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Molecules 2014, 19(5), 5650-5673; doi:10.3390/molecules19055650

Article
Chemical Profile Analysis and Comparison of Two Versions of the Classic TCM Formula Danggui Buxue Tang by HPLC-DAD-ESI-IT-TOF-MSn
Ya-Zhou Zhang 1,2, Feng Xu 3, Tao Yi 1, Jian-Ye Zhang 1, Jun Xu 1, Yi-Na Tang 1, Xi-Chen He 1, Jing Liu 1 and Hu-Biao Chen 1,*
1
School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon, Hong Kong, China
2
Guizhou College of Technology, No.1 Caiguan Road, Guiyang 550003, China
3
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China
*
Author to whom correspondence should be addressed; E-Mail: hbchen@hkbu.edu.hk; Tel.: +852-3411-2060; Fax: +852-3411-2461.
Received: 5 March 2014; in revised form: 10 April 2014 / Accepted: 19 April 2014 /
Published: 30 April 2014

Abstract

: Danggui Buxue Tang (DBT) is a Traditional Chinese Medicine (TCM) formula primarily used to treat symptoms associated with menopause in women. Usually, DBT is composed of one portion of Radix Angelicae Sinensis (RAS) and five portions of Radix Astragali (RA). Clinically, Radix Hedysari (RH) is sometimes used by TCM physicians to replace RA in DBT. In order to verity whether the chemical constituents of the DBT1 (RA:RAS = 5:1, w/w) and DBT2 (RH:RAS = 5:1, w/w) share similarities the chemical profiles of the two DBTs crude extracts and urine samples were analyzed and compared with the aid of HPLC-DAD-ESI-IT-TOF-MSn, which determines the total ion chromatogram (TIC) and multi-stage mass spectra (MSn). Then, the DBT1 and DBT2 were identified and compared on the basis of the TIC and the MSn. In the first experiment (with crude extracts), 69 compounds (C1C69) were identified from the DBT1; 46 compounds (c1c46) were identified from the DBT2. In the second experiment(with urine samples), 44 compounds (M1M44) were identified from the urine samples of rats that had been administered DBT1, and 34 compounds (m1m34) were identified from the urine samples of rats that had been administered DBT2. Identification and comparison of the chemical compositions were carried out between the DBT1 and DBT2 of the crude extracts and urine samples respectively. Our results showed that the two crude extracts of the DBTs have quite different chemical profiles. The reasons for their differences were that the special astragalosides in DBT1 and the isoflavonoid glycosides formed the malonic acid esters undergo single esterification and acetyl esters undergo acetylation in DBT1. In contrast, the urine from DBT1-treated rats strongly resembled that of DBT2-treated rats. These metabolites originate mainly from formononetin, calycosin and their related glycosides, and they were formed mainly by the metabolic process of reduction, deglycosylation, demethylation, hydrogenation and sulfation. The HPLC-DAD-ESI-IT-TOF-MSn method was successfully applied for the rapid chemical profiles evaluation of two DBTs and their related urine samples.
Keywords:
Radix Hedysari; Radix Astragali; Danggui Buxue Tang; HPLC-DAD-ESI-IT-TOF-MSn; isoflavonoid; astragaloside

1. Introduction

Danggui Buxue Tang (DBT) is a Traditional Chinese Medicine (TCM) formula primarily used to treat symptoms associated with menopause in women. It is believed to invigorate ‘Qi’ (vital energy) and nourish the ‘Blood’ (body circulation) [1]. Nowadays, it is commonly used in China as an efficacious medicinal prescription and a healthy food supplement. Pharmacological studies have found that DBT promotes hematopoietic function [2,3], regulates blood lipid and anti-inflammatory activities in diabetic atherosclerosis [4,5], anti-fibrosis effects [6], prevents osteoporosis [7,8], and increases anti-oxidation activity as well as immune response [9]. According to its original formula, DBT comprises Radix Astragali (RA) and Radix Angelicae Sinensis (RAS) (5:1, w/w). More recently, Radix Hedysari (RH) has been used to replace RA. Thus, in current clinical applications, DBT is prescribed in two forms: RA:RAS (5:1) (called DBT1), and RH:RAS (5:1) (called DBT2) [1,10].

The plants RA and RH belong to the same botanical family but different genus, and have long been widely used as the same crude herb in DBT [1]. This is always a question of whether RH can replace RA in the DBT decoction. Chemically, RA-containing DBT showed higher amounts of calycosin-7-O-β-d-glucoside, ferulic acid, ononin, calycosin, astragaloside IV, astragaloside III, and Z-ligustilide. Only formononetin was higher in RH-containing DBT. In parallel, the estrogenic, osteogenic and erythropoetic effects of RA-containing DBT1 showed better activities than that of RA-containing DBT2 [1]. So far, the chemical differences between DBT1 and DBT2 has not been investigated. Therefore, we designed a systematic comparison of the chemical ingredients of DBT1 and DBT2.

Two experiments were designed, including thorough elucidation of the chemical profiles of DBT1 and DBT2 crude extracts and illumination of the metabolites of DBT1 and DBT2 after being administrated to rats. The chemical profiles of the two DBTs were compared by determining the total ion chromatogram(TIC) and the multistage mass spectra (MSn) from HPLC-DAD-ESI-IT-TOF-MSn. Subsequently, DBT1 and DBT2 were identified and compared on the basis of the TIC and the MSn [11]. The results will be provide a solid evidence to understand the chemical profiles of the two different versions of DBT.

2. Results and Discussion

2.1. Optimization the Conditions of HPLC and Mass Spectrometry

In order to obtain desirable HPLC and mass spectrometry chromatograms, the procedures for preparation of the urine samples and crude extracted samples of the two DBTs were optimized in terms of the extraction solvents and extraction times. Methanol and acetonitrile were initially selected as the extraction solvents, but methanol is less poisonous and produced almost the same chromatograms as acetonitrile, so it was applied as the final extraction solvent. For comparison, different columns (Phenomenex RP C18, Agilent RP C18) were tested for sample separation, and Phenomenex RP C18 gave the best chromatographic resolution. The column was eluted with a gradient mobile phase that consisted of water-formic acid (100:0.1, v/v) (A), acetonitrile (B) and at a flow rate of 1.0000 mL/min, in addition, 0.1% (v/v) formic acid was added to improve the mass spectrometry ionization efficiency and enable symmetric peak shapes [12]. Both the positive ion (PI) and negative ion(NI) modes were tested for the experiment. Since MS and MSn fragmentations gave more information about the isoflavones in PI mode but about saponins in NI mode, the analysis was simultaneously conducted in both PI and NI mode.

