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Original article
2020
:14;
202101
doi:
10.1016/j.arabjc.2020.11.014

Diagnostic product ions-based chemical characterization and antioxidative activity evaluation of solid fermentation for Astragali radix produced by Paecilomyces cicadae

, , , , , , , ,
a School of Pharmacy, BIN ZHOU Medical University, 264003, China
b School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 102488, China
c Shandong Institute for Food and Drug Control, Jinan 250101, China
d Tongrentang Research Institute, Beijing 100079, China

⁎Corresponding authors. myweixia@126.com (Xia Wei), zhangjiayu0615@163.com (Jiayu Zhang)

Received: , Accepted: ,
© The Author(s) 2020
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Studies on herbal medicines and fermentation products have become increasingly essential with the development of modern industry and technology. In order to verify that fermentation can bring about changes, Paecilomyces cicadae [Paecilomyces cicadae (Miquel.) Samson] was used to ferment Astragali radix [Astragalus membranaceus (Fisch.) Bge. var. mongho-licus (Bge.) Hsiao]. After solid fermentation for Astragali radix produced by Paecilomyces cicadae (SF-AP) was established, an efficient strategy based on ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed to screen and identify the chemical transformations in SF-AP and Astragali radix according to the acquired diagnostic product ions (DPIs). As a result, 114 compounds including 45 saponins and 69 flavonoids were finally identified and validated. Moreover, two kinds of antioxidative tests corresponding to the scavenging of DPPH· and ABTS·+ were applied to evaluate the antioxidative activity of Astragali radix before and after fermentation. The results demonstrated that some significant chemical transformations such as relative content fluctuations and structural isomerism owing to the occurrence of hydrolysis and conversion reactions and the antioxidative activity of SF-AP was much higher than that of the Astragali radix. This study could provide a new method for the utilization of Astragali radix and constructive guidance for the further research of fermented herbal medicines.

Keywords

Solid fermentation for Astragali radix produced by Paecilomyces cicadae (SF-AP)
Chemical transformation
Diagnostic product ions (DPIs)
Antioxidative activity
Ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS)
1

1 Introduction

Microbial fermentation has already been applied in the processing of herbal medicines and functional food for thousands of years, such as Banxiaqu (pinellia ternata fermented mass) and Dandouchi (sojae semen praeparatum). Previous studies have demonstrated that fermentation plays an important role in toxicity reducing and efficacy enhancing (Ming et al., 2017). The prime reason was that microorganisms could generate sorts of important secondary metabolic products, and the macromolecular constituents could be decomposed into small molecules during the process (Hussain et al., 2016; Stanton et al., 2005; Xu et al., 2015).

As annual or perennial herb or shrub that is prevalently distributed in temperate and arid areas, Astragali radix [Astragalus membranaceus (Fisch.) Bge. var. mongho-licus (Bge.) Hsiao] belonging to the popular genera of plants in Leguminosae, has been widely used in herbal medicines for over 2,000 years. It contains saponins, flavonoids, etc, which are known for their anti-inflammatory, anti-oxidant, and other pharmacological effects (Fu et al., 2015). It is commonly used as food and beverage additive and nutritional dietary supplement to enhance the body's resistance against various diseases in numerous Asian countries. In terms of Paecilomyces cicadae [Paecilomyces cicadae (Miquel.) Samson], one kind of fungus owing high nutritional and medical value is formed by paecilomyces parasitizing the nymphs of cicadas. Modern pharmacological studies have showed that it had similar clinical effects to Cordyceps just like regulating immunity, improving the kidney function, strengthening with tonics, anti-oxidation, anti-tumor and anti-virus, etc (Zhao et al., 2018a,2018b; Zhang et al., 2017).

In the preliminary report, and as part of our long-term investigation for fermentation, we have described the chemical constituent profiling and lowering uric acid activity of Paecilomyces cicadae liquid fermentation for Astragli Radix (Wang et al., 2019a, Wang et al., 2019b). Compared with the previous study of Paecilomyces cicadae liquid fermentation for Astragli Radix, we found that the distinguishment between solid fermentation and liquid fermentation lies in the difference of medium state. The concept of solid fermentation covers a wide range, including the fermentation mode of suspending insoluble solid substances in liquid (also known as carrier culture) and cultivating microorganisms on wet solid materials with almost no flowable water. There are a great many advantages of solid fermentation, such as simple operation, low energy consumption, easy domination, less pollution, and so on. Nowadays, modern fermentation technology has been gradually changed from traditional natural fermentation that relying on production experience to pure strain fermentation, which represents fermentation technology and system are becoming increasingly mature (Martins et al., 2011; Singhania et al., 2009; Wang et al., 2016; Liu et al., 2004).

In order to prove that the fermentation process can some cause favorable chemical changes, an ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) method coupled with the assistance of diagnostic product ions (DPIs) analysis was developed to characterize the chemical transformation and further obtain a comprehensive knowledge about constituents in the established SF-AP system. Meanwhile, two antioxidative tests including DPPH· scavenging activity and ABTS·+ scavenging activity were utilized to evaluate the antioxidative effects of Astragali radix before and after solid fermentation.

2

2 Experimental

2.1

2.1 Chemicals and materials

The identity of Astragali radix was authenticated by histological and morphological methods according to monograph of Chinese Pharmacopoeia (version 2015) by Prof. Long Dai in BIN ZHOU Medical University (Yanai city, Shandong). Paecilomyces cicadae (Miquel) Samson (No. cfcc81169) was provided by China Forestry Culture Collection Center (Beijing, China). A total of thirteen reference substances including six triterpene saponins, i.e. β-D-Glucopyranoside,(3β, 6α, 16β, 20R, 24S)-3-[(3, 4-di-O-acetyl-β-D-xylopyranosyl)oxy]-20, 24-epoxy-16, 25-dihydroxy-9, 19-cyclolanostan-6-yl, Astragaloside I, Astragaloside II, Astragaloside IV, Isoastragaloside I, Isoastragaloside II and seven flavonoids, i.e. Calycosin, Genistin, Complanaruside, Formononetin, Ononin, Astraisoflavan-7-O-β-D-glucoside and Isoquercitrin, were all purchased from Chengdu Must Biotechnology Co. Ltd. (Sichuan, China). The structures were fully elucidated by comparing the ESI-MS, 1H NMR and 13C NMR spectra data with the published literature. All of their purities were acceptable (≥98%) according to HPLC-UV analysis.

Acetonitrile, methanol and formic acid of LC-MS grade were all purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA). 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1, 1-diphenyl-2-picrylhydrazyl (DPPH), Potassium persulfate (K2S2O4) were obtained from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). All the other chemicals of analytical grade were provided by Beijing Chemical Works (Beijing, China). Deionized water used throughout the experiment was purified by Milli-Q Gradient Å 10 System (Millipore, Billerica, MA, USA). Grace PureTM SPE C18-Low solid-phase extraction cartridges (200 mg/3 mL, 59 μm, 70 Å) were purchased from Grace Davison Discovery Science (Deerfield, IL, USA).

2.2

2.2 The preparation of SF-AP system

2.2.1

2.2.1 Fungus activation and liquid culture

Paecilomyces cicadae was inoculated on potato liquid medium in 500 mL conical flask, and then it was cultured in an incubator with constant temperature and humidity to activate it by setting parameters at 27℃ and relative humidity of 80% for 5 days. Activated Paecilomyces cicadae were selected by inoculation ring and cultured in potato liquid medium at 25℃ and 140 r/min for 7 days.

2.2.2

2.2.2 Solid-state fermentation

The powder (5 g) of Astragali radix was placed in 250 mL conical bottle and then soaked with 6 mL distilled water. After that, the conical bottle loaded with wet medicinal powder was sterilized at 121 ℃ for 30 min. 3 mL liquid spawn of activated Paecilomyces cicadae was inoculated and cultured in a solid fermentation flask with constant temperature of 26 ℃ and humidity of 90%. Astragali radix were ground into powder passing with 100 mesh sieve and cultured for 14 days at 26 ℃ under aerobic conditions.

2.3

2.3 Analytical sample preparation

SF-AP samples were taken on the 14th day for the subsequent analyses. Then they were ground into powder passing 100 mesh sieve. Furthermore, the above two powder samples were respectively dissolved in methanol at a concentration of 100 mg/mL. Samples were ultrasonic extracted for 35 min and then the solutions were evaporated. After concentration, the initial mobile phase was used to resolve these two samples.

SF-AP (1 mL) and Astragali radix (1 mL) solution was respectively added into the SPE cartridges, which were orderly pretreated with 5 mL methanol and 5 mL deionized water. Afterwards, the SPE cartridges were successively washed with 3 mL deionized water and 3 mL methanol. The methanol eluate was evaporated to dryness by water bath. Then the residue was redissolved in 200 µL methanol solution and centrifuged for 30 min (13,500 rpm, 4 ℃). The supernatant was finally used for the subsequent analysis.

