Open Access

Simultaneous determination of six active metabolites in Astragalus mongholicus (Fisch.) Bge. under salt stress by ultra-pressure liquid chromatography with tandem mass spectrometry

Contributed equally
SpringerPlus20165:927

https://doi.org/10.1186/s40064-016-2638-y

Received: 1 March 2016

Accepted: 21 June 2016

Published: 30 June 2016

Abstract

Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus, family Leguminosae) is one of the most important traditional Chinese herbs because it contains lots of bioactive metabolites, which have beneficial and pharmacological effects on health. Simultaneously, it has been proved to be a salt-tolerant plant—one of the potential species to control the soil salinization. Therefore, a sensitive and specific ultra-pressure liquid chromatography coupled with tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the simultaneous determination of six main bioactive metabolites, astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin in different organs of A. mongholicus. The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization source operating in the positive ionization mode. Calibration curves offered linear ranges of two orders of magnitude with R2 > 0.99. The method was fully validated for the linearity, intra-day and inter day precisions, accuracy, recovery, matrix effect and stability. Then this method was successfully applied to detect the content of major bioactive metabolites in different plant organs of A. mongholicus under salt stress. Significant variations in the content of six bioactive metabolites were observed after been processed by different levels of salinity in different part of plant. The results support for further exploration of the salt-tolerant mechanisms in A. mongholicus and its possibility as the species that control the soil salinization. Meanwhile, we established a UPLC-MS/MS assay of the trace components in seedling of A. mongholicus in this study.

Keywords

UPLC–MSBioactive metabolitesSalt stress Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao

Background

Radix Astragali, A widely used Chinese Herbal Medicine, is derived from the dried roots of Astragalus mongholicus (Fisch.) Bge. (Zu et al. 2009). A. mongholicus generally is mixed with other ingredients to make some medicated food in its edible aspect, which it has also traditionally been utilized in cosmetics. Pharmacological studies and clinical practice have demonstrated that A. mongholicus possess various biological activities including tonic, immunostimulant, hepatoprotective, diuretic, antidiabetic, cardioprotective, anti-oxidative and anti-tumor properties (Sun et al. 2008; Boye et al. 2015; Chen et al. 2015). More than 100 chemical constituents of A. mongholicus have been isolated and identified (Lv et al. 2015; Napolitano et al. 2013). Isoflavonoids and triterpene saponins have been considered as two types of the major bioactive metabolites found in A. mongholicus. Formononetin, ononin, calycosin and calycosin-7-O-β-d-glucoside, which boost energy, strengthen the immune system, and promote health activities and skin growth, are the major isoflavonoids in A. mongholicus (Xiao et al. 2004; Krasteva et al. 2015). Astrageloside IV has protective effects on cardiovascular system, immune, digestive and nervous system (Ren et al. 2013). Cycloastragenol is the synthetic precursor compound of astragaloside IV. And all of them could be the marker compounds for the chemical evaluation of A. mongholicus (Yesilada et al. 2005; Pu et al. 2015). In addition, formononetin, ononin, calycosin and calycosin-7-O-β-d-glucoside, are the most important metabolites in isoflavonoids biosynthesis pathway (Xu et al. 2011); astragaloside IV and cycloastragenol, are the essential metabolites of triterpene saponin biosynthesis pathway (Park et al. 2015). Synthesis or decomposition of these compounds has important implications for the quality of A. mongholicus as medicines (Zheng et al. 2015; Liu et al. 2015a).

Salinity is possibly the most imperative ecological restriction that causes extensive crop yield losses all over the world, and its threat is escalating day by day (Peng et al. 2011; AbdElgawad et al. 2016). The only way to control the soil salinization process and to maintain the sustainability of landscape and agricultural fields is to combat the salinization problems by environmentally safe and clean techniques, such as: use of salt-tolerant species (Hasanuzzaman et al. 2014; Moore 1984). A. mongholicus has been proved to be a salt-tolerant plant (Wdowiak-Wrobel et al. 2013), it was one of the potential species to control the soil salinization. So it is extremely critical to search for the salt-tolerance and the ability of physiological adaptation of A. mongholicus. When plants are exposed to salt stresses, they display intricate regulatory mechanisms to enhance their response, including cellular changes and metabolic responses (Molinier et al. 2006; Bruce et al. 2007; Chen et al. 2007). Importantly, we must focus on the content of major bioactive metabolites in A. mongholicus. under salt stress to ensure the medicinal value of A. mongholicus, then we can explore the possibility of A. mongholicus to be used as potential salinity species. As two types of the major metabolites and active components in A. mongholicus, the change in isoflavonoids and triterpene saponins level needs to be observed carefully. So we developed and applied the rapid and sensitive UPLC-MS method for simultaneous determination of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin in A. mongholicus under different levels of salt stresses.

Because of the trace amounts of compounds in the seedlings, their quantitative analysis in the seedlings was difficult. In attempts to improve the determination of these compounds in A. mongholicus, several studies have been reported. The current method for the determination of these compounds mainly uses high-performance liquid chromatography (HPLC) coupled with DAD or ESI–MS Detection (Kwon and Park 2012; Gong et al. 2015). Being the common analytical tool for various compounds, HPLC and LC–MS both are being more and more widely applied in biological research, and have been used to quantify the marked compounds in biological samples such as A. mongholicus seedlings (Liu et al. 2015a; b). However, the threshold of sensitivities of these HPLC methods for detecting these compounds in A. mongholicus seedling is high. Furthermore, the major weakness of these LC–MS methods mainly includes chromatographic running time, which at more than 18 min is considered too long. This is time consuming and not suitable for analyzing large number of sample. Even though some experiments were investigated for determination of some compounds in A. mongholicus (Lv et al. 2011; Wu et al. 2005; Qi et al. 2006), there is no established sensitive method regarding of simultaneous determination of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin in seedlings was published.