2.2. The Identification and Analysis of 19 Reference Compounds

Nineteen reference compounds which might represent the major structural types of the DBTs were analyzed. The characteristic fragment ions of 19 references are very useful for determining the structural skeleton and the substitution patterns of those related compounds in two DBTs. Their high resolution mass spectra (HRMS) data are summarized (see Table 1 and Supplementary: pages S2–S8). The base peak chromatograms (BPCs) detected in NI and PI mode were recorded (see Figure 1a). Furthermore, the structure of 19 reference compounds were shown (Figure 1b) [11,13].

According to our preliminary research, there three main types of isoflavonoids are found in Radix Astragali which were named as a: isoflavone (T1, T2, T5, T6, and T8), b: isoflavan (T4, T7, and T10) and c: pterocarpan (T3, and T9) [11,13,14,15]. To facilitate the structural identification of the isoflavonoids in the DBTs, the fragmentation behaviors of the three types of isoflavonoids were analyzed, which might represent the major structural types. We first studied the MSn fragmentation behaviors in PI and NI mode, and found that the fragmentation behaviors in PI mode could give more information about the structure than in NI modes. Then we elucidated the structure of the three types of isoflavonoids mostly from the PI mass spectra.

Isoflavone had the characteristic fragment ions 5B+-2H, 0,3B+-2H, 5A+-2H, 1,3A+-2H, 3,4A+-2H, etc., isoflavan had the characteristic fragment ions 5B+-2H, 5A+-2H, 1,3A+-2H, etc., and pterocarpan had the characteristic fragment ions 6,7B+-2H, 1,4B+-2H, 3,4A+-2H, 5,6A+-2H, etc., based on MS2 and MS3 spectra by HPLC-DAD-ESI-IT-TOF-MSn (see Figure 2).

For example, we identified the characteristic malonate-glucose-, acetyl-glucose- and glucose-binding ingredients with a neutral loss of 248 Da, 204 Da, and 162 Da, otherwise, glucuronide metabolites with a neutral loss of (−176 Da) and sulfated metabolites with a neutral loss of (−80 Da) from the molecular ion peaka in the MS2 spectra [13,16,17].

Table Table 1. The fragment ions of 19 kinds of reference compounds by HPLC-DAD-ESI-IT-TOF-MSn.

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Table 1. The fragment ions of 19 kinds of reference compounds by HPLC-DAD-ESI-IT-TOF-MSn.
NO.TR (min)[M+H]+[M−H]Predicted FormulaFragment Ions DaError (ppm)The Name of the Reference Compounds
1(T1)28.595447.1290 C22H22O10447,285,270,2250.89Calycosin-7-O-β-d-glucopyranoside
2(AW)29.162 193.0501C10H10O4193,178,134−2.59Ferulic acid
3(T2)34.545 431.1326C22H22O9431,269,253,237,213,197,163,134,107−2.55Ononin
4(T3)36.038485.1400[M+Na]+C23H26O10485,463,323,301−3.71Astrapterocarpan-7-O-β-d-glucopyranoside
5(T4)36.785487.1574[M+Na]+C23H28O10487,303,167−0.21Astraisoflavan-7-O-β-d-glucopyranoside
6(T5)38.055285.0754 C16H12O5285,270,253,225,197,137−1.40Calycosin
7(T6)40.123473.1432 C24H24O10473,269−2.116''-O-acetyl-ononin
8(T7)41.942529.1691[M+Na]+C25H30O11529,507,3032.086''-O-acetyl-astraisoflavan-7-O-β-d-glucopyranoside
9(T8)45.410269.0795 C16H12O4269,270,237−4.83Formononetin
10(T9)46.062301.1076 C17H16O5301,271,251,167,151,1341.66(6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan; Astrapterocarpan
11(T10)46.655303.1213 C17H18O5303,181,167,149,123−4.62(3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan; Astraisoflavan
12(GⅤ)41.110[M+HCOO]991.5156C47H78O19991,783,3973.73Astragaloside Ⅴ
13(GⅣ)42.775[M+HCOO]829.4615C41H68O14829,783,621,489,3832.89Astragaloside Ⅳ
14(GⅢ)43.410[M+HCOO]829.4585C41H68O14829,783,651,489−0.72Astragaloside Ⅲ
15(GⅡ)44.628[M+HCOO]871.4693C43H70O15871,765,717−0.46Astragaloside Ⅱ
16(GⅠ)50.525[M+HCOO]913.4802C45H72O16913,867,8070.00Astragaloside Ⅰ
17(HHQC)51.855513.3550[M+Na]+C30H50O5513,515,405,2290.00Cycloastragenol(HHQC)
18(ZL)55.477 191.1056 C12H14O2191,173,117−5.76Z-ligustilide
19(EL)58.102191.1066 C12H14O2191,173−0.52E-ligustilide
Molecules 19 05650 g001 1024
Figure 1. (a) LC-MS chromatogram of 19 reference compounds in PI (1BPC) and NI (4BPC) mode. (b) The chemical structures of 19 reference compounds.

Click here to enlarge figure

Figure 1. (a) LC-MS chromatogram of 19 reference compounds in PI (1BPC) and NI (4BPC) mode. (b) The chemical structures of 19 reference compounds.
Molecules 19 05650 g001 1024
Molecules 19 05650 g002 1024
Figure 2. The bond cleavage pathways of the three types of isoflavonoids in RA.

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Figure 2. The bond cleavage pathways of the three types of isoflavonoids in RA.
Molecules 19 05650 g002 1024

2.3. Profiling and Identifying Chemical Compounds of the Two Crude Extracts (DBT1, and DBT2) by HPLC-DAD-ESI-IT-TOF-MSn

2.3.1. Identification of the Chemical Profiles of DBT1 by HPLC-DAD-ESI-IT-TOF-MSn

The HRMS data of these identified compounds are summarized (see Table 2 and Supplementary: pages S9–S35). The BPCs detected in PI (1BPC) and NI (4BPC) mode were also recorded (Figure 3a) [17,18,19].

