflon reaction vessel.The samples were digested with 5.0 mL HNO 3 +1.0mL HF + 2.0 mL H 2 O 2 in a three-step program (1 -150°C/25 min, 2 -170°C/30 min, and 3 -200 °C/80 min).After digestion, each plant or soil solution was evaporated to 0.5-1.0mL on an electric hot plate at 140°C-160 °C.After cooling (25 min), the digests were diluted and transferred into a volumetric flask with 1 mL internal standard solution and up to 10 mL with deionized water.

ICP-MS measurements
Contents of essential elements in plants and soils digestion solutions were determined by ICP-MS (NexION 300D, PerkinElmer Instrument Co., USA).Instrument parameters were optimized as follows: radio frequency power of 1600 W, plasma gas flow rate of 18.0 L/min, carrier gas flow rate of 1 L/min, sweeps/reading of 20, scan mode of peak hopping, dwell time of 50 ms, integral time of 1 s, sampling depth of 8 mm, and replicates of 5.

Prediction models establishment for element transfer
To predict essential element transfer from soil to E. sinica, a regression function was used: where C plant was one specific element content in E. sinica stems, C soil referred to the total content of soil elements which are significantly associated with this element in E. sinica (P < 0.05), and a and b were regression coefficient.This regression function also applied to soil characterizations, such as sand, silt, OM content, and pH value (Cheng et al., 2015).

Statistical analysis
Data were analyzed by SPSS 21.0 software (International Business Machines Corporation, USA), and all values were expressed as mean values.P values less than 0.05 and 0.01 were considered statistically significant and statistically highly significant, respectively.

Method validation for elemental analysis
Measurements were accomplished by external calibration using aqueous mixed standard substances.Slopes of calibration curves of all analytes exhibited good sensitivity, with their correlation coefficients all reaching beyond 0.9995.Precision, which was expressed as relative standard deviation (RSD), ranged from 0.02% to 1.1%.Withinday repeatability was <3.4%.Tables 3 and 4 summarize the limits of detection (LOD) determined in digestion solutions of soils and plants samples for ICP-MS.The recoveries determined with plant or soil sample 1 ranged from 94% to 115% for ICP-MS.

Soil characteristics
Soil SMC (sand, silt, and clay), pH value, OM, and CEC were measured, and the detailed results per soil were listed in Table 2. pH values varied in a narrow range (7.36 to 8.50, i.e., neutral to moderately alkaline).pH value influenced soil protons, which were usually present in H 2 O solutions ligated to ionic exchange structures of soil components.Soil CEC ranged from 8.85 mmol/kg to 54.99 mmol/kg.CEC provided information on potential of soils to bind or to release cations (as nutrients or pollutants).Average OM content of all soil samples reached 7.39 g/kg and ranged from 0.78 g/kg to 22.11 g/kg with generally high coefficients of variation.Soil SMC (sand 2-0.05 mm, silt 0.05-0.002mm, clay <0.002 mm) exhibited significant variability in SMC distribution (37.0-211.0g/kg clay, 2.0-66.8g/kg silt, 760.2-961 g/kg sand).
All these variables were important parameters influencing elemental contents in plant samples.In this study, parameters of rhizosphere soils differed according to the collected locations.These influencing factors created a chemical environment conducive for plant growth.

Element contents of soil samples
Table 3 showed the element contents with LOD in 14 rhizosphere soil samples.The order of average contents was Ca > Na > K > Fe > Mg > Mn > N > P > S > Sr > Zn > Cl > B > Cu > Mo.Contents of total elements varied considerably from 13447.75 mg/kg to 137553.94mg/kg, with an average of 69513.62 mg/kg.The highest contents were observed for Ca with an average of 50070.91 mg/kg and accounted for 72.03% of total elements.RSD of element contents approximated 42%, which is within the acceptable range for inhomogeneous soil specimens.For each sampling site (soil samples 1-14), RSD values ranged from 2.2% to 4.9%.High variations were observed for Ca and P, and these elements were strongly related to local bedrock composition.RSD measuring less than 10% implied low variability, whereas RSD of more than 90% indicated extensive variability as reported by Zhang et al. (2007).A moderate variability was detected in most element contents of soil samples.
In general, Cu, Fe, Mn, Sr, and Zn are categorized as plant micro elements (<0.01% of plant dry weight) and play important roles in soil fertility.Normal contents of these elements in soil were of significant interest as background values, and they were needed for assessment of the degree of soil contamination to some extent.Cu content was below the limit for agricultural soil in China (100 mg/kg), France and Canada.Zn content was considerably below the maximum regulated soil contents in China and France (AFNOR, 1996;CEPA, 2006;CCME, 2012).Fe, Mn, and Sr contents were below the reference values for agricultural soil according to Kabata-Pendias and Mukhrjee ( 2007).
The study areas were not contaminated by the investigated metal elements (i.e., Cu, Fe, Mn, Sr, and Zn).All these elements and other organic compounds all serve as important pharmacodynamic material bases of medicinal plants (Qin, 2011).However, considerable element contents in plants fall within certain limits, and excessive metal elements may be harmful for humans.In our study, Zn contents ranged from 10.28 mg/kg to 28.26 mg/kg and were within the reference value of plant foodstuffs (Kabata-Pendias and Pendias, 2011).Cu contents obtained from different sites ranged from 2.07 mg/kg to 6.14 mg/ kg and were within the permissible limit proposed by WHO (1998).The contents of Mn were between 7.23 and 58.74 mg/kg.In sample 4, Mn content was above the reference value (27-50 mg/kg) of Kabata-Pendias and Mukhrjee for plants in agricultural lands.Studies found that nutritional supplementary with mineral elements, especially Cu and Mn, should be more suitable and be recommended for patients suffering chemotherapy to sustain nutrient homoeostasis (Kabata-Pendias and Mukhrjee, 2007;Akutsu et al., 2012).