Therefore, this study aimed at developing a sensitive and validated ultra-pressure liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI–MS) method with a short time for simultaneous determination of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin in A. mongholicus under different levels of salt stress.

Methods

Materials and reagents

Seeds of A. mongholicus were purchased from AnGuo Chinese Herbal Medicine Base in HeBei Province of China. Astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin, formononetin, scutellarin (IS, internal standard) and sodium chloride were purchased from Chengdu Must Bio-technology Co., LTD. (Chengdu, China). Sodium chloride (NaCl) was of analytical reagent grade. Water used for the UPLC–MS/MS analysis was prepared with a Milli-Q water purification system procured from Millipore (Milford, MA, USA). Acetonitrile (J & K Scientific Ltd. Beijing, China) was of HPLC grade. All other chemicals used in the method were of analytical grade.

UPLC–MS analysis

Chromatographic separation was performed on an Ultra-Performance LC (UPLC) system (Waters, Japan) with a LC-20AD pump, a temperature controller, column oven, SIL-20A autosampler (Waters, Japan), and ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 mm × 50 mm) with in-line filter and maintained at 25 °C was used. The mobile phase in UPLC determinations is consisted of (A) water and (B) acetonitrile. The elution program was optimized as follows: 8 % B (0–1 min), 8–34 % B (1–1.5 min), 34 % B (1.5–4 min), 34–60 % B (4–6 min), 60 % B (6–7 min), 60–8 % B (7–7.5 min), 8 % B (7.5–9 min). The flow rate was 0.25 mL/min and the injection volume was 5 μL.

Tandem mass spectrometric detection was performed on an QTRAP 5500 Ion trap mass spectrometer (AB SCIEX, USA) equipped with an electrospray ionization (ESI) source in the positive ion detection mode. MS source conditions were set as follows: Ionspray voltage: 5500 V, turbo spray temperature: 500 °C, high purity nitrogen was used in all units; nebulizer gas: 25 psi; curtain gas: 20 psi. Analytes were quantified by multiple reaction monitoring (MRM) mode. The optimized fragmentation transitions for MRM (precursor-to-product ion pair and parameters): m/z 806.9 ([M + Na]+) → 627.1 ([M–C6H11O5–OH + Na]+) with declustering potential (DP): 117 V, collision energy (CE): 81 V and collision cell exit potential (CXP): 17 V for astragaloside IV; m/z 491.3 ([M + H]+) → 143.0 [M–C22H36O3 + H]+ with DP: 85 V, CE: 20 V and CXP: 12 V for cycloastragenol, m/z 446.9 ([M + H]+) → 284.2 ([M–C6H11O5 + H]+) with DP: 104 V, CE: 24 V and CXP: 8 V for calycosin-7-O-β-d-glucoside, m/z 285.0 ([M + H]+) → 269.9 ([M–CH3 + H]+) with DP: 117 V, CE: 33 V and CXP: 17 V for calycosin, m/z 431.0 ([M + H]+) → 268.2 ([M–C6H11O5 + H]+) with DP: 111 V, CE: 28 V and CXP: 15 V for ononin, m/z 269.0 ([M + H]+) → 196.2 ([M–OH–CO–OCH3 + H]+) with DP: 80 V, CE: 50 V and CXP: 17 V for formononetin, m/z 463.0 ([M + H]+) → 287.0 ([M–C6H8O6 + H]+) with DP: 100 V, CE: 29 V and CXP: 8 V for scutellarin (IS). The analysis of astragaloside IV achieved a positive ion of m/z 806.9, referring to the [M + Na]+ precursor ion. Data acquisition was performed with Analyst 1.4.2 software version (AB SCIEX, Concord, Ontario, Canada).

Plant material, growth conditions and sample preparation

Astragalus mongholicus plants were grown in a greenhouse (ZPW-400, China), under a 14/10 light/dark photoperiod at a temperature of 25 (day)/22 °C (night) and 70 % relative humidity. Seeds of A. mongholicus were planted in pots using perlite as culture substrate and kept moistened until the seeds had germinated. 20 days seedlings were selected as initial material. Sodium chloride (NaCl) concentration in the culture substrate solution was adjusted to 0 (control), 100 and 300 mM. After 7 days, roots, stems and leaves under different salt levels were collected respectively.

The plant materials collected were pulverized to 5 μm by grinding instrument (MM 400, Retsch, GmbH, Haan, Germany), and 0.5 g tissue aliquots of roots, stems and leaves under different salt levels were extracted respectively. The plant samples were treated with 10 mL of 80 % ethanol (composed of ethanol and water in a volume ratio of 4:1, include 500 ng/mL IS), and reflux extraction was performed for 45 min. The samples extractions were filtered and the residues were treated with 10 mL of 80 % ethanol, and reflux extraction was performed for 45 min. The samples extractions were filtered and the filtrates were merged. Then the filtrates were dried under low pressure by vacuum cavitation instrument. The resultant extracted material was dissolved in mobile phase (1 mL) and filtered through micropores of 0.22 μm diameter. The purified solution was analyzed by UPLC-MS.