By comparing the fragment ions and retention times and based on the high resolution mass spectra software predicted formulas with the reference compounds from the MS and MSn, the compounds C23, C33, C36, C37, C38, C44, C46, C56, C58, C59, C49, C53, C60, C64, and C68 were identified as the reference compounds [11,13].

C27 has a RT at 30.773 min, [M+HCOO] at m/z 671.2155 in MS (predicted formula: C29H38O15: ppm error: −5.66), and characteristic fragment ions at m/z 625.2079 (−46 Da) [M-H], m/z 463.1589 [M-162-H], and m/z 301.1108 [M-162-162-H] in MS2. The neutral loss is mass 46 Da (CH2O2; identified as HCOOH), 162 Da*2 (C6H10O5; identified as glucopyranoside), and the fragment ion m/z 301.1108 predicted as C17H18O5. Then C27 was identified as astraisoflavan-di-7-O-β-d-glucoside or its isomer. The [M+H]+ or [M-H] of C24, C29, C30, and C41 shows the same neutral loss of −162 Da (C6H10O5; glucoside) in them MS2, so both of them were identified as the glycosides [17,18,19]. The characteristic fragment ions of C31, C35, C43, C42, and C51 have a neutral loss of −248 Da (C9H12O8; identified as the 6''-O-malonate-glucoside) in them MS2, so both of them were identified as glycosides of 6''-O-malonate-glucoside [13].The HRM software predicted [M+H]+ or [M-H] of C32, C40, C34, C47, and C18, whose formulas have the same characteristic fragment loss (−204 Da; C8H12O6) which was identified as 6''-O-acetylglucosides. In addition, C32, C40, C34, C47, and C18 were identified as glycosides of 6''-O-acetylglucoside [16].

For the predicted formulas of [M+H]+ or [M-H], we tentatively identified C39, C50, C62, C30, and C41 as the isoflavonoid-related constituents by referring to the literature [18,19], and C45, C55, C48, C52, C54, C61, and C65 were tentatively identified as being related to saponins [13,17,18,19].

Using the [M+H]+, [M-H] or [M+Na]+ data of C7, C8, C10, C12, C13, C14, C21, C25, C26, and C28, we predicted their formulas, which indicates that they are the ingredients of the samples. However, at this point, their exact structures could not be identified.

Table Table 2. The identified proposed compounds of the crude extract samples from Danggui Buxue Tang 1 and Danggui Buxue Tang 2 by HPLC-DAD-ESI-IT-TOF-MSn.