2.4

2.4 Instrument and conditions

UHPLC analysis was performed on DIONEX Ultimate 3000 UHPLC system (Thermo Fisher Scientific, MA, USA), which was equipped with a binary pump, an auto-sampler and a column compartment. The chromatographic separation was carried out at 40 ℃ using Waters ACQUITY HSS T3 column (2.1 × 100 mm i.d., 1.8 μm; Waters Corporation, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B) at a flow rate of 0.2 mL/min. The linear gradient procedure was described as follows: 0–6 min, 8%–25% B; 6–13 min, 25%–32% B; 13–20.5 min, 32%–48% B; 20.5–26 min, 38%–44% B; 26–30 min, 44%–92% B. The injection volume was 3 μL.

HRMS spectral analysis was executed on LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, MA, USA). The optimized operating parameters in negative and positive ion modes were set as follows: sheath gas flow rate of 40 arb, auxiliary gas flow rate of 20 arb, capillary voltage of ± 25 V, electrospray voltage of 3.0 kV, tube lens of ± 110 V, and capillary temperature of 350 °C. The components were detected using full-scan MS analysis from m/z 100–1,200 with a resolution of 30,000 in both positive and negative ion modes. The collision energy for collision induced dissociation (CID) was set to 40%.

2.5

2.5 Peak selections and data processing

Thermo Xcalibur 2.1 workstation (Thermo Fisher Scientific, MA, USA) was used for data acquisition and processing. In order to acquire as many fragment ions as possible, this method targeted the peaks with intensity over 10,000 for the subsequent structural identification. The predicted atoms for chemical formulas of all the deprotonated molecular ions were set as follows: C [0–50], H [0–100], O [0–30], N [0–1] and Ring Double Bond (RDB) equivalent value [0–15]. The maximum mass errors between the measured and calculated values were fixed within ± 5 ppm.

2.6

2.6 Determination of antioxidative capacity in vitro

2.6.1

2.6.1 Sample preparation

The two powder samples (Astragali radix and SF-AP), each weighting 3 g, were respectively suspended in 50 mL of 80% methanol solution. Each sample was sonicated for 45 min and centrifuged at 5,000 rpm for 10 min. Then the supernatant fraction was filtrated to obtain extraction solution. Subsequently, the two kinds of extraction solutions were diluted to different concentrations for the determination of antioxidative activity in vitro.

2.6.2

2.6.2 DPPH· scavenging activity assay

DPPH· solution (0.5 mmol/L) was prepared and then 1 mL was respectively added into various Astragali radix and SF-AP concentrations (1 mL). The mixed solutions were incubated at 25 °C for 30 min and protected from light. Finally, the absorbances of these sample solutions were measured at 517 nm (Zeng et al., 2012). DPPH· scavenging activity was calculated as shown in formula (1).

(1)
Scavenging of DPPH · = 1 - A 1 / A 0 × 100 %

(A0 was the absorbance of DPPH and methanol solution; A1 was the absorbance of DPPH and sample solution)

2.6.3

2.6.3 ABTS·+ scavenging activity assay

An ABTS·+ stock solution was prepared by mixing 7 mmol/L ABTS with 2.45 mmol/L K2S2O4 in water, which was placed in the dark at room temperature for 16 h to obtain a dark blue solution (Hsu et al., 2011). The ABTS·+ stock solution should be diluted with absolute ethanol before the experiment.

Two kinds of sample solutions were diluted in different concentrations, and then 1 mL of these samples were respectively taken into the ABTS·+ solution (2 mL). After the reaction lasted for 6 min at room temperature, the absorbances of these sample solutions were determined at 734 nm. ABTS·+ scavenging activity was calculated as shown in formula (2).

(2)
Scavenging of ABTS · + = 1 - A 1 / A 0 × 100 %

(A0 was the absorbance of ABTS and methanol solution; A1 was the absorbance of ABTS and sample solution.)

3

3 Results and discussion

3.1

3.1 The establishment of analytical strategy

In the present study, the analytical strategy we established included five steps. The first step was to establish the solid-state fermentation system by activating fungus and culturing liquid. Secondly, the samples of SF-AP and Astragali radix were respectively prepared into two solutions and then pretreated by SPE cartridge for the subsequent analytical experiments. Thirdly, a sensitive and validated method based on UHPLC-LTQ-Orbitrap mass spectrometer was developed for the comprehensive analysis of chemical constituents in SF-AP and Astragali radix. The structures of the representative constituents were elucidated according to the accurate mass measurement, fragmentation patterns, DPIs and literature reports. Fourthly, the chemical transformations and relative content fluctuations were compared with each other to clarify the material basis transformations brought about by solid-state fermentation. Finally, based on the summarized chemical transformations, the antioxidant activities of SF-AP and Astragali radix were also evaluated. The general procedures of the strategy were summarized into a diagram in Fig. 1.

The summary diagram of analytical strategy and methodology.
Fig. 1
The summary diagram of analytical strategy and methodology.

3.2

3.2 The characterization of chemical constituents

Structural elucidation was performed on the basis of chromatographic retention behaviors, accurate mass measurements, mass fragmentation patterns, DPIs and previous relevant literature. It should be noted that DPIs were significant to rapidly perform the structural elucidation, which were produced in by the comparable fragmentation patterns of the constituents with similar backbone (Zhao et al., 2018a,2018b). Finally, a total of 114 chemical constituents including 45 triterpene saponins (Table 1) and 69 flavonoids (Table 2) were accurately or tentatively characterized.