Preparation of calibration standards and quality control (QC) samples

The standard stock solutions of astragaloside IV (1 mg/mL), cycloastragenol (1 mg/mL), calycosin-7-O-β-d-glucoside (1 mg/mL), calycosin (1 mg/mL), ononin (1 mg/mL) and formononetin (1 mg/mL) were prepared by dissolving required amount of the reference standards in methanol. A working solution of IS was prepared in methanol at a final concentration of 100 μg/mL. A mixed stock solution containing 1 mg/mL of the six reference standards and IS in methanol for chromatographic separation was prepared. The working standard solutions of calibration curve were prepared by successive dilution of the mixed stock solution with methanol to yield the final concentration series ranged from 1.48 to 14,800 ng/mL for astragaloside IV, calycosin-7-O-β-d-glucoside and ononin, and 0.148 to 1480 ng/mL for cycloastragenol, calycosin and formononetin. QC samples including 74 (low), 740 (mid) and 7400 ng/mL (high) for astragaloside IV, calycosin-7-O-β-d-glucoside and ononin, 7.4 (low), 74 (mid) and 740 ng/mL (high) for cycloastragenol, calycosin and formononetin were prepared as the same procedure as the calibration standards. Calibration work solutions and QC samples were kept at −20 °C until UPLC-MS/MS analysis. All of these were freshly prepared before each experiment.

Method validation

The linearity of the method was generated by analysis of six calibration curves. The calibration standard curves of the tested compounds were obtained by least-squares linear regression of the peak area versus the concentrations, using the least-square linear regression with weighting factor 1/concentration2. All calibration curves were required to have a correlation value of at least 0.99. LLOQ was determined based on the signal-to-noise ratio of 10:1.

The intra- and inter-day precision and accuracy of the assay were determined by analyzing six replicates of three concentration levels of QC samples (low, mid and high concentrations) on the same day and on three consecutive days, respectively. Accuracy and precision were evaluated according to relative error (RE) and relative standard deviation (RSD), respectively. Values below ±15 % were accepted.

Extraction recoveries were evaluated at three QC levels (low, mid and high concentrations) by comparing the peak areas obtained from the samples with the analytes spiked before and after extraction. Matrix effect generally impacts in the form of either ion suppression or ion enhancement. The peak areas obtained from analytes in matrix extract (A) were then compared with corresponding peak areas obtained from the standard solutions in the mobile phase (B) at equivalent concentrations. The ratio (A/B × 100) is defined as the matrix effect. The value of matrix effect less than 85 % represented ionization suppression, while more than 115 % represented ionization enhancement.

Stability tests were performed for six replicates of QC samples at three concentrations (low, mid and high concentrations) under different conditions. The short-term and long-term stability was tested by analyzing samples exposed at room temp for 4 h and kept at −20 °C for 2 weeks, respectively. The freeze–thaw stability after three freeze–thaw cycles on three consecutive days was also determined. Stability was assessed by comparing the mean concentration of the samples with the mean concentration of freshly prepared calibration. The samples were considered stable if the assay values were within the acceptable limits of ±15 % RSD.

Statistical analysis

Results were subjected to analysis of variance (ANOVA) to determine the significant differences between different levels of salt treatment times. If ANOVA was performed, Duncan’s honestly significant difference (HSD) post hoc tests were conducted to determine the differences between individual treatments (SPSS 17.0, SPSS Inc., USA).

Results and discussions

Optimization of UPLC-MS conditions

Because of the complexity of the plant components, many analogs maybe co-eluted during analyses. It is important to obtain a sensitive and accurate UPLC-MS method including efficient chromatographic separation and appropriate ionization. The chromatographic conditions were optimized simultaneously to reduce the influence of endogenous interferences, improve the peak shape, increase the signal response of analytes, and shorten the running time. Liquid chromatographic conditions were investigated such as column, mobile phase, column temperature and flow rate that could greatly influence the separation. In the present study, ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 mm × 50 mm) was chosen for its high efficiency and improving the peak shape. Different mobile phases (water–methanol, water-acetonitrile) were examined and compared. It was found that better peak shapes and elution efficiency could be produced by water-acetonitrile. It was also found that the best separation was obtained when the column temperature was kept at 26 °C using a flow rate of 0. 25 mL/min. Full scan positive ion ESI mass spectra were obtained for each of the compounds by direct injection of the standards of astragaloside IV, cycloastragenol, calycosin, ononin, formononetin, calycosin-7-O-β-d-glucoside and IS, respectively.