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Table 2. The identified proposed compounds of the crude extract samples from Danggui Buxue Tang 1 and Danggui Buxue Tang 2 by HPLC-DAD-ESI-IT-TOF-MSn.
NO.TR (min)[M+H]+[M−H]Predicted FormulaFragment Ions DaError ppmIdentificationDBT1DBT2
12.395 173.1044C6H14N4O2 0.00ArginineC1c1
22.692 195.0502C6H12O7 −4.10Gluconic acidC2c2
32.695 341.1074C12H22O11 −4.40d(+)sucroseC3c3
43.643 191.0187C6H8O7191,173−5.24Citric acidC4c4
54.477 328.0427C11H11N3O9 1.22--C5c5
66.480346.0529 C11H11N3O10 3.47--C6c6
79.063 433.1364C18H26O12301,1914.91--C7--
89.120443.1146[M+Na]+C17H24O12 −3.16------c7
99.345267.1369 C18H18O2267,225−4.12Magnolol--c8
109.398 433.1364C18 H26O12433,351,301,2232.77--C8--
1110.773188.0688 C9 H11NO21463.19L-phenylalanineC9c9
1211.125 431.1192C18 H24O12431,299−0.70--C10--
1312.653384.1127 C13H21NO12 −2.60--C11c10
1413.840 461.1283C19H26O13461,167−3.90----c11
1513.847485.1224[M+Na]+C26H22O8485,3173.50--C12--
1617.807 205.0701C8H14O6 −8.29--C13--
1721.147 315.2004C20H28O3 12.06--C14--
1821.207 433.1129C21H22O10433,285−2.54----c12
1921.322 433.1121C21H22O10433,285,241−4.39----c13
2022.020 417.1017C17H22O12417,285,152-5.03--C15c14
2123.080389.2325 C23H32O5 0.51--C16c16
2223.018 401.1445C18H26O10401,269,161−1.99--C17c15
2323.590 503.1175C24H24O12503,443,299−3.986''-O-acetyl-pratensein-7-O-β-d-glucosideC18--
2424.347 239.0568C11H12O6 2.93--C19--
2525.662331.2296 C21H30O3331,2998.45--C20--
2626.743470.1534 C18H23N5O10 3.40--C21--
2726.967289.1747 C13H24N2O5289,272,152−3.80--C22c17
2830.653 479.1492C30H24O6479,317−1.67--C26--
2930.773[M+HCOO]671.2155C29H38O15671,625,463,301−5.66Astraisoflavan-di-7-O-β-d-glucosideC27--
3031.005 579.2062C23H36N2O15579,417,3873.28--C28--
3131.348 445.1123C22H22O10445,283−3.82Glycetein-4'-O-β-d-glucosideC29--
3232.078463.1203 C22H22O11463,301−6.91Kaempferide-7-O-β-d-glucosideC30--
3332.662533.1267 C25H24O13533,285−4.316''-O-malonate-calycosin-7-O-β-d-glucosideC31--
3434.207489.1398 C24H24O11489,2851.436''-O-acetyl-calycosin-7-O-β-d-glucosideC32--
3534.517431.1322 C22H22O9431,269,237,118−3.48OnoninC33c18
3635.348 489.1340C24H26O11489,285,271,159−12.686''-O-acetyl-isosakuranetin-7-O-β-d-glucosideC34--
3735.580549.1174 C25H24O14549,301−11.846''-O-malonate-kaempferide-7-O-β-d-glucosideC35--
3836.027463.1615 C23H26O10485(+Na+),463,3013.45Astrapterocarpan-7-O-β-d-glucopyranoside C36--
3936.773 463.1577C23H28O10463,301,271−7.13Astraisoflavan-7-O-β-d-glucopyranoside C37--
4037.480 255.0657C15H12O4255,135−2.35Isoliquiritigenin --c19
4138.035285.0744 C16H12O5285,270,225,137−4.91CalycosinC38c20
4239.917 269.0807C16H14O4269,253,227−4.46Isomer of alpinetin--c21
4339.977 255.0657C15H12O4255,237−2.35Liquiritigenin--c22
4438.653315.0844 C17H14O6 −6.034-methoxy-maackiain or the isomerC39--
4538.653473.1445 C24H24O10473,2690.63The isomer of 6''-O-acetyl-ononinC40--
4638.773447.1264 C22H22O10447,285−4.92Glycetein-7-O-β-d-glucosideC41--
4738.997549.1545 C26H28O13549,301−10.566''-O-malonate-astrapterocarpan-glucosideC42--
4839.452517.1301 C25H24O12517,269−7.736''-O-malonate-ononinC43--
4939.683 505.1699C25H30O11505,301−3.176''-O-acetyl-astraisoflavan-7-O-β-d-glucosideC44--
5039.745 957.5030C48H78O19957,541,453−3.66Soyasaponin BaC45c23
5140.138473.1424 C24H24O10473,269−3.806''-O-acetyl-ononinC46--
5240.903 927.4915C47H76O18 −4.74Akebia saponin d--c24
5340.430 503.1154C24H24O12503,299−8.156''-O-acetyl-kaempferide-7-O-β-d-glucosideC47--
5440.825 785.4629C41H70O14 −8.15Cyclocanthoside EC48--
5541.108 991.5086C48H80O21 −3.33Astragaloside VC49--
5641.273 285.0751C16H14O5285,194,109−5.96Isomer of isosakuranetin--c25
5741.772 269.0456C15H10O5269,2370.37Genistein--c26
5841.778 315.0868C17H16O6315,253−1.90Astragaluquinone or isomerC50--
5941.950533.1243 C25H24O13533,285−8.826''-O-malonate-glycetein-7-O-β-d-glucosideC51--
6042.062 867.4635C45H72O16 −13.03Isoastragaloside IC52--
6142.243 283.0602C16H12O5283,268,224−3.53Glycetein--c27
6242.612[M+HCOO]829.4572C41H68O14 −2.29Astragaloside IVC53--
6342.560 825.4532C43H70O15871,825−13.33Isoastragaloside IIC54--
6442.965 329.2319C18H34O5 −4.25----c28
6543.197 955.4857C48H76O19 −5.34----c29
6644.690 287.0577C15H12O6 5.57Dihydro-kaempferol--c30
6744.982 255.0649C15H12O4256,135−5.49Isomer of Liquiritigenin--c31
6845.315269.0791 C16H12O4269,254,237,118−6.32FormononetinC56c32
6944.190 941.5081C48H78O18941,525,437−3.61Soyasaponin BbC55c35
7045.745299.0911 C17H14O5299,284,166 Pterocarpin--c33
7145.935 329.2299C18H34O5 −10.33--C57--
7246.080 283.0599C16H12O5283,255,240−4.59The isomer of glycetein--c34
7346.158301.1052 C17H16O5 −6.31AstraoptercarpanC58--
7446.708303.1181 C17H18O5 −15.18AstraisoflavanC59--
7546.768[M+HCOO]871.4656C43H70O15871,825,603−4.70Astragaloside II C60--
7647.172[M+HCOO]911.4668C45H70O16955,9112.41--C61--
7747.772 299.0552C16H12O6 −3.01Kaempferide or isomer--c36
7847.943 909.4836C47H74O17 −1.87Acetylastragaloside I--c37
7948.347[M+HCOO]911.5011C46H74O15 0.11Castaraleside H--c38
8049.432 285.0423C15H10O6285,1636.31Kaempferol or isomerC62--
8150.065 939.4925C48H76O18 −3.62----c39
8250.435285.0748 C16H12O5285,253,152−3.51Isomer of calycosin--c40
8350.667335.2180[M+Na]+C18H32O4 −3.88--C63c41
8450.713[M+HCOO]913.4777C45H72O16 −2.74Astragaloside IC64--
8551.560193.1212 C12H16O2 −5.70Senkyunolide A--c42
8651.607[M+HCOO]953.4637C47H72O17953,909−12.06--C65--
8752.363437.3374[M+Na]+C28H46O2 −3.66--C66--
8855.212191.1043 C12H14O2 −12.56n-butyl-phthalide--c43
8959.250213.0876[M+Na]+C12H14O2403,213−4.69Z-ligustilideC67c44
9061.708403.1867[2M+Na]+C12H14O2403,381,191−3.22E-ligustilideC68c45
9169.093 283.0257C15H8O6283,203,1473.18--C69c46
Molecules 19 05650 g003 1024
Figure 3. (a) The BPC in NI and PI mode of the crude extracts of Danggui Buxue Tang 1 (RA:RAS = 5:1). (b) The BPC in NI and PI mode of the crude extracts of Danggui Buxue Tang2 (RH:RAS = 5:1). (c) The BPC in NI and PI mode of the urine samples of rats that had been administrated the Danggui Buxue Tang 1 (RA:RAS = 5:1) and (d) The BPC in NI and PI mode of the urine samples of rats had been administrated the Danggui Buxue Tang 2 (RH:RAS = 5:1).

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Figure 3. (a) The BPC in NI and PI mode of the crude extracts of Danggui Buxue Tang 1 (RA:RAS = 5:1). (b) The BPC in NI and PI mode of the crude extracts of Danggui Buxue Tang2 (RH:RAS = 5:1). (c) The BPC in NI and PI mode of the urine samples of rats that had been administrated the Danggui Buxue Tang 1 (RA:RAS = 5:1) and (d) The BPC in NI and PI mode of the urine samples of rats had been administrated the Danggui Buxue Tang 2 (RH:RAS = 5:1).
Molecules 19 05650 g003 1024

2.3.2. Identification of the Chemical Profiles of DBT2 by HPLC-DAD-ESI-IT-TOF-MSn

The HRMS data of these identified compounds are summarized (see Table 2 and Supplementary: pages S36–S51). The BPCs detected in NI and PI modes were recorded (see Figure 3b).