Table 1 Identification of saponins in Astragli radix and SF-AP.
Peak tR/min Ion mode Formula Theoretical Mass m/z Experimental Mass m/z Error (ppm) MS/MS fragment ions Identification A S
A1 7.57 P C48H79O18 943.52664 943.52582 −0.288 MS2[9 4 3]:925(1 0 0),927(76),1399(37),486(30),859(13),927(13),845(10),827(6) Soyasaponin I/isomer + +
A2 9.32 P C43H71O15 827.47875 827.47443 −4.218 MS2[8 2 7]:709(1 0 0),809(10),691(9),768(4),737(2),695(2),577(2),335(2),467(1) Astragaloside II isomer + +
A3 9.80 P C38H63O11 695.43704 695.43274 −4.391 MS2[6 9 5]:577(1 0 0),499(35),677(25),514(10),559(7),605(4),532(4),199(2) Mongholicoside II isomer + +
A4 10.38 P C38H63O11 695.43704 695.43274 −4.391 MS2[6 9 5]:577(1 0 0),677(12),519(9),499(6),559(6),636(4),578(4),605(3) Mongholicoside II + +
A5* 10.75 N C47H77O19 945.50700 945.50916 3.023 MS2[9 4 5]:783(1 0 0),489(3),621(2),765(1),651(1) Astragaloside V + +
P C47H79O19 947.52155 947.52026 −0.788 MS2[9 4 7]:437(1 0 0),455(58),419(38),587(21),785(16),473(14),599(11),535(11),738(10),472(10),277(10)
A6 11.51 P C49H81O18 957.54229 957.54187 0.134 MS2[9 5 7]:776(1 0 0),777(22),794(19),644(12),335(9),795(4),336(3),643(2),645(2),353(1) Azukisaponin V methyl ester – +
A7 11.61 N C41H69O14 785.46983 785.47198 4.834 MS2[7 8 5]:491(1 0 0),623(26),415(16),740(13),767(11),367(10),489(8) Cyclocanthoside E/isomer + +
A8 11.77 N C41H69O14 785.46983 785.46277 −4.892 MS2[7 8 5]:491(1 0 0),829(55),830(26),767(24),653(21) Cyclocanthoside E/isomer + +
A9* 11.79 N C41H67O14 783.45363 783.45612 1.578 MS2[7 8 3]:489(1 0 0),621(46),651(36),383(15),737(12),453(11),646(11),515(8),471(6) Isoastragaloside IV + +
A10 12.38 N C42H69O15 813.46474 813.46729 4.154 MS2[8 1 3]:767(1 0 0),745(78),652(47),651(30),489(30),633(27),795(26) Astramembranoside A + +
P C42H71O15 815.47930 815.47729 −1.788 MS2[8 1 5]:554(1 0 0),711(98),276(97),252(96),250(94),505(93),315(92),806(89)
A11 12.46 N C41H69O14 785.46983 785.47180 4.605 MS2[7 8 5]:491(1 0 0),623(24),767(13),741(4),653(4),701(3) Cyclocanthoside E/isomer + +
A12 12.51 P C49H81O20 989.53212 989.53296 1.404 MS2[9 8 9]:503(1 0 0),827(99),599(96),483(95),330(94),584(94),344(93),452(93),603(92) Agroastragaloside IV + +
A13 12.59 N C43H69O15 825.46419 825.46735 4.151 MS2[8 2 5]:765(1 0 0),783(45),757(17),787(12),779(11),788(5),673(5),401(4) Astragaloside II isomer + +
A14 13.19 N C36H61O11 669.42248 669.42383 4.468 MS2[6 6 9]:601(1 0 0),632(62),654(55),623(50),436(42),541(41),651(21) Mongholicoside A + +
A15 13.23 P C40H65O12 737.44760 737.44507 −2.690 MS2[7 3 7]:557(1 0 0),691(23),689(20),511(19),577(18),509(12),673(12),493(11),571(8),605(7),677(7),529(6),475(4) Huangqiyenin F – +
A16 13.79 N C43H71O15 827.48039 827.48138 3.181 MS2[8 2 7]:759(1 0 0),767(39),783(36),757(34),781(33),809(24),785(22),770(20) Agroastragaloside II isomer + +
A17# 13.95 P C43H71O15 827.47875 827.47742 −1.605 MS2[8 2 7]:639(1 0 0),558(98),232(94),443(94),640(91),294(90),579(90),231(89),295(83),371(83) Isoastragaloside II + +
N C43H69O15 825.46419 825.46710 2.849 MS2[8 2 5]:765(1 0 0),783(63),644(31),762(19)
A18# 14.31 P C41H69O14 785.46818 785.46722 −1.226 MS2[7 8 5]:782(1 0 0),720(78),237(74),575(74),526(73),434(72),480(72),248(70) Astragaloside IV + +
N C41H67O14 783.45363 783.45813 2.144 MS2[7 8 3]:621(1 0 0),489(50),490(31),651(23),708(23),553(18),700(17),471(14),
A19 14.56 N C41H67O14 783.45363 783.45654 4.115 MS2[7 8 3]:489(1 0 0),383(13),651(12),453(4),401(2),471(2),381(2),760(2) Astragaloside III + +
A20 14.56 N C41H69O14 785.46983 785.46246 −4.286 MS2[7 8 5]:491(1 0 0),489(53),385(15),383(11),491(11),622(11),718(5) Isoastragaloside IV isomer + +
A21 16.01 P C36H59O10 651.41082 651.40857 −2.616 MS2[6 5 1]:177(1 0 0),199(62),269(44),234(42),180(38),574(37),663(37),379(36),229(35),300(32) Huangqiyenin A – +
A22 16.10 N C51H81O21 1029.52758 1029.52173 −4.619 MS2[1029]:985(1 0 0),984(18),967(2) Agroastragaloside III + +
P C51H83O21 1031.54214 1031.54199 −0.141 MS2[1031]:984(1 0 0),494(57),558(52),331(50),667(49),936(48),323(47),482(46),300(45)
A23 16.23 N C47H73O17 909.48532 909.48804 4.192 MS2[9 0 9]:891(1 0 0),613(99),523(80),453(76),849(61),569(58),435(36),746(25),495(18) Acetylastragaloside I/isomer + +
A24 16.29 N C48H77O18 941.51209 941.50549 −4.259 MS2[9 4 1]:922(1 0 0),524(56),873(36),923(32),615(27),523(26),879(20),456(18), Soyasaponin I/isomer + +
A25 16.36 P C42H67O14 795.45308 795.45203 −0.632 MS2[7 9 5]:421(1 0 0),597(86),214(81),295(74),429(74),512(72),233(72),625(71) Huangqiyenin E – +
A26 16.71 N C43H69O15 825.46419 825.46796 4.890 MS2[8 2 5]:765(1 0 0),633(30),744(18),634(17),736(11),717(9),536(8),703(7) Astragaloside II isomer + +
P C43H71O15 827.47875 827.47729 −1.762 MS2[8 2 7]:269(1 0 0),592(67),629(66),351(64),296(63),632(60),709(60),247(59),277(57)
A27 16.74 N C36H59O11 667.40683 667.40820 4.512 MS2[6 6 7]:649(1 0 0),449(82),623(81),299(80),450(74),485(54) Mongholicoside B + +
A28 16.84 N C48H77O18 941.51209 941.51392 3.694 MS2[9 4 1]:923(1 0 0),525(73),615(51),744(49),879(41),457(40),795(37),437(35),597(16) Soyasaponin I/isomer + +
P C48H79O18 943.52664 943.52496 −1.200 MS2[9 4 3]:599(1 0 0),797(88),441(79),423(48),617(28),581(23),520(10),269(8),454(8),867(8),448(8)
A29# 16.91 N C43H69O15 825.46419 825.46631 3.892 MS2[8 2 5]:783(1 0 0),765(49),633(24),795(10),697(9),758(7) Astragaloside II + +
A30 17.25 N C43H71O15 827.48039 827.48267 4.740 MS2[8 2 7]:809(1 0 0),757(69),781(40),758(38),769(25),783(20),767(19) Agroastragaloside II + +
A31 17.53 N C42H69O15 813.46474 813.46686 4.625 MS2[8 1 3]:725(1 0 0),455(43),633(30),651(29),767(28),523(25),407(22),795(21) Astramembranoside A + +
A32 18.80 N C42H65O14 793.43853 793.44080 4.937 MS2[7 9 3]:631(1 0 0),775(24),663(8),724(7),747(5),718(5),697(4) Huangqiyenin E/isomer + +
A33 18.83 N C42H69O15 813.46474 813.46692 4.699 MS2[8 1 3]:745(1 0 0),767(36),489(20),729(18),726(15),651(14),305(9) Astramembranoside A + +
A34# 18.94 N C45H71O16 867.47476 867.47809 3.104 MS2[8 6 7]:807(1 0 0),821(63),765(53),783(22),849(21),687(17) Isoastragaloside I + +
A35 19.16 N C47H73O17 909.48532 909.48846 4.654 MS2[9 0 9]:891(1 0 0),849(48),763(47),453(46),569(29),523(27),613(19),407(16) Acetylastragaloside I/isomer + +
A36 19.21 N C48H77O18 941.51209 941.50427 −4.555 MS2[9 4 1]:922(1 0 0),524(44),879(37),614(36),523(36),613(32),732(31) Soyasaponin I + +
A37 19.27 P C42H67O14 795.45308 795.45209 −0.557 MS2[7 9 5]:439(1 0 0),597(89),421(44),600(43),528(36),253(35),299(33),245(31) Huangqiyenin E + +
A38 19.37 N C45H71O16 867.47476 867.47766 4.608 MS2[8 6 7]:821(1 0 0),799(34),731(23),717(16),343(15),787(11),831(8) Astragaloside I isomer + +
A39 20.15 N C42H65O14 793.43853 793.44073 4.849 MS2[7 9 3]:725(1 0 0),455(43),631(30),663(29),747(28),775(21),689(20),279(19),636(14),588(13),753(11) Huangqiyenin E/isomer + +
A40 20.20 P C36H63O11 671.43704 671.43341 −4.586 MS2[6 7 1]:479(1 0 0),461(33),478(8),443(8),611(8),653(6),177(4),417(3),460(3),199(2) Mongholicoside A + +
A41# 20.37 N C45H71O16 867.47476 867.47662 2.410 MS2[8 6 7]:849(1 0 0),799(85),783(81),821(77),747(39),687(33) Astragaloside I + +
A42# 20.92 N C45H71O16 867.47476 867.47943 3.684 MS2[8 6 7]:703(1 0 0),747(80),599(73),821(53),783(44),799(35),807(32),687(24) β-D-Glucopyranoside,(3β,6α,16β,20R,24 s)-3-[(3,4-di-O-acetyl-β-D-xylopyranosyl)oxy]-20,24-epoxy-16,25-dihydroxy-9,19-cyclolanostan-6-yl + +
A43 22.12 N C45H73O16 869.49096 869.49335 4.644 MS2[8 6 9]:823(1 0 0),851(46),599(18),767(15),536(11),809(10),749(8),705(8) Agroastragaloside I + +
A44 22.76 N C47H73O17 909.48532 909.48846 4.654 MS2[9 0 9]:849(1 0 0),867(27),711(10),453(8),891(7),803(2) Acetylastragaloside I/isomer + +
A45 22.77 P C48H75O19 955.48853 955.48853 −1.231 MS2[9 5 5]:742(1 0 0),1884(96),478(96),406(95),864(95),561(94),701(94),567(94),919(91) Malonylastragaloside I + +
N C48H73O19 953.47570 953.47968 4.898 MS2[9 5 3]:935(1 0 0),5379(67),627(40),891(23),469(22),907(15),849(3),807(14)

#: Unambiguously identification by comparing with the reference substances; *: Structural validation by using the reference substances.

+: detected; -: undetected; A: Astragli radix; S: SF-AP.