To optimize mass spectrometric conditions for detection of these compounds, several conditions were investigated such as scan mode, the precursor and product ions of these analytes, source temperature and ions spray voltage. Under ESI ion source condition, the response of these compounds observed in positive ion mode was higher than that in negative ion mode. Thus, positive ion mode was finally employed. The precursor and product ions of these analytes were ascertained by injecting standard solutions in Q1 scan and product ions (MS2) mode, respectively. In the precursor ion scan mass spectra, most analytes formed molecular ions [M + H]+, except that the ions of astragaloside IV were [M + Na]+. Astragaloside IV predominantly formed sodium adduct ions [M + Na]+, at m/z of 806.9 with the response higher than [M + H]+. However, both of these precursor ions formed the same fragment ion, with [M + H]+ being the most abundant. The [M + H]+ and [M + Na]+ ions were therefore chosen as the precursor ions to obtain their major fragment ions for MRM analysis. The fragment ions at m/z values of 627.1, 143.0, 284.2, 269.9, 268.2, 196.2 and 287.0 were present in the highest abundance for astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin, formononetin and IS, respectively. The instrument was tuned to yield the maximum product ion for each compound. The MS/MS product ion spectra of these analytes are shown in Fig. 1. Then, the MS parameters, including ionspray voltage, decluster potential (DP), collision energy (CE) and collision cell injection potential (CXP), were optimized through the product ion mode. The optimized MS/MS transitions and energy parameters of these analytes were shown in Table 1. In order to obtain the maximum abundance of the molecular ions of the compounds, the source temperature, the nebulizing gas (N2) pressure and the curtain gas flow were set at 500 °C, 25 and 20 psi, respectively. A typical MRM chromatogram of the standards and the sample is shown in Fig. 2. The retention times of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin, formononetin and IS were 3.90, 6.27, 2.23, 2.80, 2.53, 4.75 and 2.06 min, respectively.
Fig. 1

The production scan spectra and chemical structure of the tested compounds and scutellarin (IS): a astragaloside IV; b cycloastragenol; c calycosin-7-O-β-d-glucoside; d calycosin; e ononin; f formononetin; g IS

Table 1

The optimized MS/MS transitions and energy parameters of analytes

Analyte

DP (V)

CE (V)

CXP (V)

Precursor (amu)

Product (amu)

Astragaloside IV

117

81

17

806.9 [M + Na]+

627.1 [M–C6H11O5–OH + Na]+

cycloastragenol

85

20

12

491.3 [M + H]+

143.0 [M–C22H36O3 + H]+

Calycosin-7-O-β-d-glucoside

104

24

8

446.9 [M + H]+

284.2 [M–C6H11O5 + H]+

Calycosin

117

33

17

285.0 [M + H]+

269.9 [M–CH3 + H]+

Ononin

111

28

15

431.0 [M + H]+

268.2 [M–C6H11O5 + H]+

Formononetin

80

50

17

269.0 [M + H]+

196.2 [M–OH–CO–OCH3 + H]+

Scutellarin

100

29

8

463.0 [M + H]+

287.0 [M–C6H8O6 + H]+

Fig. 2

Representative MRM chromatograms of a six tested analytes and IS standard; b seed sample (A1, B1—astragaloside IV; A2, B2—cycloastragenol; A3, B3—calycosin-7-O-β-d-glucoside; A4, B4—calycosin; A5, B5—ononin; A6, B6—formononetin; A7, B7—scutellarin)

Method validation

The typical equations of calibration curves, the linearity ranges, correlation coefficients and LLOQs for the six analytes are shown in Table 2. Calibration data for each analyte were obtained using the optimized UPLC-MS method. The response profile was determined and observed to be linear for each of the compounds within the linear ranges. All calibration curves exhibited good linearity with correlation coefficient (r) within the range of 0.999–0.9999. It was observed that the LLOQs were 1.25 ng/mL for astragaloside IV, calycosin-7-O-β-d-glucoside and ononin, 0.125 ng/mL for cycloastragenol, calycosin and formononetin. These LLOQs are sufficient for content detection of the six compounds.
Table 2

Regression equations, linear ranges, correlation coefficients, and LLOQ of the tested compounds

Analyte

Linear range

(ng/mL)

Calibration curves

r2

(n = 6)

LLOQ

(ng/mL)

Astragaloside IV

1.48–14,800

y = 0.4672x + 0.00004

0.9995

1.25

Cycloastragenol

0.148–1480

y = 0.1243x + 0.00002

0.9998

0.125

Calycosin-7-O-β-d-glucoside

1.48–14,800

y = 0.7575x + 0.0017

0.9999

1.25

Calycosin

0.148–1480

y = 4.836x + 0.038

0.9992

0.125

Ononin

1.48–14,800

y = 0.1166x + 0.0001

0.9994

1.25

Formononetin

0.148–1480

y = 3.0566x + 0.0042

0.9995

0.125

The intra- and inter-day precisions and accuracies of QC samples are presented in Table 3. The intra- and inter-day precisions (RSD) of these analytes were all less than 2.33 and 4.16 %, while the accuracy was within ±6.87 % for all the analytes. The values for accuracy and precision demonstrated that the method is reliable and reproducible.
Table 3

The intra-day and inter-day accuracies and precisions of the tested analytes (n = 6)

Analyte

Nominal concentration (ng/mL)

Intra-day(n = 6)

Inter-day (n = 6)

Observed concentration (ng/mL)

Precision (RSD%)

Accuracy (RE%)

Observed concentration (ng/mL)

Precision (RSD%)

Accuracy (RE%)

