Evidence-Based Complementary and Alternative Medicine

Volume 2014 (2014), Article ID 170959, 14 pages

http://dx.doi.org/10.1155/2014/170959

Research Article

Investigation of the Chemical Changes from Crude and Processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma Herbal Pair Extracts by Using Q Exactive High-Performance Benchtop Quadrupole-Orbitrap LC-MS/MS

Gang Cao,1,2 Qinglin Li,³ Hao Cai,¹ Sicong Tu,⁴ and Baochang Cai1,2

¹College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

²Research Center of TCM Processing Technology, Zhejiang Chinese Medical University, Hangzhou 310053, China

³Zhejiang Cancer Hospital, Hangzhou 310022, China

⁴Faculty of Medicine, University of New South Wales, Sydney, NSW 2031, Australia

Received 20 January 2014; Revised 7 March 2014; Accepted 20 March 2014; Published 4 May 2014

Academic Editor: Shi-Biao Wu

Copyright © 2014 Gang Cao et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair is mainly used for regulating the functions of liver and spleen, benefiting qi, and nourishing blood. However, the bioactive compounds for the pharmacological activities of the crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair extracts are still unclear to date. In the present study, Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS was applied to identify the complicated components from crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts. 123 and 101 compounds were identified in crude and processed Paeoniae Radix Alba samples, respectively. Meanwhile, 32 and 26 compounds were identified in crude and processed Atractylodis Macrocephalae Rhizoma samples, respectively. In the crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair extracts, co-decoction could significantly change the chemical composition of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma in solution. The developed method may provide a scientific foundation for deeply elucidating the processing and compatibility mechanism of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma.

1. Introduction

Traditional Chinese medicine (TCM) processing is regarded as a pharmaceutical technology based on TCM theory, the requirements of different syndrome treatment, the quality nature of medicine, and different demands of clinical dispensing and preparations [1]. It is one of the characteristics in application of TCM. The compatible components of prescription are composed of prepared Chinese crude drugs after TCM processing.

The prescription compatibility and TCM processing are not only two major features of clinical medication in TCM, but are also critical to distinguish TCM from natural medicine. The research on structural features, compatible effect, and material basis of the herbal pair is the important support in the study of the prescription compatibility since the herbal pair is the minimum unit in prescription of TCM [2, 3]. They play a guidance and significant role in reveal of the compatibility rule and the scientific connotation. The herbal pair compatibility theory can explain the relationship of the prescription compatibility to some extent. The research on the relationship between the herbal pair compatibility and the prescription compatibility contributes to the elucidation of the prescription compatibility mechanism and the action mechanism of treatment. There are many herbal pairs commonly used in the clinical practice of TCM, such as the herbal pairs of Paeonia Lactiflora-Liquorice, Ginseng-Aconite, and Aconite-Rhizome Zingiberis [4, 5] besides the Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair frequently used in all China dynasties [6, 7]. Paeoniae Radix Alba nourishes blood and liver, and Atractylodis Macrocephalae Rhizoma helps invigorate spleen and eliminate dampness [8–12]. Thus, the compatibility of these two medicines could help achieve the goal of purging wood from the earth, regulating the functions of liver and spleen, benefiting qi, and nourishing blood [13–15]. Although the compositions of these two medicines have been extensively studied, the appropriate processing method of them, such as frying, which is believed by the practitioners of traditional medicine to have the effects for enhancing the efficacy of the medicine, and their underlying compatibility mechanism are still under investigation.

The objective of this study is to investigate the qualitative, preprocessing, and postprocessing changes in the composition and compatibility of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma by using Q Exactive hybrid quadrupole-Orbitrap mass spectrometer combined with high-performance quadrupole precursor selection with high-resolution and accurate-mass Orbitrap detection. The work could serve as a theoretical basis for the development of medicines from Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma, and the reasonable clinical medication. Furthermore, it provides new insights into the investigation of the herbal pair and for the study of the appropriate processing method for Chinese herbal medicines and their underlying compatibility mechanism.

2. Experimental

2.1. Chemicals, Solvents, and Herbal Materials

Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma samples were acquired from Zhejiang suppliers. All of these herbal samples were authenticated by Professor Jianwei Chen (College of Pharmacy, Nanjing University of Chinese Medicine). HPLC-grade acetonitrile and formic acid were obtained from Merck (Darmstadt, Germany). Deionized water was purified using the Milli-Q system (Millipore, Bedford, MA, USA). All other reagents and chemicals were analytical grade.