Using their fragment ions and retention times in MS data, c18, c20, c32, c44, and c45 were identified as the reference compounds [11,13].

Based the MS data, c12 shows RT at 21.207 min, [M-H] at m/z 433.1129 in MS (predicted the formula: C21H22O10: ppm error: −2.54), and characteristic fragment ions at m/z 285.0744 (−148 Da; C5H8O5; identified as the ribonic acid) and predicted as C16H14O. Compound c12 was identified as the isomer of isosakuranetin-ribonic acid. Moreover, c13 shows a characteristic neutral loss at −148 Da (C5H8O5) with the same as c12 [16].

By the formulas predicted of [M-H] or [M+H]+ and referring to literature [13,17,18,19], c23, c24, c29, c35, c38, and c39 were tentatively identified as saponin-related constituents, and c7, c8, c11, c19, c21, c22, c25, c26, c27, c31, c33, c34, c36, c40, and c43 were tentatively identified (see in Table 2).

By using the HRMS data (RT, Predicted the formulas and characteristic fragment ions) compared with the BDT1 crude extract samples, C1, c1; C2, c2; C3, c3; C4, c4; and C9, c9 were identified as the same constituents [17,18,19].

The groups of C5, c5; C6, c6; C11, c10; C15, c14; C16, c16; C17, c15; C22, c17; C57, c28; C63, c41; and C69, c46 between DBT1 and DBT2 were tentatively identified as the same compounds with uncertain structures.

From the analysis based on the comparison of TIC and MSn: 69 compounds (C1–C69) were identified from the crude extracts of DBT1, 46 compounds (c1–c46) were identified from the crude extracts of DBT2. The isoflavonoids glycosides had experienced acetylation (seven compounds, C18, C32, C34, C40, C44, C46, and C47), formed the malonate acid esters (five compounds, C31, C35, C42, C43, and C51) and with special astragalosides (six compounds, C49, C52, C53, C54, C60, and C64) in DBT1. Thus, the number of identified components in DBT1 was significantly more than in DBT2 (the chemical structural diversity of isoflavonoids which were detected in DBT1 more than in DBT2 are shown in Figure 4). Among these, the 24 common chemical constituents accounted for approximately 27% to the total 91 identified compounds. However, and the proportion of the total isoflavonoids and saponins to the total identified ingredients accounted for nearly 62% (see Table 3).

Molecules 19 05650 g004 1024
Figure 4. The chemical structures of the main proposed different isoflavonoids in Danggui Buxue Tang 1 more than Danggui Buxue Tang 2.

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Figure 4. The chemical structures of the main proposed different isoflavonoids in Danggui Buxue Tang 1 more than Danggui Buxue Tang 2.
Molecules 19 05650 g004 1024
Table Table 3. The number comparison of the identified compounds between Danggui Buxue Tang 1 and Danggui Buxue Tang 2.

Click here to display table

Table 3. The number comparison of the identified compounds between Danggui Buxue Tang 1 and Danggui Buxue Tang 2.
N.ID.S-ID.T.ID.S%H + SS%
DBT16924115−24 = 9126.375661.54
DBT246
DBT1-U441978−19 = 5932.204881.35
DBT2-U34

ID. Total identified proposed compounds number; S-ID. Identified the common proposed compounds number between two Danggui Buxue Tangs; T.ID. Identified the unfamiliar proposed compounds number between two Danggui Buxue Tangs; H + S: The total isoflavones and the total saponins number; S% The ratio about the selective compounds in the total identified proposed compounds number.

2.4. Profiling and Identifying Chemical Profiles of the Urine Samples after Administration of the DBT1 and DBT2 Performed by HPLC-DAD-ESI-IT-TOF-MSn

In the study of the existing literature, ingredients such as isoflavones, saponins in the two DBTs had no obvious differences in chemical profiles between the serum and bile samples collected from enterohepatic circulation. In addition, they had a lower concentration in serum samples, even when giving at dosages of 60–120 g/kg (w/w) several times to rats within 24 h [13,17,20]. Thus, this approach is not conducive to tracing these minor components. This study chose the normal usage of 10 g/kg (w/w) by comparing the urine samples of rats that were administrated two different DBTs, so as to improve the detection through enrichment of the treatments.

2.4.1. Identification of the Chemical Profiles of Urine Sample after Administration of the DBT1 by HPLC-DAD-ESI-IT-TOF-MSn

The HRMS data of these identified metabolites are summarized (see Table 4 and Supplementary: pages S52–S66). The BPCs detected in NI mode were recorded (see Figure 3c). In addition, the main proposed structures of these metabolites identified from the urine samples of rats that had been administrated DBT1 were showed (see Figure 5).

Using MS data with the reference compounds, M15 was identified as calycosin, and M23 was identified as formononetin [11,13].

With the predictions of [M-H] or [M+HCOO], and the characteristic fragment ions, M9, M7, M19, M8, M10, M11, M13, M14, M22, M16, M18, M20, M21, M24, M32, and M36 were tentatively identified as the metabolites of isoflavonoids [13,17,18,19,21].

M25 shows RT at 46.290 min, [M+H]+ at m/z 335.0201 in MS (predicted formula: C15H10O7S: ppm error: −5.67), and characteristic fragment ions at m/z 255.0637 (−80 Da; SO3; identified as the sulfonyl hydroxide) and predicted as C15H10O4. Then M25 was identified as daidzein after sulfation. M27, M26, M34, M28, M29, M30, M31, M33, M35, M37, M38, M43, and M44 have the same neutral loss of −80 Da, which was identified as the sulfonyl hydroxide (SO3), so they were identified as the sulfated products [13,16].

When predicting their formulas, M39, M41, and M42 were identified as related metabolites of saponins [13,17].

Table Table 4. The identified proposed metabolites from the urine samples of rats that had been administrated Danggui Buxue Tang1 and Danggui Buxue Tang 2.