Astragaloside IV

74

73.1 ± 0.11

1.46

−1.22

73.8 ± 0.14

3.25

−0.25

740

737 ± 1.23

1.64

−0.47

746 ± 1.73

2.28

0.79

7400

7395 ± 13.2

1.78

0.34

7505 ± 31.2

4.16

4.24

Cycloastragenol

7.4

7.39 ± 0.11

1.43

0.52

7.47 ± 0.21

2.85

4.35

74

73.7 ± 1.21

1.64

−0.47

73.1 ± 1.31

1.72

−1.22

740

742.8 ± 6.63

0.89

0.79

726.3 ± 2.43

3.24

0.88

Calycosin-7-O-β-d-glucoside

74

73.7 ± 1.29

1.64

0.89

73.1 ± 1.3

1.72

−3.18

740

742.8 ± 6.67

0.89

0.79

726.3 ± 2.36

3.24

0.88

7400

7383 ± 14. 7

0.67

−0.89

7393 ± 18.18

2.46

0.59

Calycosin

7.4

7.40 ± 0.17

2.33

−0.02

7.26 ± 0.15

2.01

−1.89

74

74.37 ± 0.79

1.06

0.90

72.63 ± 2.48

3.41

0.88

740

740.83 ± 5.85

0.79

−0.56

738.50 ± 15.15

2.05

0.48

Ononin

74

73.1 ± 0.13

1.72

−0.88

74.4 ± 0.15

2.06

6.87

740

737 ± 1.2

1.64

−0.87

728 ± 1.7

2.40

−1.67

7400

7440 ± 6.7

0.90

0.95

7363 ± 2.01

2.73

2.27

Formononetin

7.4

7.37 ± 0.09

1.27

0.29

7.49 ± 0.21

2.81

4.59

74

73.7 ± 0.12

1.64

−1.14

73.4 ± 1.2

1.64

−0.77

740

741.3 ± 5.6

0.76

0.59

731.3 ± 1.97

2.70

1.57

The average extraction recoveries of the QC samples are presented in Table 4. Mean recoveries of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin, and formononetin were 96.46–99.64, 98.21–100.54, 99.2–99.33, 93.63–98.63, 98.64–99.57, and 95.39–97.55 % at three QC levels. The data indicated that the extraction recoveries of these analytes were efficient, consistent and reproducible. The mean matrix effect values of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin, and formononetin were 99.65–99.81, 99.96–102.70, 97.71–101.50, 95.33–98.89, 97.47–101.47, and 99.65–104.36 %, respectively. In addition, the RSD showed that there was no severe variation across all QC concentration levels. These results indicated that these analytes did not exhibit obvious matrix effect under this UPLC-MS condition.
Table 4

Mean extraction recoveries and matrix effects of the tested analytes (n = 6)

Analyte

Spiked concentration (ng/mL)

Recovery (%)

Matrix effect (%)

Mean ± SD

RSD

Mean ± SD

RSD

Astragaloside IV

74

99.64 ± 0.002

0.28

99.81 ± 0.004

0.49

740

99.61 ± 0.004

0.49

99.65 ± 0.012

1.27

7400

96.46 ± 0.066

6.91

99.71 ± 0.024

2.41

Cycloastragenol

7.4

100.54 ± 0.031

3.17

102.70 ± 0.09

8.80

74

98.21 ± 0.058

6.00

100.74 ± 0.063

6.25

740

100.13 ± 0.004

0.49

99.96 ± 0.011

1.18

Calycosin-7-O-β-d-glucoside

74

99.20 ± 0.016

1.68

97.71 ± 0.05

5.20

740

99.21 ± 0.01

1.10

98.53 ± 0.022

2.27

7400

99.33 ± 0.015

1.54

101.50 ± 0.028

2.76

Calycosin

7.4

98.63 ± 0.026

2.71

98.89 ± 0.018

1.88

74

97.17 ± 0.063

6.50

98.48 ± 0.042

4.31

740

93.63 ± 0.081

8.73

95.33 ± 0.111

11.64

Ononin

74

99.57 ± 0.006

0.69

100.69 ± 0.013

1.36

740

98.95 ± 0.017

1.77

101.47 ± 0.024

2.46

7400

98.64 ± 0.021

2.17

97.47 ± 0.052

5.37

Formononetin

7.4

95.49 ± 0.07

7.36

99.65 ± 0.059

5.97

74

97.55 ± 0.039

4.01

100.44 ± 0.007

0.78

740

95.39 ± 0.081

8.59

104.36 ± 0.068

6.57

The results of stability experiments were summarized in Table 5 and it’s indicate that these analytes were stable in the QC sample at room temp for 4 h, −20 °C for 2 weeks and three freeze–thaw cycles. the RSD were found between 0.35 and 2.05 % for astragaloside IV, between 0.59 and 4.07 % for cycloastragenol, between 0.68 and 2.75 % for calycosin-7-O-β-d-glucoside, between 0.9 and 3.79 % for calycosin, between 0.92 and 3.11 % for ononin, and between 0.38 and 1.47 % for formononetin. No significant degradation of these analytes was observed during the 4 h storage at room temp, −20 °C for 2 weeks and the three freeze–thaw cycles. The responses varied from 0.35 to 4.07 % at all tested concentrations.
Table 5

The stability of the tested analytes (n = 6)

Analyte

Nominal concentration (ng/mL)

Short-term

Freeze–thaw cycles

Long-term

25 °C for 4 h (RSD%)

Three freeze–thaw cycles (RSD%)

Frozen for 2 weeks (RSD%)