2.2. Preparation of the Sample Solutions

The dried and powdered samples of crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts (1 : 1, g/g) were prepared. A total of 2.0 g of each sample powder was accurately weighed and transferred into a 50 mL round bottom flask with 20 mL of 70% methanol aqueous solution (v/v) and refluxed in a 80°C water bath for 1 h. The filtrate was collected after filtration and the residue was then refluxed with 20 mL of 70% methanol aqueous solution in a 80°C water bath for 1 h, the filtrate was collected again after filtration and the residue was removed. Finally, the combined filtrates were treated by rotary evaporation concentration and the resultant residue was dissolved and transferred into a 25 mL volumetric flask with 70% methanol aqueous solution to make it up to a final concentration of 0.08 g·mL⁻¹. All solutions were stored at 4°C and filtered through a 0.22 μm filter membrane before injection into the HPLC system.

2.3. Liquid Chromatography and Mass Spectrometry

Analyses were performed by using Dionex UltiMate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) with a diode array detector. Detection wavelengths were set at 255 nm. A Thermo Scientific Hypersil Gold C₁₈ column (100 mm × 2.1 mm, 1.9 μm) was used with a flow rate of 0.35 mL·min⁻¹. The injection volume was 5 μL, and the column temperature was maintained at 30°C. The sample separation was performed according to the previous reports with minor modification [16–18]. The mobile phase was composed of (a) aqueous formic acid (0.1%, v/v) and (b) acetonitrile under following gradient elution: 10–55% B from 0 to 40 min, 55–90% B from 40 to 51 min, 90% B from 51 to 56 min, 90–10% B from 56 to 56.1 min, and 10% B from 56.1 to 60 min. Mass spectrometry was performed on a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, USA) using a heated electrospray ionization (HESI-II) source for ionization of the target compounds in positive and negative ion modes. The key parameters were as follows: ionization voltage, +3.0 kV/−2.8 kV; sheath gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; heat temperature, 300°C; and capillary temperature, 300°C. For the compounds of interest, a scan range of m/z 150–1500 was chosen. Resolution for higher energy collisional dissociation cell (HCD) spectra was set to 17,500 at m/z 150 on the Q Exactive.

3. Results and Discussion

3.1. Identification of the Main Components in Crude and Processed Paeoniae Radix Alba

Tentative identification of the main compounds in crude and processed Paeoniae Radix Alba samples was generated based on elemental composition data determined from accurate mass measurements and comparison with the literature data. The total ion chromatograms of crude and processed Paeoniae Radix Alba samples obtained from both positive and negative ion modes were shown in Figure 1. In the preliminary study, the Q Exactive mass spectrometer was confirmed to be highly selective and sensitive. Under the present chromatographic and MS conditions, 123 and 101 compounds were identified in crude and processed Paeoniae Radix Alba samples, respectively. Compounds 16, 30, 31, 42, 45, 58, 59, 61, 62, 63, 64, 75, 78, 80, 87, 90, 91, 94, 95, 103, 112, and 120 were not detected in processed Paeoniae Radix Alba sample. Meanwhile, the ESI-MS data of crude and processed samples demonstrated that the peak areas of components 8, 113, and 122 varied significantly, and their amounts were dramatically increased in processed sample. The results were shown in Table 1.

Table 1: Major chemical constituents identified in crude and processed Paeoniae Radix Alba and in crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair.

No.
(min)
Compound name
Formula
Paeoniae

Radix Alba
Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair

(Measured area)
(Measured area)