Table 2 Identification of flavanoids in Astragli radix and SF-AP.
Peak tR/min Ion mode Formula Theoretical Mass m/z Experimental Mass m/z Error (ppm) MS/MS fragment ions Identification A S
B1 1.24 P C23H27O10 463.16042 463.15952 −0.763 MS2[4 6 3]:268(1 0 0),330(13),398(8),365(3),398(3),453(3),136(3) Astraisoflavan-7-O-β-D-glucoside/isomer – +
B2# 1.54 P C21H21O12 465.10330 465.09921 −4.617 MS2[4 6 5]:429(1 0 0),303(59),398(23),314(23),285(21),363(18),199(17),366(17) Isoquercitrin + +
B3 3.71 N C23H23O11 475.12513 475.12048 −4.331 MS2[4 7 5]:257(1 0 0),275(92),437(65),179(46),180(45),276(42),419(39),438(38),457(26),283(16) Odoratin-7-O-β-D-glucoside/isomer + +
B4 3.98 P C23H27O10 463.16042 463.15796 −4.131 MS2[4 6 3]:205(1 0 0),415(93),266(68),267(36),378(33),433(32),301(27) Astraisoflavan-7-O-β-D-glucoside/isomer + +
B5 4.37 N C29H37O16 641.20926 641.21063 4.708 MS2[6 4 1]:479(1 0 0),317(75),595(35),611(30),623(26),379(24),610(22) 5′-hydroxy isomucronulatol 2′,5′-di-O-glucoside + +
B6 4.37 P C24H25O12 505.13460 505.13318 −1.727 MS2[5 0 5]:333(1 0 0),335(41),306(33),373(26),438(21),281(21),343(13),282(11),317(9),181(7),487(6) Neocomplanoside/isomer + +
B7 4.76 N C28H31O16 623.16231 623.16388 4.165 MS2[6 2 3]:299(1 0 0),284(31),604(7),283(6),461(6),605(5),415(5),577(4) Complanatuside isomer + +
B8 4.86 P C23H29O10 465.17607 465.17184 −4.919 MS2[4 6 5]:303(1 0 0)446(6),429(6),432(5),302(2),346(1),301(1) Astraisoflavan-7-O-β-D-glucoside/isomer + +
B9# 5.23 N C28H31O16 623.16231 623.16364 3.780 MS2[6 2 3]:299(1 0 0),284(32),461(10),240(4),461(3),577(2),605(2),211(2),239(2) Complanatuside + +
B10 5.37 N C22H21O11 461.10948 461.11050 4.773 MS2[4 6 1]:299(1 0 0),284(9) Kaempferol- 4′- methylether-3-D-glucoside + +
P C22H23O11 463.12404 463.12265 −1.809 MS2[4 6 3]:445(1 0 0),371(29),253(19),285(19),344(4),401(3),301(3)
B11 5.53 P C16H17O5 289.10760 289.10645 −2.076 MS2[2 8 9]:271(1 0 0),205(91),270(68),233(41),207(16),261(15),231(13),247(10),163(8),219(7),184(7),177(6),229(5),213(5) (3R)-7,2′,3′-Trihydroxy-4′-methoxy isoflavonone/isomer + –
B12 5.92 P C16H13O5 285.07630 285.07529 −1.614 MS2[2 8 5]:270(1 0 0),253(43),225(19),137(8),229(7),257(3),181(2),271(1) Calycosin isomer + -+
N C16H11O5 283.06175 283.06198 4.642 MS2[2 8 3]:268(1 0 0),269(3),255(1)
B13 6.19 N C22H21O12 477.10440 477.10532 4.382 MS2[4 7 7]:315(1 0 0),301(18),300(14),347(13),431(11),459(5),297(4) Isorhamnetin-3-O-β-D-glucoside + +
B14 6.19 P C22H23O10 447.12912 447.12695 −3.630 MS2[4 4 7]:300(1 0 0),283(19),255(7),167(5),301(5),259(4),138(3),269(3),168(2),297(1) Calycosin-7-O-β-D-glucoside isomer
B15 6.19 N C24H23O12 503.12005 503.12112 4.401 MS2[5 0 3]:299(1 0 0),284(23),443(4),467(2),488(1),240(1) Neocomplanoside/isomer + +
B16 6.21 P C16H13O5 285.07630 285.07526 −1.719 MS2[2 8 5]:270(1 0 0),253(42),225(18),137(8),229(6),271(4),257(3),181(2) Calycosin isomer + +
N C16H11O5 283.06175 283.06180 4.006 MS2[2 8 3]:268(1 0 0),269(5),239(2),265(1),255(1)
B17 6.34 P C17H15O6 315.08686 315.08603 −0.903 MS2[3 1 5]:300(1 0 0),283(20),255(8),167(5),259(4),301(2),287(2),175(2) 7,3′-dihydroxy-8,4-dimethoxyisoflavone isomer + +
B18 6.34 P C23H25O11 477.13969 477.13779 −2.825 MS2[4 7 7]:458(1 0 0),356(98),398(41),361(26),305(14),459(11),289(8),357(7),445(7),333(7),287(6),272(5),169(5) Odoratin-7-O-β-D-glucoside isomer + +
B19 6.35 N C17H13O6 313.07231 313.07236 4.415 MS2[3 1 3]:298(1 0 0),285(2),295(1),269(1),283(1) 7,3′-Dihydroxy-8,4-dimethoxyisoflavone/isomer + +
B20 6.72 N C15H9O5 269.04610 269.04605 4.947 MS2[2 6 9]:225(1 0 0),241(36),197(23),181(22),236(16),226(11),183(11),251(9),213(9),201(8),254(8) 5,7,4′-trihydroxy- isoflavonone/isomer + +
B21 7.00 N C16H11O5 283.06175 283.06192 4.430 MS2[2 8 3]:268(1 0 0),269(3),265(1),239(1) Calycosin isomer + +
P C16H13O5 285.07630 285.07529 −1.614 MS2[2 8 5]:270(1 0 0),253(43),225(20),285(17),137(9),229(7),286(4),257(3),181(2)
B22 7.01 P C16H17O5 289.10760 289.10651 −1.868 MS2[2 8 9]:270(1 0 0),271(22),184(8),252(8),166(7),205(4),182(2) (3R)-7,2′,3′-trihydroxy-4′-methoxy isoflavonone/isomer + –
B23* 7.10 N C22H21O10 445.11457 445.11575 3.351 MS2[4 4 5]:283(1 0 0),268(17),255(9) Calycosin-7-O-β-D-glucoside + +
B24 7.19 N C17H13O6 313.07231 313.07230 4.224 MS2[3 1 3]:298(1 0 0),181(17),245(8),137(6),295(6),269(5),285(5),194(3) 7,3′-dihydroxy-8,4-dimethoxyisoflavone/isomer + +
B25# 7.25 N C21H19O10 431.09892 431.09961 2.421 MS2[4 3 1]:268(1 0 0),269(48),311(8),162(6), Genistin + +
B26 7.30 N C24H23O11 487.12513 487.12631 4.793 MS2[4 8 7]:193(1 0 0),178(15),161(13),179(11),323(10),163(8),355(5),203(5),293(4) Calycosin-7-O-β-D-glucoside-6″-O-acetate/isomer + +
B27 7.35 N C16H11O5 283.06175 283.06189 4.324 MS2[2 8 3]:268(1 0 0),269(1) Calycosin isomer + +
B28 7.37 P C16H13O5 285.07630 285.07571 −0.140 MS2[2 8 5]:270(1 0 0),253(43),225(20),255(14),137(8),229(7),268(5),257(3),181(2),197(1) Calycosin isomer
B29 7.39 P C17H15O6 315.08686 315.08575 −1.792 MS2[3 1 5]:300(1 0 0),283(19),255(9),269(8),297(5),167(5),259(4),138(3) Kumatakenin + +
B30 7.52 N C23H27O10 463.16152 463.16220 4.023 MS2[4 6 3]:301(1 0 0),283(40),273(37),191(36),341(11),176(9),268(3) Astraisoflavan-7-O-β-D-glucoside/isomer + +
B31 7.69 P C15H11O5 271.06065 271.05978 −1.180 MS2[2 7 1]:151(1 0 0),250(78),251(12),66(8),252(7),215(7),153(6),243(5),256(5),137(4),253(4) 5,7,4′-trihydroxy- isoflavonone/isomer + –
B32 7.70 N C24H23O11 487.12513 487.12631 4.793 MS2[4 8 7]:283(1 0 0),268(50),427(14),193(11),419(10),253(3) Calycosin-7-O-β-D-glucoside-6″-O-acetate + +
B33 7.88 N C16H11O4 267.06683 267.06693 4.533 MS2[2 6 7]:252(1 0 0),253(5),249(2) Formononetin isomer + +
P C16H13O4 269.08138 269.08051 −1.209 MS2[2 6 9]:254(1 0 0),237(51),213(35),253(13),107(9),118(6),241(6),136(5)
B34 7.89 N C15H9O5 269.04610 269.04617 4.393 MS2[2 6 9]:225(1 0 0),254(88),241(78),201(64),181(53),197(43),180(38),223(30) 5,7,4′-trihydroxy- isoflavonone/isomer + +
P C15H11O5 271.06065 271.05972 −1.402 MS2[2 7 1]:243(1 0 0),153(87),215(85),239(50),66(41),149(36),253(34),211(30),221(25),159(16),199(14)
B35 7.93 N C29H37O15 625.21434 625.21527 4.116 MS2[6 2 5]:301(1 0 0),463(9),286(4),445(3),607(2),271(2),473(1) Isomucronulatol-7,2′-di-O-glucoside + +