Astragaloside IV

74

0.35

1.21

2.05

740

0.47

1.17

0.89

7400

0.96

0.74

1.15

Cycloastragenol

7.4

0.59

2.1

0.78

74

1.47

0.99

1.04

740

0.85

4.07

1.54

Calycosin-7-O-β-d-glucoside

74

0.76

1.59

2.19

740

0.97

2.75

0.68

7400

2.41

1.34

1.3

Calycosin

7.4

1.02

3.52

2.11

74

0.9

1.57

2.54

740

1.53

3.79

1.15

Ononin

74

1.2

1.45

0.93

740

0.92

2.97

1.06

7400

2.36

3.11

2.35

Formononetin

7.4

1.14

0.82

1.47

74

0.88

1.09

0.38

740

0.56

1.18

1.02

Accumulation of active metabolites in different organs under salt stress

In the present study, the established UPLC-MS method has been successfully applied for the quantitative analysis studies of six bioactive compounds in A. mongholicus samples. Cycloastragenol and astragaloside IV are triterpene saponins; formononetin, ononin, calycosin and calycosin-7-O-β-d-glucoside are the major isoflavonoids in A. mongholicus. Representative MRM chromatograms of analytes standard (A) and samples (B) have shown in Fig. 2. Content of target compounds in root, stem and leaf under salt stress of different levels have shown in Table 6. Accumulation of target compounds in seedlings grown under control showed tissue specificity, Calycosin-7-O-β-d-glucoside, ononin, formononetin, astragaloside IV and cycloastragenol all have the highest content in root, which were 11,189.37, 1429.63, 6.78, 21,022.63 and 27.10 ng/g for fresh weight (Plant samples be excised and weighed immediately). Calycosin have the highest concentration in stem, which was 646.46 ng/g for fresh weight. Because of the numerous accumulation of the major bioactive metabolites in root, the root of A. mongholicus always been used as medicinal material. Significant variations in the content of 6 bioactive metabolites were observed after been processed by different levels of salinity in different part of plant (Fig. 3).
Table 6

Content of six active metabolites in different organs of A. mongholicus under different salinity levels

NaCl (mM)

Organs

Content of six active metabolites (ng/g, FW)

Calycosin-7-O-β-d-Glc

Calycosin

Ononin

Formononetin

Astragaloside IV

Cycloastragenol

0

Root

11,189.37

646.4608

1429.63

6.775

21,022.63

27.10247

Stem

783.368

999.7942

286.63

6.375

92.12638

18.92748

Leaf

87.168

13.79417

899.63

0.25

10.62637

24.85248

100

Root

3549.368

401.4608

3649.63

0.8525

34,497.63

17.72748

Stem

294.368

376.4608

455.63

0.26

163.8764

11.67747

Leaf

70.668

16.1275

1029.63

0.1925

131.1264

13.15247

300

Root

130.368

80.1275

156.63

0.16375

39,247.63

5.302475

Stem

181.368

233.1275

806.63

0.102

6297.626

5.827475

Leaf

57.468

1.6275

1509.63

0.06275

169.3764

6.752475

Fig. 3

Content of six active metabolites in different organs of A. mongholicus under different salinity levels. Mean values with the same letter in the single columns were not significantly different according to Duncan’s multiple comparison test with a family error rate of 0.05

In the triterpene saponins pathway, cycloastragenol is the synthetic precursor compound of astragaloside IV. Cycloastragenol is relatively uniformly distributed in roots, stems and leaves. As the main active ingredients in A. mongholicus, astragaloside IV has massive accumulation in roots. Under the action of salt stress, cycloastragenol accumulated in root, stem and leaves was decreased. This suggests the possibility of two aspects: one is the synthesis of cycloastragenol being inhibited by salt stress; the other is the cycloastragenol consumption because of its glycosylation process being promoted by salt stress; while, the accumulation of astragaloside IV was significantly increased in roots, stems and leaves. This might means that astragaloside IV could extenuate the salt damage for plant, and also reveal the possibility of cycloastragenol conversion to astragaloside IV via glycosylation under salt stress.

In the isoflavonoids pathway, calycosin is the synthetic precursor compound of Calycosin-7-O-β-d-glucoside, formononetin is the common synthetic precursor of calycosin and ononin. As the main active ingredients in A. mongholicus, calycosin-7-O-β-d-glucoside has massive accumulation in roots. The content of calycosin-7-O-β-d-glucoside and its synthetic precursors calycosin and formononetin were declined with salinity in root, stem and leaves. This might be the conversion from formononetin to calycosin being inhibited, and leading to decline of calycosin and calycosin-7-O-β-d-glucoside contents. Meanwhile, ononin, synthesized by formononetin via glycosylation, showed contents increment in roots, stems and leaves under salt stress. This reveals that conversion of formononetin to ononin via glycosylation process was promoted; while its hydroxylation process to calycosin was inhibited, decreasing the synthesis of calycosin and calycosin-7-O-β-d-glucoside. A probable mechanism was prompted, glycosylation process of flavonoids pathway was promoted under salt stress. Ononin, a compound of flavonoids and phenolics in A. mongholicus, was synthesized in large quantities by formononetin via glycosylation to play roles in adapt salt ambience. Flavonoids usually accumulate transiently, as a plastic response to biotic or abiotic stressors (Del Valle et al. 2015). Flavonoids suppress ROS (reactive oxygen species) levels in guard cells and thereby modulate the dynamics of stomatal aperture (Watkins et al. 2014). Plant phenolics, like other natural compounds, provide the plant with specific adaptations to changing environmental conditions and, therefore, they are essential for plant defense mechanisms (Caretto et al. 2015).