Click here to display table

Table 4. The identified proposed metabolites from the urine samples of rats that had been administrated Danggui Buxue Tang1 and Danggui Buxue Tang 2.
NO.TR (min)[M+H]+[M-H]Predicted FormulaFragment IonsError ppmIdentificationDBT1DBT2
14.132 287.0065C10H8O10 6.97--M1m1
228.200 231.0768C12H12N2O3463,231−3.03--M2m2
329.298 233.0115C12H2N4O2233,1694.29--M3m3
429.298 337.1408C16H22N2O6337,2530.89--M4m4
531.083268.1164266.1021C13H17NO5 −4.89----m5
631.338 275.0209C13H8O7275,1954.36--M5m6
732.927 273.0056C13H6O7273,1935.49--M6m7
833.038271.0585 C15H10O5 −5.90HydroxydaidzeinM7--
934.518[M+HCOO]475.1244C22H22O9475,267−0.42Isomer of ononin--m8
1036.155 303.0863C16H16O6303,151−3.63Hydroxylcalycosin, direduction(C2=C3; C4=O)M8--
1136.705 253.0492C15H10O4 −5.53DaidzeinM9m9
1236.808 477.1372C23H26O11477,301−6.29Astraisoflavan, glucuronidationM10--
1336.868 255.0662C15H12O4255,149−0.39Daidzein, reduction(C2=C3)M11m10
1437.255385.1478 C18H24O9 −3.89Hydroligustilide, glucuronidationM12--
1537.442 255.0655C15H12O4 −3.14Daidzein, reduction(C4=O)--m11
1637.502 285.0751C16H14O5285,269,149−5.96Calycosin, reduction(C2=C3)M13m12
1737.195 257.0809C15H14O4 −3.89Daidzein, direduction(C2=C3; C4=O)M14--
1838.033 283.0608C16H12O5283,268−1.41CalycosinM15m13
1938.362 285.0751C16H14O5285,270−5.96Calycosin, reduction(C4=O)M16--
2039.047 273.0761C15H14O5273,240,109−2.56Hydroxydaidzein, direduction (C2=C3; C4=O)--m14
2139.367 233.0098C12H2N4O2 −3.00--M17m15
2240.260[M+HCOO]363.0748C16H14O7 7.16Dihydroxycalycosin, reduction(C2=C3)M18--
2341.630 283.0609C16H12O5283,268,224−1.06Isomer of calycosinm16
2441.755 269.0441C15H10O5 −5.20HydroxydaidzeinM19--
2542.180 283.0603C16H12O5283,268,224−3.18Isomer of calycosin--m17
2642.365 299.0554C16H12O6299,284−2.34Hydroxycalycosin, or isomerM20--
2742.923 299.0556C16H12O6299,284−1.67HydroxycalycosinM21--
2844.315 257.0819C15H14O4 0.00Isoliquiritigenin, reduction(C=C)M20m18
2945.322269.0796 C16H12O4269,253,237−4.46FormononetinM23m19
3045.733 269.0804C16H14O4269,254,135−5.57Formononetin, reduction(C2=C3)M24m20
3146.290335.0201 C15H10O7S335,255−5.67Daidzein, sulfationM25m21
3247.775 299.0556C16H12O6299,256−1.67Hydroxycalycosin--m22
3347.862 363.0174C16H12O8S363,268−1.65Calycosin, sulfationM26--
3447.922 333.0059C15H10O7S333,253,225−4.50Daidzein, sulfationM27--
3548.747 365.0347C16H14O8S365,2852.74Calycosin, reduction(C2=C3), sulfationM29--
3649.072 333.0056C15H10O7S333,253,208−5.41Daidzein, sulfation--m23
3749.543 333.0059C15H10O7S333,253−4.50Isomer of daidzein, sulfation--m24
3849.193 365.0360C16H14O8 S365,2856.30Calycosin, reduction(C2=C3), sulfationM30--
3949.810 349.0033C15H10O8S349,269,2252.58Hydroxydaidzein, sulfationM28m25
4049.623 351.0187C15H12O8S351,271,1491.99Hydroxydaidzein, reduction(C2=C3), sulfationM31--
4150.325 337.0395C15H14O7S337,2572.37Daidzein, direduction(C2=C3; C4=O), sulfation--m27
4250.467 283.0595C16H12O5283,268−6.01Isomer of calycosinM32--
4350.973 367.0483C16H16O8S367,272,150−2.72Calycosin, direduction(C2=C3; C4=O), sulfationM33--
4451.515 363.0180C16H12O8S363,2830.00Calycosin, sulfationM34m26
4552.563 335.0250C15H12O7S335,255,1355.67Daidzein, reduction(C2=C3), sulfationM35--
4653.637 343.0835C18H16O7 3.50--M36--
4755.693 351.0172C15H12O8S351,271−2.28Hydroxydaidzein, reduction(C2=C3), sufaltion--m28
4855.165 321.0417C15H14O6S321,241−6.54Equol, sulfationM37m29
4959.468 337.0388C15H14O7S337,257,2430.30Daidzein, direduction(C2=C3; C4=O), sulfationM38--
5059.728619.3669[M+Na]+C36H52O7 10.33Related to astragaloside--m30
5161.838683.4277 C35H64O11 −9.57Related to astragalosideM39--
5262.002639.4061 C35H58O10 −6.57Related to astragalosideM40--
5362.113595.3742 C33H54O9 −16.63Related to astragalosideM41m31
5463.292 509.3599C33H50O4 −7.26Related to astragaloside--m32
5562.414507.3296 C29H46O7 −3.94Related to astragalosideM42--
5671.557 353.0324C15H14O8S353,273−3.68Hydroxydaidzein, direduction(C2=C3;C4=O) sulfationM43--
5771.557 397.0250C16H14O10S397,3173.78Dihydroxycalycosin, reduction(C2=C3), sulfationM44--
5872.797 363.0197C16H12O8S363,2834.68Calycosin, sulfation--m33
5973.543 347.0213C16H12O7S347,267−5.19Formononetin, sulfation--m34
Molecules 19 05650 g005 1024
Figure 5. The main proposed metabolites identified from the urine samples of rats that had been administrated Danggui Buxue Tang 1.