Crude
Processed
Crude
Processed

1
0.84
6-O-galloylsucrose
C₁₉H₂₆O₁₅

2
0.84
Glucogallin
C₁₃H₁₆O₁₀

—

3
1.05
Desbenzoylpaeoniflorin
C₁₆H₂₄O₁₀

—

4
1.06
1′-O-galloylsucrose
C₁₉H₂₆O₁₅

—
—

5
1.07
1-O-glucopyranosyl paeonisuffrone
C₁₆H₂₄O₉

—

6
1.13
Gallic acid
C₇H₆O₅

7
1.18
Oxypaeoniflorin sulfonate
C₂₃H₂₈O₁₄S

8
1.22
Ethyl gallate
C₉H₁₀O₅

9
1.22
6-O-galloyl desbenzoylpaeoniflorin
C₂₃H₂₈O₁₄

—

10
1.26
6-O-glucopyranosyl-lactinolide
C₁₆H₂₆O₉

—

11
1.30
Paeoniflorin sulfonate I
C₂₃H₂₈O₁₃S

12
1.30
Mudanpioside E sulfonate
C₂₄H₃₀O₁₅S

13
1.43
6-O-glucopyranosyl-lactinolide
C₁₆H₂₆O₉

14
1.64
Mudanpioside F
C₁₆H₂₄O₈

15
1.76
Isomaltopaeoniflorin sulfonate
C₂₉H₃₈O₁₈S

16
1.81
Pedunculagin
C₃₄H₂₄O₂₂

—

17
1.97
Paeoniflorin sulfonate I
C₂₃H₂₈O₁₃S

18
2.25
Oxypaeoniflorin
C₂₃H₂₈O₁₂

19
2.36
Gallotannin
C₂₇H₂₄O₁₈

20
2.37
1-O-benzoylsucrose
C₁₉H₂₆O₁₂

21
2.41
d-catechin
C₁₅H₁₄O₆

22
2.63
Methyl gallate
C₈H₈O₅

—

23
2.63
Salicylic acid
C₇H₆O₃

24
2.72
Albiflorin R1
C₂₃H₂₈O₁₁

25
3.00
Kaempferol-3,7-di-O-glucoside
C₂₇H₃₀O₁₆

26
3.00
Paeonoside
C₂₇H₃₀O₁₆

27
3.46
Galloylpaeoniflorin
C₃₀H₃₂O₁₅

28
3.47
Paeonolide
C₂₀H₂₈O₁₂

—

29
3.58
6-O-glucopyranosyl-lactinolide
C₁₆H₂₆O₉

30
3.68
Oxypaeoniflorin
C₂₃H₂₈O₁₂

—

31
3.76
6-O-glucopyranosyl-lactinolide
C₁₆H₂₆O₉

—

32
3.88
Paeonilactone B
C₁₀H₁₂O₄

33
3.93
Isomaltopaeoniflorin
C₂₉H₃₈O₁₆

34
4.07
Albiflorin
C₂₃H₂₈O₁₁

35
4.32
Glucopyranosylalbiorin
C₂₉H₃₈O₁₆

36
4.34
Galloylpaeoniflorin sulfonate
C₃₀H₃₂O₁₇S

37
4.34
Galloylpaeoniflorin isomer
C₃₀H₃₂O₁₅

38
4.38
1,2,3,6-tetra-O-galloylglucose
C₃₄H₂₈O₂₂

39
4.38
Tetragalloyl glucose A
C₃₄H₂₈O₂₂

40
4.56
Mudanpioside F
C₁₆H₂₄O₈

41
4.60
Oxypaeoniflorin isomer
C₂₃H₂₈O₁₂

42
4.65
Gallotannin
C₂₇H₂₄O₁₈

—
—
—

43
4.77
Paeoniflorin
C₂₃H₂₈O₁₁

44
4.89
Paeoniflorin sulfonate II
C₂₃H₂₈O₁₃S

45
4.98
Isogalloylpaeoniflorin sulfonate
C₃₀H₃₂O₁₇S

—
—
—

46
5.05
Ethyl gallate
C₉H₁₀O₅

47
5.05
Methyl salicylate
C₈H₈O₃

48
5.15
Benzoic acid
C₇H₆O₂

49
5.25
Paeonol
C₉H₁₀O₃

50
5.25
4-hydroxy-3-methoxy acetophenone
C₉H₁₀O₃

51
5.31
ortho-oxypaeoniflorin
C₂₃H₂₈O₁₂

52
5.63
Ethyl gallate
C₉H₁₀O₅

53
5.63
Methyl salicylate
C₈H₈O₃

54
5.66
Kaempferol-3-O-glucoside
C₂₁H₂₀O₁₁

—
—

55
5.66
Astragalin
C₂₁H₂₀O₁₁

—
—

56
6.01
Eugeniin
C₄₁H₃₀O₂₆

57
6.01
Dihydroxymethyl benzoyl tetragalloyl glucose
C₄₁H₃₀O₂₆

58
6.03
1,2,3,6-tetra-O-galloylglucose isomer A
C₃₄H₂₈O₂₂

—

59
6.03
Tetragalloyl glucose B
C₃₄H₂₈O₂₂

—
—
—

60
6.08
Astragalin
C₂₁H₂₀O₁₁

61
6.09
Isomaltopaeoniflorin isomer
C₂₉H₃₈O₁₆

—
—
—

62
6.47
1,2,3,6-tetra-O-galloylglucose isomer B
C₃₄H₂₈O₂₂

—
—

63
6.47
Tetragalloyl glucose C
C₃₄H₂₈O₂₂

—
—

64
6.85
3,6-di-O-galloyl paeoniorin