The results presented the response of major active metabolites in different organs of A. mongholicus to different salinity levels, and provided support for further exploration of the salt-tolerant mechanisms in A. mongholicus and its possibility of being the species that control the soil salinization.

Conclusions

A sensitive UPLC-MS method for the determination of astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin was developed, validated, and applied for their quantitative analysis in A. mongholicus. The described UPLC-MS method was sensitive, with high accuracy and a short run time of 9 min, and met all the requirements for effective plant extract analysis. With no reports on UPLC-MS methods for the quantitative determination of these six compounds, this is the first report of the simultaneous quantitative study of Astragaloside IV, cycloastragenol, calycosin-7-O-β-d-glucoside, calycosin, ononin and formononetin in different organs of A. mongholicus under different levels of salinity. The results provided basis and support for further exploration of the potential salt-tolerant species development. The method presented is valuable for providing a procedure for the future analysis of trace components in seedlings of A. mongholicus under salt stress.

Notes

Declarations

Authors’ contributions

LY, LJ and TZH designed the experiment; LY, LJ and WY conducted the field experiment and analyzed samples; LY, AA, and TZH finalized the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was financially supported by National Natural Science Foundation of China (31570520).

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Key Laboratory of Plant Ecology, Northeast Forestry University
(2)
Botany Department, Faculty of Science, Menoufia University