Click here to enlarge figure

Figure 5. The main proposed metabolites identified from the urine samples of rats that had been administrated Danggui Buxue Tang 1.
Molecules 19 05650 g005 1024

2.4.2. Identification of the Chemical Profiles of Urine Sample after Administration of the DBT2 by HPLC-DAD-ESI-IT-TOF-MSn

The HRMS data of these identified metabolites are summarized (see Table 4 and Supplementary: pages S67–S77). The BPCs detected in NI model were recorded (see in Figure 3d). The main proposed structures of metabolites identified from the urine samples of rats that had been administrated DBT2 were showed (see Figure 6).

The MS data of m13, and m19 show that they are the reference compounds. In addition, m13 was identified as calycosin, and m19 was identified as formononetin [11,13].

Moreover, m8 has a RT at 34.518 min, [M+HCOO] at m/z 475.1244 in MS (predicted formula: C22H22O9: ppm error: −0.42), and characteristic fragment ions at m/z 267.0656 that are predicted as C16H12O4, the neutral loss is 46 Da (HCOOH) + 162 Da (C6H10O5; identified as glucoside). Thus, m8 was identified as the isomer of ononin [13].

In their MS and MS2 Data, m21, m23, m22, m24, m25, m26, m33, m27, m28, m29, and m34 have the same neutral loss of −80 Da, which was predicted as the sulfonyl hydroxide (SO3), so they were identified as the sulfated products [13,16].

Molecules 19 05650 g006 1024
Figure 6. The main proposed metabolites identified from the urine samples of rats that had been administrated Danggui Buxue Tang 2.

Click here to enlarge figure

Figure 6. The main proposed metabolites identified from the urine samples of rats that had been administrated Danggui Buxue Tang 2.
Molecules 19 05650 g006 1024

By the MS data of [M-H] or [M+HCOO] and the characteristic fragment ions, m9, m10, m11, m12, m14, m16, m17, m18, and m20 were identified as the metabolites of isoflavonoids [13,14,15,17,18,21]. Using their predictions of [M+Na]+ or [M+H]+, m30, m31, and m32 were tentatively identified as saponin-related metabolites [13,17].

Between DBT1 and DBT2, the groups M1, m1; M2, m2; M3, m3; M4, m4; M5, m6; M6, m7; and M15, m17 were identified as the same compounds, respectively.

In this part of the experiment, decoctions of DBT1 and DBT2 were administered to rats, and an analysis was conducted on the rats’ urine for metabolites: 44 compounds (M1–M44) were identified from the urine samples after DBT1 was administrated, 34 compounds (m1–m34) were identified from the urine samples after DBT2 was administrated. The number of the chemical components in the urine samples from DBT1 was administrated to rats are slightly more than these of DBT2. The proportion of the 19 common constituents to the total 59 identified compounds accounted for approximately 33%. The proportion of 48 total isoflavonoids and saponins to the total identified compounds, however, reached approximately 82% (See Table 3).

The phase Ⅱ metabolites from the urine samples of DBT1 and DBT2 were dominantly sulfated products, with rare or no glucuronide metabolites. This result still requires further research as the literature reports the chief presence of mainly glucuronide metabolites [13,14,15,16]. Metabolites that originated from RAS are relatively rare or not detected. This is likely due to the low proportion of RAS in DBT and even the low dosage that was given to rats in this study, those chemical constituents of RAS being easy to volatilize, or a loss when sampling was conducted by concentrated processes [22,23]. At this normal dosage of DBT and due to poor absorption, the content levels of astragalosides are much lower. In addition, saponins and their metabolites that originated from astragalosides are rarely detected [24,25,26,27].

The HPLC-DAD-ESI-IT-TOF-MSn method adopted in our research was confirmed to be a powerful method to evaluate the chemical profiles of the crude extracts and the related urine samples. As we know, the chemical composition found in a Chinese herbal decoction is rather complicated. In this study, the chemical profile analysis of DBT1 and DBT2 was conducted, which provided a comprehensive understanding of that those isoflavonoids that play an important role in the main common chemical basis when they are used in clinical practice. Some identified metabolites are known to have many bioactivities, such as calycosin, formononetin, daidzein and equol (which are well-known phytoestrogens), and most of them displayed many beneficial effects to humans [28,29,30].

Through the comparison of chemical profiles of two DBTs at our used normal dosage, the similarity of urine samples is higher than that of the crude extract samples. This leads us to believe that the main chemical basis of the chemical constituents is almost the same. Whether the DBT2 of RH:RAS can replace the DBT1 of RA:RAS, we need to further investigate different ratios of RH:RAS with the RA: RAS (5:1) when using equivalent pharmacological research.

3. Experimental

3.1. Materials and Reagents

Radix Astragali was collected from Shanxi Province (Voucher No. 130401, Specimen No. 1167), Radix Hedysari was collected from Neimeng Province (Voucher No. 130401, Specimen No. 1168) and Radix Angelica sinensis was collected from Gansu Province (Voucher No. 130401, Specimen No. 1169). All of those medicinal materials were purchased from Guangzhou Zixing Herbal Company in Guangzhou at June in 2013 by Liu Jing and they are identified by Prof. Chen Hu-Biao. The following reference compounds: (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan (astrapterocarpan), ononin, formononetin, 6''-O-acetyl-ononin, calycosin, ferulic acid, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan (astraisoflavan), calycosin-7-O-β-d-glucoside, astrapterocarpan-7-O-β-d-glucoside, Z-ligustilide, E-ligustilide, astragaloside I, astragaloside II, astragaloside III, astragaloside IV, astragaloside V, astraisoflavan-7-O-β-d-glucoside and 6''-O-acetyl-astraisoflavan-7-O-β-d-glucoside were prepared and identified in our preliminary work [10,11,12,13,14]. Acetonitrile (Merck Co., Darmstadt, Germany) and formic acid (Mreda Technology Inc., Beijing, China) were of HPLC grade. Ultra-pure water was prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA).

3.2. Sample Preparation

In clinical use, DBT is typically boiled with water twice, then the two decoctions are combined and applied [1]. Therefore, in our study, DBT1, consisting of RA 100 g and RAS 20 g was boiled in 1,000 mL of water (w:v) for 45 min, and then the decoction was filtered. The residue was again boiled in 700 mL of water (w:v) for 30 min. The two decoctions were evaporated to dryness under reduced pressure at 50 °C to 100 mL volume. Samples of DBT2, consisting of RH 100 g and RAS 20 g, were prepared in the same way.