C₃₇H₃₆O₁₉

—
—
—

65
6.96
1,2,3,6-tetra-O-galloylglucose
C₃₄H₂₈O₂₂

66
6.96
Tetragalloyl glucose D
C₃₄H₂₈O₂₂

67
7.35
Galloylpaeoniflorin isomer I
C₃₀H₃₂O₁₅

68
7.60
1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone
C₂₃H₂₈O₁₀

69
7.71
Glucopyranosylalbiorin isomer I
C₂₉H₃₈O₁₆

—

70
8.18
1-O-glucopyranosyl-8-O-benzoyl paeonisuffrone
C₂₃H₂₈O₁₀

71
8.31
Ortho-oxypaeoniflorin
C₂₃H₂₈O₁₂

72
8.45
1,2,3,4,6-Penta-O-galloyl–D-glucopyranose
C₄₁H₃₂O₂₆

73
8.45
Pentagalloyl glucose
C₄₁H₃₂O₂₆

74
8.64
Lactiflorin
C₂₃H₂₆O₁₀

—

75
8.80
Galloylalbiroin
C₃₀H₃₂O₁₅

—
—
—

76
9.17
Astragalin
C₂₁H₂₀O₁₁

77
9.25
Lactinolide
C₁₀H₁₆O₄

78
9.29
Galloylpaeoniflorin isomer II
C₃₀H₃₂O₁₅

—

79
9.68
Glucopyranosylalbiorin isomer II
C₂₉H₃₈O₁₆

80
9.84
Hexagalloyl glucose
C₄₈H₃₆O₃₀

—

81
9.95
Oxybenzoyl-oxypaeoniflorin
C₃₀H₃₂O₁₄

82
10.07
1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone
C₂₃H₂₈O₁₀

83
10.29
Albiflorin R1 isomer I
C₂₃H₂₈O₁₁

84
10.74
Hexagalloyl glucose
C₄₈H₃₆O₃₀

85
10.76
Lactiflorin
C₂₃H₂₆O₁₀

86
10.84
Benzoylpaeoniflorin Sulfonate
C₃₀H₃₂O₁₄S

87
10.88
3,6-di-O-galloyl paeoniorin
C₃₇H₃₆O₁₉

—
—
—

88
10.95
Ortho-oxypaeoniflorin isomer
C₂₃H₂₈O₁₂

89
11.52
3,6-di-O-galloyl paeoniorin
C₃₇H₃₆O₁₉

90
11.72
3,6-di-O-galloyl paeoniorin isomer
C₃₇H₃₆O₁₉

—

91
11.75
Galloylalbiroin isomer I
C₃₀H₃₂O₁₅

—
—
—

92
11.84
Oxypaeoniflorin sulfonate isomer
C₂₃H₂₈O₁₄S

93
12.15
1-O-glucopyranosyl-8-O-benzoylpaeonisuffrone
C₂₃H₂₈O₁₀

94
12.15
Oxybenzoyl-oxypaeoniflorin
C₃₀H₃₂O₁₄

—
—

95
12.18
Benzoyloxypaeoniflorin
C₃₀H₃₂O₁₃

—

96
13.42
Benzoyloxypaeoniflorin isomer
C₃₀H₃₂O₁₃

97
13.44
Oxybenzoyl-oxypaeoniflorin isomer I
C₃₀H₃₂O₁₄

98
13.85
Galloylalbiroin isomer II
C₃₀H₃₂O₁₅

99
14.05
Oxybenzoyl-oxypaeoniflorin isomer II
C₃₀H₃₂O₁₄

100
14.13
Benzoyloxypaeoniflorin
C₃₀H₃₂O₁₃

101
15.07
Benzoyloxypaeoniflorin isomer I
C₃₀H₃₂O₁₃

—
—

102
15.38
Benzoyloxypaeoniflorin isomer II
C₃₀H₃₂O₁₃

—
—

103
16.01
Oxybenzoyl-paeoniflorin
C₃₀H₃₂O₁₂

—

—

104
16.95
Isobenzoylpaeoniflorin
C₃₀H₃₂O₁₂

105
16.95
Oxybenzoyl-paeoniflorin isomer I
C₃₀H₃₂O₁₂

106
17.23
Benzoylpaeoniflorin Sulfonate
C₃₀H₃₂O₁₄S

107
17.48
Isobenzoylpaeoniflorin isomer I
C₃₀H₃₂O₁₂

108
17.48
Oxybenzoyl-paeoniflorin isomer II
C₃₀H₃₂O₁₂

109
17.86
Benzoyloxypaeoniflorin
C₃₀H₃₂O₁₃

110
18.55
Benzoyloxypaeoniflorin isomer
C₃₀H₃₂O₁₃

111
18.69
Albiflorin R1 isomer II
C₂₃H₂₈O₁₁

—

112
19.30
Albiflorin R1 isomer III
C₂₃H₂₈O₁₁

—
—

113
21.79
Palbinone
C₂₂H₃₀O₄

114
21.93
Isobenzoylpaeoniflorin isomer II
C₃₀H₃₂O₁₂

115
21.93
Oxybenzoyl-paeoniflorin isomer III
C₃₀H₃₂O₁₂

116
22.15
Paeonilactinone
C₁₀H₁₆O₂

117
36.46
Hederagenin
C₃₀H₄₈O₄

118
37.31