References

  1. AbdElgawad H, Zinta G, Hegab MM, Pandey R, Asard H, Abuelsoud W (2016) High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Front Plant Sci 7:276Google Scholar
  2. Boye A, Wu C, Jiang Y, Wang J, Wu J, Yang X, Yang Y (2015) Compound Astragalus and Salvia miltiorrhiza extracts modulate MAPK-regulated TGF-beta/Smad signaling in hepatocellular carcinoma by multi-target mechanism. J Ethnopharmacol 169:219–228View ArticleGoogle Scholar
  3. Bruce TJA, Matthes MC, Napier JA, Pickett JA (2007) Stressful “memories” of plants: evidence and possible mechanisms. Plant Sci 173:603–608View ArticleGoogle Scholar
  4. Caretto S, Linsalata V, Colella G, Mita G, Lattanzio V (2015) Carbon fluxes between primary metabolism and phenolic pathway in plant tissues under stress. Int J Mol Sci 16:26378–26394View ArticleGoogle Scholar
  5. Chen DS, Li YG, Zhou JC (2007) The symbiosis phenotype and expression patterns of five nodule-specific genes of Astragalus sinicus under ammonium and salt stress conditions. Plant Cell Rep 26:1421–1430View ArticleGoogle Scholar
  6. Chen Y, Song M, Wang Y, Xiong W, Zeng L, Zhang S, Xu M, Du H, Liu J, Wang D, Wu Y, Hu Y (2015) The anti-DHAV activities of Astragalus polysaccharide and its sulfate compared with those of BSRPS and its sulfate. Carbohydr Polym 117:339–345View ArticleGoogle Scholar
  7. Del Valle JC, Buide ML, Casimiro-Soriguer I, Whittall JB, Narbona E (2015) On flavonoid accumulation in different plant parts: variation patterns among individuals and populations in the shore campion (Silene littorea). Front Plant Sci 6:939Google Scholar
  8. Gong AG, Li N, Lau KM, Lee PS, Yan L, Xu ML, Lam CT, Kong AY, Lin HQ, Dong TT, Tsim KW (2015) Calycosin orchestrates the functions of Danggui Buxue Tang, a Chinese herbal decoction composing of Astragali Radix and Angelica sinensis Radix: an evaluation by using calycosin-knock out herbal extract. J Ethnopharmacol 168:150–157View ArticleGoogle Scholar
  9. Hasanuzzaman M, Nahar K, Alam MM, Bhowmik PC, Hossain MA, Rahman MM, Prasad MN, Ozturk M, Fujita M (2014) Potential use of halophytes to remediate saline soils. Biomed Res Int 2014:589341Google Scholar
  10. Krasteva I, Bratkov V, Bucar F, Kunert O, Kollroser M, Kondeva-Burdina M, Ionkova I (2015) Flavoalkaloids and flavonoids from Astragalus monspessulanus. J Nat Prod 78:2565–2571View ArticleGoogle Scholar
  11. Kwon H-J, Park Y-D (2012) Determination of astragalin and astragaloside content in Radix Astragali using high-performance liquid chromatography coupled with pulsed amperometric detection. J Chromatogr A 1232:212–217View ArticleGoogle Scholar
  12. Liu M, Li P, Zeng X, Wu H, Su W, He J (2015a) Identification and pharmacokinetics of multiple potential bioactive constituents after oral administration of radix astragali on cyclophosphamide-induced immunosuppression in Balb/c mice. Int J Mol Sci 16:5047–5071View ArticleGoogle Scholar
  13. Liu XH, Zhu RJ, Hu F, Guo L, Yang YL, Feng SL (2015b) Tissue distribution of six major bio-active components after oral administration of Zhenqi Fuzheng capsules to rats using ultra-pressure liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 986–987:44–53View ArticleGoogle Scholar
  14. Lv Y-W, Hu W, Wang Y-L, Huang L-F, He Y-B, Xie X-Z (2011) Identification and determination of flavonoids in Astragali Radix by high performance liquid chromatography coupled with DAD and ESI-MS detection. Molecules 16:2293–2303View ArticleGoogle Scholar
  15. Lv GP, Hu DJ, Cheong KL, Li ZY, Qing XM, Zhao J, Li SP (2015) Decoding glycome of Astragalus membranaceus based on pressurized liquid extraction, microwave-assisted hydrolysis and chromatographic analysis. J Chromatogr A 1409:19–29View ArticleGoogle Scholar
  16. Molinier J, Ries G, Zipfel C, Hohn B (2006) Transgeneration memory of stress in plants. Nature 442:1046–1049View ArticleGoogle Scholar
  17. Moore CV (1984) An economic analysis of plant improvement strategies for saline conditions. In: Staples RC, Toenniessen GH (eds) Salinity tolerance in plants, strategies for crop improvement. Wiley, New York, pp 381–397Google Scholar
  18. Napolitano A, Akay S, Mari A, Bedir E, Pizza C, Piacente S (2013) An analytical approach based on ESI-MS, LC-MS and PCA for the quali-quantitative analysis of cycloartane derivatives in Astragalus spp. J Pharm Biomed Anal 85:46–54View ArticleGoogle Scholar
  19. Park YJ, Thwe AA, Li X, Kim YJ, Kim JK, Arasu MV, Al-Dhabi NA, Park SU (2015) Triterpene and flavonoid biosynthesis and metabolic profiling of hairy roots, adventitious roots, and seedling roots of Astragalus membranaceus. J Agric Food Chem 63:8862–8869View ArticleGoogle Scholar
  20. Peng J, Li Y, Shi P, Chen X, Lin H, Zhao B (2011) The differential behavior of arbuscular mycrorrhizal fungi in interaction with Astragalus sinicus L. under salt stress. Mycorrhiza 21:27–33View ArticleGoogle Scholar
  21. Pu W, Wang D, Zhou D (2015) Structural characterization and evaluation of the antioxidant activity of phenolic compounds from Astragalus taipaishanensis and their structure–activity relationship. Sci Rep 5:13914View ArticleGoogle Scholar
  22. Qi L-W, Yu Q-T, Li P, Li S-L, Wang Y-X, Sheng L-H, Yi L (2006) Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors. J Chromatogr A 1134:162–169View ArticleGoogle Scholar
  23. Ren S, Zhang H, Mu Y, Sun M, Liu P (2013) Pharmacological effects of Astragaloside IV: a literature review. J Tradit Chin Med 33:413–416View ArticleGoogle Scholar
  24. Sun WY, Wei W, Gui SY, Wu L, Wang H (2008) Protective effect of extract from Paeonia lactiflora and Astragalus membranaceus against liver injury induced by Bacillus Calmette-Guérin and Lipopolysaccharide in Mice. Basic Clin Pharmacol Toxicol 103:143–149View ArticleGoogle Scholar
  25. Watkins JM, Hechler PJ, Muday GK (2014) Ethylene-induced flavonol accumulation in guard cells suppresses reactive oxygen species and moderates stomatal aperture. Plant Physiol 164:1707–1717View ArticleGoogle Scholar
  26. Wdowiak-Wrobel S, Leszcz A, Malek W (2013) Salt tolerance in Astragalus cicer microsymbionts: the role of glycine betaine in osmoprotection. Curr Microbiol 66:428–436View ArticleGoogle Scholar
  27. Wu T, Bligh SA, Gu L-H, Wang Z-T, Liu H-P, Cheng X-M, Branford-White CJ, Hu Z-B (2005) Simultaneous determination of six isoflavonoids in commercial Radix Astragali by HPLC-UV. Fitoterapia 76:157–165View ArticleGoogle Scholar
  28. Xiao HB, Krucker M, Albert K, Liang XM (2004) Determination and identification of isoflavonoids in Radix astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection. J Chromatogr A 1032:117–124View ArticleGoogle Scholar
  29. Xu RY, Nan P, Yang Y, Pan H, Zhou T, Chen J (2011) Ultraviolet irradiation induces accumulation of isoflavonoids and transcription of genes of enzymes involved in the calycosin-7-O-beta-d-glucoside pathway in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao. Physiol Plant 142:265–273View ArticleGoogle Scholar
  30. Yesilada E, Bedir E, Calis I, Takaishi Y, Ohmoto Y (2005) Effects of triterpene saponins from Astragalus species on in vitro cytokine release. J Ethnopharmacol 96:71–77View ArticleGoogle Scholar
  31. Zheng L, Wang M, Ibarra-Estrada E, Wu C, Wilson EG, Verpoorte R, Klinkhamer PG, Choi YH (2015) Investigation of chemomarkers of astragali radix of different ages and geographical origin by NMR profiling. Molecules 20:3389–3405View ArticleGoogle Scholar
  32. Zu Y, Yan M, Fu Y, Liu W, Zhang L, Gu C, Efferth T (2009) Determination and quantification of astragalosides in Radix Astragali and its medicinal products using LC-MS. J Sep Sci 32:517–525View ArticleGoogle Scholar

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© The Author(s) 2016