B36 7.99 P C17H17O5 301.10760 301.10669 −1.196 MS2[3 0 1]:167(1 0 0),284(66),269(54),241(19),191(19),147(17),267(10),163(9),245(9) 3,9-dimethoxy-10-hydroxypterocarpan/isomer + +
B37 8.12 N C16H11O5 283.06175 283.06168 4.582 MS2[2 8 3]:268(1 0 0),269(3),255(1) Calycosin isomer + +
B38 8.15 P C24H25O11 489.13969 489.13794 −2.449 MS2[4 8 9]:285(1 0 0),177(5),471(5),387(4),470(4),471(3),294(3),443(2),371(2) Calycosin-7-O-β-D-glucoside-6″-O-acetate/isomer + +
B39 8.24 N C16H11O4 267.06683 267.06702 4.870 MS2[2 6 7]:252(1 0 0),253(1) Formononetin isomer + +
B40 8.24 N C23H23O11 475.12513 475.12625 4.813 MS2[4 7 5]:267(1 0 0),456(1),252(1) Odoratin-7-O-β-D-glucoside/isomer + +
B41# 8.26 P C22H23O9 431.13421 431.13263 −2.386 MS2[4 3 1]:269(1 0 0),343(0.3),413(0.2) Ononin + +
B42 8.27 P C16H13O4 269.08138 269.08038 −1.692 MS2[2 6 9]:254(1 0 0),237(51),213(40),241(17),66(14),252(12) Formononetin isomer + +
B43 8.27 P C26H27O11 515.15534 515.15076 −4.819 MS2[5 1 5]:339(1 0 0),321(3),497(2),199(1) Calycosin-7-O-β-D-glucoside-6″-O-butylene ester/isomer + –
B44 8.43 N C17H15O5 299.09305 299.09293 4.115 MS2[2 9 9]:284(1 0 0),269(1),255(1) 3,9-dimethoxy-10-hydroxypterocarpan/isomer + +
P C17H17O5 301.10760 301.10641 −2.126 MS2[3 0 1]:167(1 0 0),269(26),191(21),147(19),163(12),273(11),207(9),286(6),241(6),270(3)
B45 8.49 N C16H15O5 287.09305 287.09317 4.165 MS2[2 8 7]:135(1 0 0),272(91),165(46),177(29),121(22),147(19) (3R)-7,2′,3′-trihydroxy-4′-methoxy isoflavonone + +
B46 8.49 N C29H37O15 625.21434 625.21716 4.139 MS2[6 2 5]:323(1 0 0),367(71),324(70),343(48),325(36),445(26),547(24),366(17) Isomucronulatol-7,2′-di-O-glucoside/isomer + +
B47 8.70 N C29H37O15 625.21434 625.21558 −4.782 MS2[6 2 5]:323(1 0 0),301(30),245(5),263(3),268(3),283(3),341(2),607(2) Isomucronulatol-7,2′-di-O-glucoside/isomer + +
B48 8.78 N C16H11O4 267.06683 267.06683 4.158 MS2[2 6 7]:252(1 0 0),253(3),249(2),223(1) Formononetin isomer + +
B49 8.92 N C17H15O5 299.09305 299.09314 4.817 MS2[2 9 9]:284(1 0 0),269(4) 3,9-dimethoxy-10-hydroxypterocarpan/isomer + +
P C17H17O5 301.10760 301.10641 −2.126 MS2[3 0 1]:167(1 0 0),269(22),191(20),147(15),163(10),273(9),207(7),241(6),286(2),270(2)
B50 9.02 N C17H17O5 301.10870 301.10870 4.479 MS2[3 0 1]:286(1 0 0),109(14),135(12),147(10),283(8),271(6),179(3) (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan/isomer + +
P C17H19O5 303.12325 303.12225 −1.485 MS2[3 0 3]:167(1 0 0),149(32),123(19),284(16),181(14),168(7),219(6),270(5),193(5)
B51 9.13 P C16H13O4 269.08138 269.08041 −1.581 MS2[2 6 9]:269(1 0 0),252(51),237(28),213(22),270(21),253(7) Formononetin isomer + +
B52 9.19 N C16H11O4 267.06683 267.06680 4.046 MS2[2 6 7]:252(1 0 0),253(5) Formononetin isomer + +
P C16H13O4 269.08138 269.08023 −2.250 MS2[2 6 9]:254(1 0 0),237(52),269(51),213(40),253(15),270(13),107(10),136(6)
B53 9.23 N C17H17O5 301.10870 301.10880 4.811 MS2[3 0 1]:286(1 0 0),135(19),109(15),147(10),121(8),283(6),271(6),179(6) (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan/isomer + +
P C17H19O5 303.12325 303.12219 −1.683 MS2[3 0 3]:167(1 0 0),149(29),123(22),181(16),193(6),285(2),219(1),168(1)
B54# 9.23 N C23H27O10 463.16152 463.16254 2.757 MS2[4 6 3]:301(1 0 0),286(5),299(1) Astraisoflavan-7-O-β-D-glucoside + +
B55 9.37 N C17 H13O5 297.07740 297.07748 4.823 MS2[2 9 7]:282(1 0 0),283(4),279(3),267(2),253(2),254(1),167(1) Afromosin + +
P C17 H15O5 299.09195 299.09119 −0.702 MS2[2 9 9]:284(1 0 0),166(23),243(21),239(11),267(11),285(10),137(4)
B56 9.44 P C16H13O4 269.08138 269.08035 −1.804 MS2[2 6 9]:269(1 0 0),254(75),237(39),213(31),270(17),252(11) Formononetin isomer + +
N C16H11O4 267.06683 267.06699 4.757 MS2[2 6 7]:252(1 0 0),253(1)
B57# 9.60 N C16H11O5 283.06175 283.06183 2.112 MS2[2 8 3]:268(1 0 0),255(5) Calycosin + +
P C16H13O5 285.07630 285.07520 −1.929 MS2[2 8 5]:270(1 0 0),253(43),225(20),137(9),229(7),257(3),181(2),175(1)
B58 9.80 P C17H19O5 303.12325 303.12247 −0.759 MS2[3 0 3]:167(1 0 0),149(30),123(28),181(19),193(6) (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan/isomer + +
N C17H17O5 301.10870 301.10886 4.011 MS2[3 0 1]:286(1 0 0),109(17),135(12),147(8),271(7),283(7),259(3),121(3)
B59 9.98 N C17 H15O5 299.09305 299.09329 4.319 MS2[2 9 9]:284(1 0 0),269(4) 3,9-dimethoxy-10-hydroxypterocarpan + +
P C17H17O5 301.10760 301.10657 −1.594 MS2[3 0 1]:167(1 0 0),269(22),191(20),147(16),163(10),273(10),207(7),241(6),270(3)
B60 10.00 P C17H15O6 315.08686 315.08588 −1.379 MS2[3 1 5]:300(1 0 0),283(19),138(11),255(5),186(7),168(7),294(5),167(5),259(4),296(3) Kumatakenin + +
B61 10.01 N C15H9O5 269.04610 269.04617 4.393 MS2[2 6 9]:241(1 0 0),225(20),213(17),123(9),251(8),145(6),197(5) 5,7,4′-trihydroxy- isoflavonone + +
B62 10.25 N C17H15O5 299.09305 299.09323 4.118 MS2[2 9 9]:284(1 0 0),269(6),267(6),165(4),271(4),281(2) 3,9-dimethoxy-10-hydroxypterocarpan/isomer + +
B63 10.34 P C26H27O11 515.15534 515.15393 −1.666 MS2[5 1 5]:411(1 0 0),353(19),497(13),455(13),393(10),369(10),597(10),337(9),335(8),395(7),167(6) Calycosin-7-O-β-D-glucoside-6″-O-butylene ester/isomer + +
B64 10.38 N C17H17O5 301.10870 301.10886 4.011 MS2[3 0 1]:286(1 0 0),135(38),121(17),109(13),147(10),283(8),179(7),271(6) (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan/isomer + +
P C17H19O5 303.12325 303.12247 −0.759 MS2[3 0 3]:167(1 0 0),149(29),123(23),181(16),193(7),285(2),261(1),167(1)
B65 10.99 N C16H11O4 267.06683 267.06693 4.533 MS2[2 6 7]:252(1 0 0),253(5),249(2) Formononetin isomer + +
B66 11.75 P C17H17O5 301.10760 301.10690 −0.499 MS2[3 0 1]:167(1 0 0),269(22),191(20),147(16),163(10),281(10),207(7),241(6),267(4) 3,9-dimethoxy-10-hydroxypterocarpan/isomer + +
B67# 14.00 N C16H11O4 267.06683 267.06699 2.757 MS2[2 6 7]:252(1 0 0),253(3) Formononetin + +
P C17H13O5 269.08138 269.08041 −1.581 MS2[2 6 9]:254(1 0 0),237(45),251(36),213(26),253(14),107(10),118(5)
B68 14.69 P C17H17O5 301.10760 301.10666 −1.296 MS2[3 0 1]:167(1 0 0),269(81),147(46),191(45),163(28),273(24),267(20),241(18),281(7),270(6),284(4) 3,9-dimethoxy-10-hydroxypterocarpan/isomer + +
B69 15.28 P C17H19O5 303.12325 303.12262 −0.264 MS2[3 0 3]:167(1 0 0),149(33),123(22),181(15),193(7)280(7),199(2) (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan + +

#: Unambiguously identification by comparing with the reference substances; *: Structural validation by using the reference substances;

+: detected; -: undetected; A: Astragli radix; S: SF-AP.

3.2.1

3.2.1 Structural identification of triterpenoid saponins in SF-AP and Astragali radix

Saponins are important effective components existing in Astragali radix, most of which are tetracyclic triterpenoids. Based on the retention time, ESI-MS and ESI-MS/MS data, a total of 45 constituents attributed to triterpene saponins were screened and identified from SF-AP sample and Astragali radix. These constituents mostly belong to cycloartane-type triterpenoids, which were mainly derivatives of 9, 19-cyclolanostane cycloastragenol or 9, 19-cyclolanostane cyclocanthogenin, 9, 10-secocycloartane and oleanane-type triterpenoid saponins (Chu et al., 2010).

In addition, Astragaloside I and Astragaloside IV were selected as subjects to determine their DPIs for the subsequent structral identification. Both reference standards possessed the same backbone structure while the quantity of acetyl groups connected to xylose were different. There are two acetyl groups at 2 and 3 position of xylose in Astragaloside I, while zero acetyl group in Astragaloside IV. Owing to the special structure of acetyl group (Ac), Astragaloside I could generate some characteristic fragment ions by loss of Ac (42 Da), Ac + H2O (60 Da) and 2Ac (84 Da). Moreover, by comparing with the characteristic dissociation pathways in the MS/MS spectra of the other reference standards, some DPIs of triterpene saponins could be summarized in Fig. 2, which provided a basis for further characterization of the others candidates. Taking negative ion mode as an example, the mass spectrometry cleavage of triterpene saponins usually lose glucose (Glc, 162 Da), xylose (Xyl, 132 Da), H2O (18 Da), CO2 (44 Da), malonyl (Ma, 86 Da) and acetyl group (Ac, 42 Da) to generate the corresponding DPIs.

The summary structures and DPIs for the triterpenoid saponins in SF-AP and Astragali radix.
Fig. 2
The summary structures and DPIs for the triterpenoid saponins in SF-AP and Astragali radix.

A7, A8 and A11 all possessed the [M−H]- ions at m/z 785.46983 (C41H69O14, mass error within ± 5 ppm). In their ESI-MS2 spectra, they could yield a wide range of DPIs just like [M−H−Glc−Xyl]- ion at m/z 491, [M−H−Glc]- ion at m/z 623, [M−H−Xyl]- ion at m/z 653, [M−H−H2O]- ion at m/z 767, and [M−H−CO2]- ion at m/z 741. Hence, A7, A8 and A11 were tentatively judged as Cyclocanthoside E or its isomers.

A10, A31 and A33 showed the identical [M−H]- ions at m/z 813.46474 (C42 H69 O15, mass error within ± 5 ppm). In the ESI-MS2 spectra, a number of DPIs such as m/z 651 [M−H−Glc]-, m/z 633 [M−H−Glc−H2O]-, m/z 795 [M−H−H2O]- and m/z 767 [M−H−H2O−CO]- were all observed. Meanwhile, combined with the bibliography data and fragmentation pathways, A10, A31 and A33 were tentatively characterized as Astramembranoside A or its isomers.

A13, A17, A26 and A29 all possessed the [M−H]- ions at m/z 825.46419 (C43H69O15, mass error within ± 5 ppm). Owing to the successive loss of acetyl, acetyl + H2O and xylose + acetyl + H2O, the [M−H]- ion generated a serial of DPIs at m/z 783, m/z 765 and m/z 633 in the ESI-MS2 spectra. Based upon the comparison of ESI-MS/MS spectra and retention time with the corresponding reference standards, A29 was positively identified as Astragaloside II, while A17 was unambiguously characterized as Isoastragaloside II. The accurate mass weight and major product ions of A13 and A26 were broadly similar to those of A29 and A17, which indicated that A13 and A26 could be deduced as the isomers of Astragaloside II or Isoastragaloside II.

A14 afforded [M−H]- ion at m/z 669.42248 (C36H61O11) with mass error of 4.47 ppm. Due to the loss of H2O (18 Da), 2H2O (36 Da) and glucose (162 Da), the product ions in its ESI-MS2 spectrum at m/z 651, m/z 633 and m/z 507 were respectively yielded. Therefore, according to the fragmentation pathways and literature data (Wang et al., 2019a), A14 could be deduced as Mongholicoside A.

A23, A35 and A44 all afforded the [M−H]- ions at m/z 909.48532 (C47H73O17, mass error within ± 5 ppm). In the MS/MS spectra, there were some product ions such as [M−H−H2O]- at m/z 891, [M−H−Ac−H2O]- at m/z 849 and m/z 453 [M−H−3Ac−Xyl−Glc−2H2O]- at m/z 453. And thus, A23, A35 and A44 were tentatively characterized as Acetylastragaloside I or its isomers.

Both A32 and A39 provided the deprotonated [M−H]- ions at m/z 793.43853 (C42H65O14, mass error within ± 5 ppm). The characteristic product ions such as [M−H−Glc]- ion at m/z 631, [M−H−H2O]- ion at m/z 775 and [M−H−2Ac−H2O−CO]- ion at m/z 663 were all illustrated in the ESI-MS2 spectra. Hence, A32 and A39 were deduced to be Huangqiyenin E or its isomer.

A34, A38, A41 and A42 gave the identical [M−H]- ions at m/z 867.47476 (C45H71O16, mass error within ± 5 ppm). Based upon the obtained high-resolution mass spectrometry data, they yielded the DPIs at m/z 849, m/z 807, m/z 783, m/z 747 and m/z 687 by the respective loss of H2O (18 Da), acetyl + H2O (60 Da), 2acetyl (84 Da), 2acetyl + 2H2O (120 Da) and glucose + H2O (180 Da) in the ESI-MS2 spectra. Compared with the standard substances, A41 was identified as Astragaloside I and A34 was characterized as Isoastragaloside I, while A38 was characterized as Isoastragaloside I isomer. Moreover, A42 was deduced as β-D-Glucopyranoside-(3β, 6α, 16β, 20R, 24 s)-3-[(3, 4-di-O-acetyl-β-D-xylopyranosyl)oxy]-20, 24-epoxy-16, 25-dihydroxy-9, 19-cyclolanostan-6-yl.

A43 possessed the [M−H]- ion at m/z 869.49096 (C45H73O16, mass error of 4.644 ppm). In the ESI-MS2 spectrum, it yielded some DPIs at m/z 851, m/z 809, m/z 767 and m/z 749 through the successive loss of H2O, acetyl + H2O, 2acetyl + H2O and 2acetyl + 2H2O, respectively. Hence, A43 was tentatively interpreted as Agroastragaloside I.

A45 generated its [M−H]- ion at m/z 953.47570 (C48H73O19) with mass error of 4.898 ppm. It further produced a series of fragment ions at m/z 935 [M−H−H2O]-, m/z 627 [M−H−Glc−Ma−Ac−2H2O]-, m/z 891 [M−H−CO2−H2O]-, and m/z 807 [M−H−Ma−Ac−H2O]- in its ESI-MS2 spectrum. Therefore, A45 was tentatively interpreted as Malonylastragaloside I. In addition, the ESI-MS2 spectra of A9, A17, A44 and A45 were illustrated in Fig. 3.

The ESI-MS2 spectra and chemical structures of A9, A17, A44 and A45.
Fig. 3
The ESI-MS2 spectra and chemical structures of A9, A17, A44 and A45.