23-hydroxybetulinic acid
C₃₀H₄₈O₄

119
38.14
Astrantiagenin D
C₃₀H₄₆O₄

120
43.00
Astrantiagenin D isomer
C₃₀H₄₆O₄

—

—

121
45.65
Oleanolic acid
C₃₀H₄₈O₃

122
46.10
Betulinic acid
C₃₀H₄₈O₃

123
52.48
Daucosterol
C₃₅H₆₀O₆

(a)

(b)

Figure 1: Total ion chromatograms of crude (a) and processed (b) Paeoniae Radix Alba obtained from both positive and negative ion modes.

From ESI-MS information, it was found that the sensitivities for all kinds of components in Paeoniae Radix Alba were high in both positive and negative ion modes. In present study, we chose peaks 1, 2, and 3 to explain the identification process using Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS. Peaks 1, 2, and 3 were eluted at retention times of 4.08, 4.79, and 8.47 min, respectively. Peak 1 showed the [M+H]⁺ m/z 481.16986, [2 M+NH₄]⁺ m/z 978.35950, [M–H]⁻ m/z 479.15591, [M–H+HCOOH]⁻ m/z 525.16101, and [2 M−H+HCOOH]⁻ m/z 1005.32404 and the corresponding elemental compositions were C₂₃H₂₉O₁₁, C₄₆H₆₀O₂₂N, C₂₃H₂₇O₁₁, C₂₄H₂₉O₁₃, and C₄₇H₅₇O₂₄, respectively. On the basis of above data we deduced that the elemental composition of peak 1 was C₂₃H₂₈O₁₁. The molecular ion of peak 1 could lead to seven main MS² ions at m/z 319.11731, 197.08075, 133.06473, and 105.03342 in positive ion mode, and m/z 479.15594, 283.08231, and 121.02956 in negative ion mode. On the basis of the elemental compositions of fragment ions, peak 1 was assigned as albiflorin. Peaks 2 and 3 were therefore identified as paeoniflorin, and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose with above mentioned method. The mass spectra and proposed fragmentations of albiflorin, paeoniflorin, and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose were shown in Figure 2.

(a)

(b)

(c)

Figure 2: Mass spectra and proposed fragmentations of albiflorin (a), paeoniflorin (b), and 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucopyranose (c).

3.2. Identification of the Main Components in Crude and Processed Atractylodis Macrocephalae Rhizoma

Figure 3 showed the total ion chromatograms of crude and processed Atractylodis Macrocephalae Rhizoma samples obtained from both positive and negative ion modes. 32 and 26 compounds were identified in crude and processed Atractylodis Macrocephalae Rhizoma samples, respectively. Compounds 2, 4, 13, 14, 17, and 29 were not detected in processed Atractylodis Macrocephalae Rhizoma sample. Moreover, the amounts of compounds 3, 7, 9, 10, 21, 23, and 27 were substantially decreased, and the amounts of compounds 8, 18, and 22 were increased in processed sample compared with crude one. The results were shown in Table 2.