3.3. Animals and Administration

12 male Sprague-Dawley (SD) rats (220–250 g) were provided by the Experimental Animal Center of Peking University Health Science Center (Beijing, China) and divided into two groups. They were housed in metabolic cages (Type: DXL-DL, Suzhou Fengshi Laboratory Animal Equipment Co. Ltd., Suzhou, China), and kept in an environmentally controlled breeding room for one-week acclimation. Throughout the experiments, rats had unrestricted access to laboratory chow and water. The DBT1 and DBT2 were administrated by oral at a dose of raw medicinal material 10 g/kg body weight once a day (at 17:30 pm) respectively. Totally for 2 days. All procedures used in the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institute of Health. The experiments were reviewed by the Biomedical Ethical Committee of Peking University (Approval No. LA2013-193).

3.4. Urine Sample Collection and Pretreatment

Urine samples in each group (n = 6) were collected during the first 48 h after administration of the drugs began(Blank urine collected by self-control.); Finally, all urine samples from the same group were merged into one sample, then dried under vacuum at 50 °C using a Heidolph Laborota 4001 rotatory evaporator (Heidolph Instruments GmbH & Co., Schwabach, Germany), and then 1.00 g of the dried samples were reconstituted in 10 mL methanol, followed by 30 min ultrasonic extraction and 15 min centrifugation at 5,000 rpm. Afterward, the supernatant was collected for detection.

3.5. Instrumentations and Conditions

HPLC analysis was performed on a Shimadzu HPLC (Shimadzu, Kyoto, Japan) equipped with two LC-20AD pumps, aCTO-20A column oven, an SIL-20AC autosampler, an SPD-M20A PDA detector and a CBM-20A system controller. The chromatographic separation was carried out on a Phenomenex Gemini C18 column (250 × 4.6 mm, 5 μM) protected with a Phenomenex Security Guard column (4 × 3.0 mm, 5 μM) (Phenomenex, Torrance, CA, USA). For each sample, an aliquot of 20 μL was injected with needle wash. The thermostatted auto-sampler was maintained at 15 °C; column oven temperature was kept at 30 °C. The column was eluted with a gradient mobile phase consisted of water-formic acid (100:0.1, v/v) (A) and acetonitrile (B) at the flow rate of 1.0000 mL/min. Gradient program was adopted in the following manner: 5% B at 0–10 min, 5%–15% B at 10–20 min, 15%–40% B at 20–40 min, 40%–65% B at 40–55 min, 65%–100% B at 55–65 min, 100% B at 65–75 min, 5% B at 75–85 min.

High resolution mass spectra were recorded on an IT-TOF mass spectrometer (Shimadzu). The ESI source was operated both in negative and positive ion mode. The mass spectrometry was programmed to carry out full scan over m/z 100–1000 Da (MS1), m/z 50–1000 Da (MS2 and MS3). A trifluoroacetic acid sodium solution (2.5 mM) was used to calibrate the mass range from 50 to 1000 Da. The other parameters were set as follows: flow rate, 0.20 mL/min (split from 1.00 mL/min HPLC effluent); heat block and curved desolvation line temperature, 200 °C; nebulizing nitrogen gas flow, 1.5 L/min; interface voltage: (+), 4.5 kV; (−), −3.5 kV; detector voltage, 1.70 kV; relative collision-induced dissociation energy (50%) [15].

3.6. Data Analysis

All data were recorded and processed by Shimadzu software LCMS solution version 3.60, Formula Predictor version 1.2 and Accurate Mass Calculator (Shimadzu).

4. Conclusions

A comparison was conducted on the similarities and differences of crude extracts and urine samples of DBT1 and DBT2. The chemical profiles of the crude extracts comprised a total of 115 proposed chemical components. There were 24 common ingredients, which was accounted for 27% in the total 91 identified components. There were a total of 56 isoflavonoids and saponins identified, which accounted for nearly 62% in the total identified components. Since isoflavonoid glycosides had acetylation (C18, C32, C34, C40, C44, C46, and C47), the formation of malonate acid esters (C31, C35, C43, C42, and C51) and special astragalosides (C49, C52, C53, C54, C60, and C64) in DBT1, the identified compounds from DBT1 were significantly greater than DBT2. Of these, C18, C34, C35, C42, C47, and C51 were identified from DBT for the first time.

In total, 78 proposed chemical components in the urine samples of rats that had been administrated DBT1 and DBT2, respectively, were found. These included 19 common ingredients, which accounted for approximately 33% in the total identified constituents. In addition, 48 of total isoflavonoids and saponins were found, which accounted for nearly 82% of the total 59 identified components. The differences between those metabolites in the urine samples were revealed to be less than the crude extracts. These identified metabolites are mainly originated from formononetin, calycosin and their related glycosides, which are formed mainly through the metabolic processes of reduction, deglycosylation, demethylation, hydrogenation and sulfation. Through the comparison of chemical profiles of two DBTs at our used doses, the similarity of urine samples is higher than that of the crude extract samples, one can think the main chemical constituents are almost the same as when administrated to rats.

The HPLC-DAD-ESI-IT-TOF-MSn method was successfully applied for the chemical profile comparison of two different DBTs and its related medicinal materials. The proposed assay provides an important reference and can be a suitable method for the rapid and accurate chemical basis evaluation of TCM or their related prescriptions.

Supplementary Materials

Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/5/5650/s1.

Acknowledgements

This study was financially supported by Hong Kong Baptist University Faculty Research Foundation (Grant No. FRG2/12-13/058). We thank to Li Jun for HPLC-MS analysis in this work.

Authors Contributions

Ya-Zhou Zhang, who presided the research work, finished the data analysis, and written this paper; Feng Xu, The main works by the writer are the data analysis, and participated in the writing of this paper; Tao Yi, who participated in the design of research and modified the paper; Jian-Ye Zhang, who participated the research design, and the data analysis; Jun Xu, who participated in the research design, and modified the paper; Yi-Na Tang, who participated in the research design and data analysis; Xi-Chen He, who participated in the data analysis of LC-MS; Jing Liu, who participated in the experiment work; Hu-Biao Chen, who is providing fund for this research work, the design of the work and modified the paper.

Conflict of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds are available from the authors.
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