In order to verify the fragmentation regularities, the other two reference substances were conducted. Take Astragaloside V and Isoastragaloside IV as examples, which would make structural validation clear. Based on the obtained high-resolution mass spectrometry data, it could be seen that Astragaloside V (A5*) produced the [M−H]- ion at m/z 945.50700 (C47H77O19). Then the [M−H]- ion generated a series of characteristic product ions at m/z 783 [M−H−Glc]-, m/z 651 [M−H−Glc−Xyl]-, m/z 621 [M−H−2Glc]- and m/z 489 [M−H−Xyl−2Glc]- in its ESI-MS2 spectrum. Isoastragaloside IV (A9*) gave rise to [M−H]- ion at m/z 783.45363 (C41H67O14). In its ESI-MS2 spectrum, the [M−H]- ion at m/z 783 further generated several product ions at m/z 489, m/z 651, m/z 621, m/z 471, and m/z 453 by the subsequent losing xylose + glucose, xylose moiety, glucose moiety, xylose + glucose + H2O, and xylose + glucose + 2H2O. By referring to the cracking mode of these two reference substances, some similar rules in cracking could be found. For the special structures of flavonoid glycoside, the ions of [M−H−162]- and [M−H−132]- were usually produced via the loss of glucose moiety and xylose moiety in their ESI-MS2 spectra.

3.2.2

3.2.2 Structural identification of flavonoids in SF-AP and Astragali radix

Flavonoids are the other major category of components existing in Astragali radix. The DPIs for flavonoids have previously summarized on the basis of high-resolution MS data acquired in Fig. 4. The characteristic DPIs just like [M−H−CH3]- (15 Da), [M−H−H2O]- (18 Da), [M−H−CO2]- (44 Da) and [M−H−CO]- (28 Da) were obviously observed according to the ESI-MS/MS data of the obtained reference standards. Meanwhile, flavonoid glycosides usually first split up the glycosidic bond, and then produce the corresponding aglycone ions (Ren et al., 2007; Es-Safi et al., 2007). Generally speaking, the glucose group is usually replaced at the C-7 or C-3 position of the ligand ketone. Subsequently, the aglycone ions would be cleaved to form a series of fragment ion. In addition, they will ordinarily lose Glc + H2O + CO (208 Da) and Glc + CO + H2O + CH3 (223 Da). For polymethoxylated flavones, the fragment ions produced by loss of one or more methyl radicals from the protonated molecule ions are usually detected, which could be regarded as their DPIs (Zhang et al., 2011; Shang et al., 2017). Finally, 64 flavonoids including 33 isoflavnones, 15 isoflavans, 7 pterocarpans and 9 flavnones were identified from SF-AP while 69 flavonoids including 35 isoflavnones, 18 isoflavans, 7 pterocarpans and 9 flavnones were characterized from Astragali radix.

The summary structures and DPIs for the flavonoids in SF-AP and Astragali radix.
Fig. 4
The summary structures and DPIs for the flavonoids in SF-AP and Astragali radix.

Both B3 and B40 yielded [M−H]- ions at m/z 475.12513 (C23H23O11, mass error within ± 5 ppm). In the ESI-MS2 spectra, a number of DPIs were observed such as m/z 267 [M−H−Glc−H2O−CO]-, m/z 252 [M−H−Glc−CO−H2O−CH3]- and m/z 429 [M−H−H2O−CO]-. Based on this, B3 and B40 were tentatively characterized as Odoratin-7-O-β-D-glucoside or its isomer.

B5 possessed the [M−H]- ion at m/z 641.20926 (C29H37O16) with mass error of 4.708 ppm. Due to the presence of ESI-MS2 product ions at m/z 479, m/z 317, m/z 433 and m/z 611 by losing glucose, 2glucose, CO + H2O and 2CH3, B5 was finally characterized as 5′-hydroxy-isomucronulatol-2′, 5′-di-O-glucoside.

Both B7 and B9 generated the same [M−H]- ions at m/z 623.16231 (C28H31O16, mass error within ± 5 ppm). Moreover, a range of characteristic DPIs at m/z 415 [M−H−Glc−CO−H2O]-, m/z 461 [M−H−Glc]-, m/z 299 [M−H−2Glc]- and m/z 577 [M−H−CO−H2O]- were detected in the ESI-MS/MS spectra. As a result, B9 was tentatively characterized as Complanatuside, while B7 was judged as Complanatuside isomer.

B10 gave rise to the [M−H]- ion at m/z 461.10948 (C22H21O11) with mass error of 4.773 ppm. Based on the obtained high-resolution mass spectrometry data, it yielded respective base peak ions at m/z 446 [M−H−CH3]-, m/z 299 [M−H−Glc]-, m/z 267 [M−H−Glc−CH2−H2O]- and m/z 271 [M−H−Glc−CO]- in the ESI-MS2 spectra. Therefore, according to the fragmentation pathways, B10 was characterized as Kaempferol-4′-methylether-3-D-glucoside.

Six isomeric constituents, B12, B16, B21, B27, B37 and B57, afforded the same theoretical [M−H]- ions at m/z 283.06175 (C16H11O5, mass error within ± 5 ppm), respectively. In the ESI-MS spectra, they showed the characteristic ESI-MS2 product ions at m/z 268 and m/z 255 by lossing of CH3 (15 Da) and CO (28 Da). Combined with the standard substance, B57 was unambiguously identified as Calycosin. Meanwhile, the other five constituents including B12, B16, B21, B27 and B37 were tentatively characterized as Calycosin isomers.

B13 possessed the [M−H]- ion at m/z 477.10440 (C22H21O12) with mass error of 4.382 ppm. In the ESI-MS2 spectrum, it produced many DPIs just like [M−H−Glc]- ion at m/z 315, [M−H−H2O−CO]- ion at m/z 431, [M−H−Glc−H2O]- ion at m/z 297, [M−H−Glc−CH3]- ion at m/z 300 and [M−H−H2O]- ion at m/z 459. Combined with the standard substance, B13 was positively identified as Isorhamnetin-3-D-glucoside.

B15 afforded the [M−H]- ion at m/z 503.12005 (C24H23O12, mass error of 4.401 ppm). In the ESI-MS/MS spectrum, it showed the product ions at m/z 299 [M−H−acetyl−Glc]-, m/z 488 [M−H−CH3]- and m/z 467 [M−H−2H2O]-. And thus, it indicated that B15 could be characterized as Neocomplanoside or its isomer.

B19 and B24 afforded the same [M−H]- ions at m/z 313.07231 (C17H13O6, mass error within ± 5 ppm). They further produced DPIs at m/z 298 ([M−H−CH3]-), m/z 295 ([M−H−H2O]-), m/z 285 ([M−H−CO]-) and m/z 269 ([M−H−CO2]-) in the ESI-MS/MS spectra. Hence, according to the proposed fragmentation patterns, B19 and B24 could be assumed as 7,3′-dihydroxy-8,4-dimethoxyisoflavone or its isomer.

B20, B34 and B61 afforded the same [M−H]- ions at m/z 269.04610 (C15H9O5, mass error within ± 5 ppm), respectively. There were a battery of DPIs at m/z 254 ([M−H−CH3]-), m/z 241 ([M−H−Glc−CO]-), m/z 225 ([M−H−CO2]-), m/z 197 ([M−H−CO−CO2]-) and m/z 181 ([M−H−2CO2]-) in the ESI-MS/MS spectra. Therefore, B20, B34 and B61 could be characterized as 5,7,4′-trihydroxy- isoflavonone or its isomers.

B25 generated the [M−H]- ion at m/z 431.09892 (C21H19O10) with mass error of 2.421 ppm. In the ESI-MS2 spectrum, it further possessed the product ions at m/z 269 by the loss of 162 Da (glucose). By Combing with the standard substance, B25 was positively characterized as Genistin.

Seven constituents containing B33, B39, B48, B52, B56, B65 and B67, which respectively afforded the same identical [M−H]- ions at m/z 267.06683 (C16H11O4, mass error within ± 5 ppm). In the ESI-MS/MS spectra, they further yielded the product ion at m/z 252 by the loss of CH3 radical. Combined with the standard substance, B67 was unambiguously identified as Formononetin, while B33, B39, B48, B52, B56 and B65 could be deduced as Formononetin isomers.

B45 afforded the [M−H]- ion at m/z 287.09305 (C16H15O5, mass error of 4.165 ppm). In the ESI-MS/MS spectrum, it further generated the product ions at m/z 272 [M−H−CH3]-, m/z 269 [M−H−H2O]- and m/z 255 [M−H−CH2−H2O]-. Based upon this, B45 was deduced to be (3R)-7,2′,3′-trihydroxy-4′-methoxy isoflavonone.

B50, B53, B58 and B64 all gave rise to the same [M−H]- ions at m/z 301.10870 (C17H17O5, mass error within ± 5 ppm), respectively. In the ESI-MS/MS spectra, the DPIs at m/z 286 ([M−H−CH3]-), m/z 283 ([M−H−H2O]-)and m/z 271 ([M−H−2CH3]-) were all observed. Combined with the obtained fragmentation pathways, B50, B53, B58 and B64 were characterized as (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan or its isomers.