Table 2: Major chemical constituents identified in crude and processed Atractylodis Macrocephalae Rhizoma and in crude and processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair.

No.
(min)
Compound name
Formula
Atractylodis Macrocephalae Rhizoma
Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair

(Measured area)
(Measured area)

Crude
Processed
Crude
Processed

1
1.72
Protocatechuic acid
C₇H₆O₄

2
2.67
Protocatechuic acid isomer I
C₇H₆O₄

—
—
—

3
3.24
Caffeic acid
C₉H₈O₄

4
3.73
Protocatechuic acid isomer II
C₇H₆O₄

—
—

5
4.21
Dictamnoside A isomer I
C₂₁H₃₆O₉

6
4.70
Dictamnoside A isomer II
C₂₁H₃₆O₉

7
5.63
Scopoletin
C₁₀H₈O₄

8
5.82
Dictamnoside A
C₂₁H₃₆O₉

9
8.77
Atracetylentriol
C₁₄H₁₆O₃

—
—

10
9.33
Ferulic acid
C₁₀H₁₀O₄

11
25.81
Atractylenolide I isomer
C₁₅H₁₈O₂

12
25.83
Atractylenolide III
C₁₅H₂₀O₃

13
26.17
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol
C₂₁H₂₆O₅

—
—
—

14
26.95
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer
C₂₁H₂₆O₅

—
—
—

15
31.10
Atractylenolide II isomer
C₁₅H₂₀O₂

16
31.66
Atractylenolide II
C₁₅H₂₀O₂

17
33.44
Atractylodin
C₁₃H₁₀O

—

—

18
35.07
Atractylenolide I isomer
C₁₅H₁₈O₂

19
35.94
Atractylenolide I
C₁₅H₁₈O₂

20
39.03
12-methylbutyryl-14-acetyl-2E,8EZ,10E-atractylentriol isomer I
C₂₁H₂₆O₅

—

21
39.81
Dibutyl phthalate
C₁₆H₂₂O₄

22
40.00
12-methylbutyryl-14-acetyl 2E,8EZ,10E-atractylentriol isomer II
C₂₁H₂₆O₅

23
40.26
Dibutyl phthalate isomer
C₁₆H₂₂O₄

24
41.50
14-methylbutyryl-2E,8EZ,10Es-atractylentriol
C₁₉H₂₄O₄

25
46.43
Spinasteryl
C₂₉H₄₈O

26
47.32
Atractylon
C₁₅H₂₀O

—

27
47.37
Biatractylolide
C₃₀H₃₈O₄

—

28
47.96
Linoleic acid
C₁₈H₃₂O₂

29
48.25
Linoleic acid isomer
C₁₈H₃₂O₂

—
—
—

30
48.59
Biepiasterolid isomer
C₃₀H₃₈O₄

—

31
48.90
Atractylon isomer
C₁₅H₂₀O

32
49.42
Palmitic acid
C₁₆H₃₂O₂

(a)

(b)

Figure 3: Total ion chromatograms of crude (a) and processed (b) Atractylodis Macrocephalae Rhizoma obtained from both positive and negative ion modes.

Atractylenolide I, atractylenolide II, and atractylenolide III are the main active compounds that belong to the sesquiterpenes in Atractylodis Macrocephalae Rhizoma. The mass spectra of atractylenolide I showed a [M+H]⁺ ion at m/z 231.13799, which could lead to four MS² ions at m/z 213.12740, 185.13251, 157.10127, and 143.08569. The molecular ion of atractylenolide II ([M+H]⁺ m/z 233.15358) could lead to six MS² ions at m/z 215.14310, 187.14818, 159.08055, 151.07541, 133.10117, and 95.08547. Meanwhile, the MS² spectrum of m/z 249.14836 from atractylenolide III contained six major fragment ions at m/z 231.13802, 213.12758, 189.09108, 163.07541, 135.04411, and 105.06989. The mass spectra of the above three compounds were shown in Figure 4.

(a)

(b)

(c)

Figure 4: Mass spectra of atractylenolide I (a), atractylenolide II (b), and atractylenolide III (c).