Element uptake and accumulation of E. sinica
Availability of mineral elements to plants is regulated by soil characteristics, plant biological properties, climate conditions, etc. Soil characteristics, such as sand, silt, OM, and pH value, have important effects on absorption and transfer of specific elements in rhizosphere soil.We investigated the relationships between contents of essential elements in the soils and E. sinica grown on them.The measured results were shown in Table 5. Correlation analysis showed that sand, silt, and OM of the soils were more importance than other factors for E. sinica.For example, content of soil silt was positively related with the contents of N, K, Cl, Na, Mn, B, Cu, and Mo in plant, but content of soil sand was negatively proportional to N, K, Cl, Sr, Na, B, and Cu contents.OM content was positively correlated with N, K, Na, Mn, B, Cu, and Mo contents in plants, whereas Mn content was affected by pH value of soil.
Among the 240 correlations analyzed between element contents from E. sinica and their rhizosphere soils (Table 6), 111 were statistically significant, and N, K, Ca, Sr, Mn, Zn, and Cu contents in plants were correlated to those in soils.
Given the high variation of soil composition, the exact calculation of plant enrichment coefficients (enrichment coefficient = average element content in plants/ average element content in soils) was not considered justifiable.Nevertheless, general conclusions can be drawn regarding mineral uptake and accumulation behavior upon comparison of soil and plant elemental contents.
The order for element enrichment coefficients was N > S > P > Cl > Sr > Mg > K > Mo > B > Zn > Cu > Ca = Fe > Mn > Na.Mean contents of N, S, P, Cl, Sr, and Mg in plants were higher than those in soils, whereas higher mean contents of the other nine minerals were observed in soils.Results demonstrated that E. sinica could hyperaccumulate N (>12,500 mg/kg), S (>1,890 mg/kg), P (>463 mg/kg), Cl (>102 mg/kg), Sr (>80 mg/kg), and Mg (>1,488 mg/kg), and mean enrichment coefficients of E. sinica to the six elements totaled 42.88, 34.37, 7.81, 4.38, 2.16, and 1.56, respectively, and the other elements yielded mean enrichment coefficients of <1.
Prediction models for element transfer from soil to E. sinica Table 7 showed the prediction models for the essential elements.Multiple linear regression analysis identified C soil , pH, sand, silt, and OM as factors that best explained variability in C plant (R 2 = 39.2%-96.6%,P < 0.05).More than 76% of Cu, B, Mo, Na, Mn, and P contents in E. sinica were explained by soil factors.These prediction models could be used to obtain reliable predictions of element contents in E. sinica herbaceous stems and therefore used to assess potential value or risk to humans.These models also contributed to regulation of E. sinica rhizosphere soils to ensure herb growth and quality safety.For example, based on the regression model 2, when grown on soil with high Mn factors, Mn content of E. sinica herbaceous stem can be reduced by raising soil pH.
Fig. 1 showed the interrelation between the identified log [C plant ] and its predicted value.Most predicted values fell within the 95% prediction interval, displaying the good accuracy for these models.Mean square error (MSE) values ranged from 0.029 to 0.679 for these prediction models (Table 7).Thus, these models were responsible predictors of element contents in E. sinica herbaceous stems.
These regression equations could also be used to evaluate the suitability of soils for herb safe production (Römkens et al., 2011).For example, according to Equation 6, an  6, Cu content of 5.73 mg/kg from E. sinica was calculated, this value is considerably lower than the green standard limit (≤20 mg/kg) for medicinal plants (MOC, 2004).Therefore, when soil Cu soil content reaches 123,764.69mg/kg, Cu content in E. sinica will not exceed the safety limit.This finding further confirms the very low risk of producing E. sinica with Cu contents exceeding the safety limit.Therefore, Equation 6 can provide scientific basis for Cu monitoring of E. sinica.

CONCLUSIONS
The present study showed that 15 essential element characteristics in wild E. sinica and its rhizosphere soil, revealed the influences of rhizosphere soil on the elements in the herbs, and established prediction models for transfer of elements from soil to plant.The study results will assist in regulation of mineral elemental contents in E. sinica herbaceous stems.7.
ZHANG performed statistical analysis and helped in interpretation of data and discussion of results.Junyu LIANG and Hongling TIAN collected the samples.

Fig 1 .
Fig 1. Relationship between the measured log[C plant ] and the predicted log[C plant ]. y is measured logarithm value, and x is predicted logarithm value; R 2 is the regression equations coefficient of determination in Table7.

Table 4
listed the elemental contents in plant samples along with LOD.The order of average element contents was N > K > S > Ca > Mg > P > Fe > Cl > Sr > Na > Mn > B > Zn > Cu > Mo.Contents of total elements varied from 21,216.91 mg/kg to 54,426.02mg/kg, with an average of 34,863.55mg/kg.The highest contents

Table 5 : Correlation coefficients between the basic compositions in soils and the elemental contents in plants
*. Correlation is significant at the 0.05 level (2-tailed).**.Correlation is significant at the 0.01 level (2-tailed).