Five isomeric constituents, including B44, B49, B59, B62 and B72, produced the same [M−H]- ions at m/z 299.09305 (C17H15O5, mass error within ± 5 ppm), respectively. There were a series of DPIs at m/z 284 [M−H−CH3]-, m/z 269 [M−H−2CH3]-, m/z 267 [M−H−CH2−H2O]- and m/z 281 [M−H−H2O]- in the ESI-MS2 spectra. And thus, B44, B49, B59, B62 and B72 were tentatively characterized as 3,9-dimethoxy-10-hydroxypterocarpan or its isomers.

B54 gave rise to the identical [M−H]- ion at m/z 463.16152 (C23H27O10, mass error 2.757 ppm). In the ESI-MS/MS spectrum, it showed the characteristic product ions at m/z 301 and m/z 286 by the loss of 162 Da (glucose) and 177 Da (glucose + CH3). Combined with the corresponding standard substance, B54 was identified as Astraisoflavan-7-O-β-D-glucoside.

B55 afforded the [M−H]- ion at m/z 297.07740 (C17 H13O5) with mass error of 4.823 ppm. In its ESI-MS2 spectrum, it generated some characteristic product ions such as m/z 282, m/z 253, m/z 267 and m/z 279 through the successive loss of CH3, CO2, 2CH3, H2O, orderly. Based upon this, B55 was concluded to be Afromosin isomer. In addition, the ESI-MSn spectra of B9, B23, B57 and B61 were all illustrated in Fig. 5.

The ESI-MS2 spectra and chemical structures of B9, B23, B57 and B61.
Fig. 5
The ESI-MS2 spectra and chemical structures of B9, B23, B57 and B61.

For the reference substance with flavonoid structure, Calycosin-7-O-β-D- glucoside (B23*) yielded the [M−H]- ion at m/z 445.11457 (C22H21O10) with mass error of 3.351 ppm. In the ESI-MS2 spectra, a series of DPIs such as m/z 283, m/z 268 and m/z 255 were observed in negative mode. The existence of these molecular weights verified the loss of glucose (162 Da), glucose + CH3 (177 Da) and glucose + CO (190 Da), orderly. Its characteristic ions just like [M−H−Glu]-, [M−H−Glu−CH3]- and [M−H−Glu−CO]- could also be verified from the DPIs for flavonoids which have previously summarized. Therefore, DPIs mentioned above could be summarized the fragmentation regularities of group compositions and utilized for deducting the structures of related compounds from abundant complex constituents.

3.3

3.3 Comparative analysis of the main constituents existing in Astragali radix and SF-AP

Our previous study of liquid fermentation found that 42 constituents were attributed to saponins while the remaining 65 were identified as flavonoids [7]. However, in this report, coupled with the high-resolution mass data, obtained DPIs, retention time, standard references and related literatures, a total of 110 chemical constituents including 45 triterpene saponins and 65 flavonoids, while 109 components containing 41 triterpene saponins and 68 flavonoids were screened and identified from SF-AP and Astragali radix, respectively (Fig. 6). After comparing the results from SF-AP and Astragali radix, it could be found that the newly generated constituents after fermentation could be attributed to Azukisaponin V methyl ester, Huangqiyenin F, Huangqiyenin A, Huangqiyenin E and Astraisoflavan-7-O-β-D-glucoside. In the meantime, Astragalus flavonoids such as Calycosin-7-O-β-D-glucoside-6″-O-butylene ester, 5,7,4′-trihydroxy-isoflavonone, (3R)-7,2′,3′-trihydroxy-4′-methoxy isoflavonone and Calycosin were undetected after fermentation. Movever, by comparing these two fermentation methods, it was illustrated that many more isomeric constituents could be generated from solid fermentation, while some constituents such as Pratensein and Calycosin-7-O-β-D-glucoside-6″-O-butylene ester were only observed in liquid fermentation.

The composition of constituents existing in Astragli radix and SF-AP.
Fig. 6
The composition of constituents existing in Astragli radix and SF-AP.

It is worth noting that the most obvious change during the fermentation transversion is the relative content of some representative components. According to the experimental results (Fig. 7), it could be seen that the relative content of some constituents were increased, such as Malonylastragaloside I, Soyasaponin I, Astragaloside IV, (3R)-8,2′-dihydroxy-7,4′-dimethoxy-isoflavan, Cyclocanthoside E, Astraisoflavan-7-O-β-D-glucoside, Odoratin-7-O-β-D-glucoside, Isoquercitrin, 5′-hydroxy isomucronulatol 2′,5′-di-O-glucoside, while some constituents including Astragaloside II, Astragaloside V, Formononetin, 3,9-dimethoxy-10- hydroxypterocarpan were observed with decreased relative content after the process of fermentation. Compared with the previous study of liquid fermentation, there were similar changes about the increase content of Astragaloside IV, but some other components like Cyclocanthoside E/isomers have no significant same change trend in fact.

The changes of representative constituents including flavonoids and triterpene saponins before and after fermentation.
Fig. 7
The changes of representative constituents including flavonoids and triterpene saponins before and after fermentation.

Among them, Astragaloside IV has various pharmacological activities especially in cardiovascular diseases, digestive diseases, cancer and the other modern high incidence, high-risk diseases (Ren et al., 2013; Zhang et al., 2006). Meanwhile, Astragaloside IV is officially used as a quality-marker for Astragali Radix in Chinese Pharmacopoeia (2015 version). The increased content of Astragaloside IV may be due to the loss of acetyl group or glucose moiety in the fermentation transversion of Astragaloside II and Astragaloside V and the other components (Fig. 8). In this sense, it could be deduced that the transformation during the fermentation process was more conducive to playing a therapeutic effect in clinical application.

The proposed transformations after fermentation.
Fig. 8
The proposed transformations after fermentation.

3.4

3.4 The antioxidative activity of Astragali radix and SF-AP

For further study, two kinds of antioxidative tests were chosen to evaluate the antioxidative activity of Astragali radix before and after solid fermentation. According to the results, the scavenging ability of the SF-AP to DPPH· was significantly improved by comparing with Astragali radix. By selecting the mass concentration range of the samples as 0.06 ∼ 0.84 mg/mL, the scavenging activity of Astragali radix increased from 6.21% to 36.02% while that of the SF-AP increased from 8.23% to 45.65%. In addition, with the increase of samples’ mass concentration, the scavenging ability of Astragali radix and SF-AP to ABTS·+ were enhanced. When the mass concentration was between 0.06 and 0.96 mg/mL, it could be seen that the scavenging ability of ABTS·+ of SF-AP was much higher than Astragali radix. While the mass concentration reached 0.96 mg/mL, the scavenging ability of Astragali radix to ABTS·+ was 49.5% while the scavenging ability of SF-AP was 57.4% (shown in Fig. 9).

The evaluation of antioxidative activity in vitro.
Fig. 9
The evaluation of antioxidative activity in vitro.

The improvement of antioxidative activity of SF-AP is complex and a great many factors are attributed to it. What’s more, some physical and chemical changes during the fermentation process played the decisive part on the antioxidative activity of Astragali radix. For instance, a varieties of secondary metabolites produced after fermentation: some methoxylated flavones lost methoxy(s) and then produced a great deal of OH-flavones; some flavonoid glycosides were hydrolyzed into aglycones, which could also increase the antioxidative activity.

4

4 Conclusion

In this study, UHPLC-LTQ-Orbitrap MS was used to acquire chemical profiles of Astragali radix and SF-AP. Combining with the fragmentation rules, chromatographic behavior, DPIs and related literature data, 114 compounds including 45 saponins and 69 flavonoids were finally identified in both positive and negative ion modes. By comparison with Astragali radix, some components contained in SF-AP had significant chemical changes such as content fluctuation and isomerism owing to the occurrence of hydrolysis and other conversion reactions. Moreover, two kinds of antioxidative tests were applied to evaluate the antioxidative activity of Astragali radix before and after fermentation. The antioxidative activity of SF-AP in two kinds of antioxidative tests corresponding to the scavenging of DPPH· and ABTS·+ were both significantly higher than that of Astragali radix.

Based on the comparison of the above two aspects, fermentation can improve the internal conversion efficiency and the content of compounds, so as to improve the therapeutic effect. Although the specific chemical transformation mechanism during the fermentation process still needs further exploration, this study set a good example for the comprehensive chemical identification and much more in-depth pharmacodynamics study of the fermentation system between microbiota and Chinese herbal medicines.

Acknowledgments

This work has been financially supported by Young and Creative Team for Talent Introduction of Shandong Province, Binzhou Medical University Scientific Research Fund for High-level Talents (2019KYQD06), Locality-University Cooperation Project of Yantai City (2019XDRHXMPT18), and Independent Topic Selection of Beijing University of Chinese Medicine (2019-JYB-XSCXCY-06).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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© Copyright 2025 – Arabian Journal of Chemistry.

Published by Scientific Scholar on behalf of King Saud University together with Saudi Chemical Society, Riyadh, Saudi Arabia.

ISSN (Print): 1878-5352
ISSN (Online): 1878-5379
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