3.3. Analysis of Chemical Changes of Paeoniae Radix Alba after Compatibility with Atractylodis Macrocephalae Rhizoma

In the present study, the Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS based on chemical profiling approach was used to evaluate chemical constitution between co-decoction and single decoction of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. For crude Paeoniae Radix Alba, the relative contents of most compounds were dramatically decreased except those of compounds 80, 90, 98, 113, 119, and 122 were significantly increased and 19 compounds were not detected after its compatibility with crude Atractylodis Macrocephalae Rhizoma. For processed Paeoniae Radix Alba, the relative contents of compounds 12, 36, 84, and 86 were remarkably increased except 12 compounds including pedunculagin, oxypaeoniflorin, 6-O-glucopyranosyl-lactinolide, 1, 2, 3, 6-tetra-O-galloylglucose isomer A, 1, 2, 3, 6-tetra-O-galloylglucose isomer B, tetragalloyl glucose C, galloylpaeoniflorin isomer II, hexagalloyl glucose, 3, 6-di-O-galloyl paeoniorin isomer, oxybenzoyl-oxypaeoniflorin, benzoyloxypaeoniflorin, and albiflorin R1 isomer III were newly generated and 13 compounds were not found after its compatibility with processed Atractylodis Macrocephalae Rhizoma. The results were presented in Figure 5 and Table 1.

(a)

(b)

Figure 5: Total ion chromatograms of crude (a) and processed (b) Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair obtained from both positive and negative ion modes.

3.4. Analysis of the Chemical Changes of Atractylodis Macrocephalae Rhizoma after Compatibility with Paeoniae Radix Alba

For crude Atractylodis Macrocephalae Rhizoma, the relative contents of compounds 17, 18, and 25 were increased clearly except those of compounds 6, 23, and 30 decreased considerably and six compounds including protocatechuic acid isomer I, protocatechuic acid isomer II, atracetylentriol, 12-methylbutyryl-14-acetyl-2E, 8EZ, 10E-atractylentriol, 12-methylbutyryl-14-acetyl-2E, 8EZ, 10E-atractylentriol isomer, and linoleic acid isomer were lost after its compatibility with crude Paeoniae Radix Alba. For processed Atractylodis Macrocephalae Rhizoma, compounds 9, 20, 26, 27, and 30 were not found except the relative contents of compounds 5, 6, and 8 were decreased while those of compounds 15, 19, 21, and 31 were increased after its compatibility with processed Paeoniae Radix Alba. Furthermore, compound 4 (protocatechuic acid isomer II) was not found in processed Atractylodis Macrocephalae Rhizoma but could be detected in processed Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair by using Exact Finder and MassFrontier softwares. The above results illustrated that Paeoniae Radix Alba significantly changed the components of Atractylodis Macrocephalae Rhizoma in solution when they decocted together. The corresponding results were presented in Figure 5 and Table 2.

4. Conclusions

Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS is a powerful tool for discriminating the chemical changes between single herbal and co-decocting medicines. In our present study, the Q Exactive high-performance benchtop quadrupole-Orbitrap LC-MS/MS based on chemical profiling approach to investigate and evaluate chemical changes from crude and processed Paeoniae Radix Alba, crude and processed Atractylodis Macrocephalae Rhizoma, and their crude and processed herbal pair extracts was proposed. The results showed that processing and compatibility of TCM could significantly change the chemical composition of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. The developed method is considered to provide a scientific foundation for deeply elucidating the processing and compatibility mechanism of Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Gang Cao, Qinglin Li, Hao Cai, and Sicong Tu contributed equally to this work.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (nos. 81202918, 81173546, and 30940093), the Natural Science Foundation of Jiangsu Province, China (no. BK2009495), the International Science and Technology Cooperation Project of Jiangsu Province, China (no. BZ2011053), the Open Project of National First-Class Key Discipline for Science of Chinese Materia Medica, Nanjing University of Chinese Medicine (no. 2011ZYX2-006), the Project of Science and Technology for Chinese Medicine of Zhejiang Province, China (no. 2013KYB183), the Science and Technology Project of Hangzhou, China (nos. 20130533B68, 20131813A23), the Chinese Medicine Research Program of Zhejiang Province, China (nos. 2014ZQ008, 2008ZA002), the Project of Science Technology Department of Zhejiang Province, China (no. 2013C33SA1C0003), and the Science Foundation of Zhejiang Chinese Medical University (nos. 2011ZY25, 2013ZZ12).

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