Identification Of The Major Constituents Biology Essay

This work describes a sensitive and specific LC-ESI-MSn methodological analysis for designation of the major components in Zhimu-Huangqi herb-pair infusion and their metabolites in rats after unwritten disposal. A sum of 30 compounds were characterized from the infusion. 13 were unequivocally identified by comparing the keeping times and mass spectra with those of mention criterions, and 17 were tentatively identified on the footing of their MSn atomization behaviours and molecular weight information from literatures. In add-on, the metabolites in vivo were besides identified. The Zhimu-Huangqi herb-pair was actively metabolized in rats, including 3 paradigms and 9 metabolites in serum, while 7 paradigms and 23 metabolites in piss. This survey proposed a serious of possible bioactive constituents of this herb-pair in traditional Chinese medical specialty ( TCM ) and could ease further pharmacokinetic surveies of the Zhimu-Huangqi herb-pair.

Keywords: LC-ESI-MSn ; Zhimu-Huangqi ; herb-pair ; major components ; metabolites


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Compatibility of Chinese medicative herbs refers to the combination of two or more herbs with intent in the visible radiation of the clinical demand and medicative belongingss and actions [ 1 ] . Unique combinations of the traditionally defined herbal belongingss of traditional Chinese medical specialty ( TCM ) are often used for accomplishing common support, common aid, common restraint, and common detoxication [ 2, 3 ] . The Zhimu-Huangqi herb-pair is a combination of Rhizoma Anemarrhenae ( RA ) and Astragalus Membranaceus ( AM ) . Nowadays, as a celebrated Chinese medical specialty expression, legion surveies have demonstrated the Zhimu-Huangqi herb-pair have attractive pharmacological activities, including tonic, diuretic, immunostimulant, antioxidant, hepatoprotective, anti-diabetic, anti-cancer and expectorant effects [ 4-7 ] .

Assorted analytical techniques presently used for designation of petroleum drugs of RA or AM include thin bed chromatography [ 8 ] , high-performance liquid chromatography coupled with evaporative visible radiation dispersing sensing ( HPLC-ELSD ) [ 9 ] , and HPLC-diode array sensing ( DAD ) [ 10 ] . However, the saponins in Zhimu-Huangqi herb-pair with hapless UV soaking up ( at short wavelengths 200-210 nanometer ) are hard to observe utilizing DAD. Although it is a cosmopolitan sensing suitable for the analysis of non-chromophoric compounds, ELSD merely provides information about keeping clip for intents of designation, taking to hapless truth.

Recently, the development of high public presentation liquid chromatography coupled with electrospray ionisation multistage tandem mass spectroscopy ( LC-ESI-MSn ) has demonstrated its value in analysis of the complex mixtures like the Traditional Chinese medical specialty ( TCM ) and their metabolites in vivo [ 11-13 ] . The compounds in a mixture can be expeditiously separated by high-performance liquid chromatography ( HPLC ) , and so be characterized by MS. Due to its high sensitiveness and functional versatility, MS sensing can supply more valuable structural information than conventional sensors like UV, and allows for fast and specific structural elucidation of unknown compounds at reasonably low degrees, even when criterion samples are non available [ 14 ] .

Here, we report a simple and robust LC-ESI-MSn method for the designation of the major components and their metabolites in rat piss after unwritten disposal of decoction of Zhimu-Huangqi herb-pair. A sum of 30 compounds were identified or tentatively characterized which chiefly included xanthones, isoflavonoids and saponins. In add-on, the metabolites in vivo were besides identified, including 3 paradigms and 9 metabolites in serum while 7 paradigms and 23 metabolites in piss. Our consequences will ease the comprehensive research on the Zhimu-Huangqi herb-pair and aid in the better understanding the mechanism of action of this TCM.


Chemicals and Reagents

Mention criterions ( Timosaponin A-III, Timosaponin B-III, Timosaponin B-II, Calycosin-7-O-I?-glucoside, Calycosin and Formononetin-7-O-I?-glucoside ) were prepared in our research lab and their chemical constructions were unequivocally identified by comparing of their 1H-NMR, 13C-NMR and MS informations with the literature informations [ 15-18 ] . The Mangiferin ( NO.111607-200301 ) , the Neomangiferin ( NO.111597-200308 ) , the Astragaloside IV ( NO.110781-200613 ) and the Formononetin ( NO.111703-200501 ) were purchased from the National Institutes for Food and Drug Control ( Beijing, China ) . The Astragaloside I, Astragaloside II and Astragaloside III were purchased from DELTA information Centre for Nature Organic Compounds ( Anhui, China ) . The purenesss of all ingredients were supra 98 % harmonizing to HPLC analysis.

RA and AM were purchased from Shanghai Kangqiao Traditional Chinese Medicine Co. Ltd. , Shanghai, China and identified by Professor C. G. Huang of the Shanghai Institute of Meteria Medica, Chinese Academy of Sciences.

Acetonitrile and formic acid of HPLC class purchased from Dikma Company ( Dikma, USA ) . All other analytical chemical reagents of analytical class purchased from Sinopharm Chemical Reagent Co. Ltd. , Shanghai, China. Deionized H2O was purified utilizing a Milli-Q system ( Millipore, Billerica, MA, USA ) , Double-distilled H2O was used for all the readyings.

Chromatographic System and Mass Spectrometry Conditions

The chromatographic separations were performed utilizing a reversed-phase column ( Inertsil ODS-3, 4.6A-250 millimeter ID, 5 I?m, GL Science, Tokyo, Japan ) connected to an Easy-Guard Kit C18 ( 4A-2 millimeter, Grace, USA ) guard column, with the column temperature set at 25°C. The nomadic stage consisted of additive gradients of 0.2 % ( v/v ) formic acid ( A ) and acetonitrile ( B ) : 0-10 min, 10-27 % B ( v/v ) ; 10-18 min, 27-29 % B ; 18-40 min, 29-95 % B ; 40-55 min, held at 95 % B. The nomadic stage flow rate was 1.0mL/min and the analysis clip was 55 min.

The LC-MSn analysis was performed on the 6300 Series Ion Trap LC/MS ( Agilent engineering, Pala Alto, CA, USA ) . The hardware including an Agilent 1200 Series LC, the ion trap mass spectrometer and the information system. The package ( version 6.1 ) included the Trap Control Program for trap control, informations acquisition, information analysis, quantitative analysis and the LC control. MSn analyses were conducted in negative-ion manner and the instrument was operated under the undermentioned optimized conditions: hit gas, ultrahigh-purity Helium ( He ) ; nebulizing gas, high-purity N ( N2 ) ; capillary electromotive force, 3.5 kilovolt ; stop home base beginning, 500 V ; atomizer, 30 pounds per square inch ; drying gas flow rate, 10 LA·min-1 ; drying gas temperature, 350 °C. For full-scan MS analysis, the spectra were recorded in the scope of 50-1500 m/z.

Extract readying

To 50 g of RA and 150 g AM petroleum herbs were added 3000 milliliter of H2O. The mixture was refluxed at 65 °C twice for 1.5 h each. The sample was instantly cooled to room temperature, and was so centrifuged at 12,000 revolutions per minute for 10 min. The supernatant was concentrated under decreased force per unit area to afford 400 milliliter residue, 2 milliliter of the residue was filtered through a 0.22 I?m micro-porous membrane before usage. Samples of single herbs were prepared with the same process to do 0.5 gA·mL-1 infusions. An aliquot of 10 I?L of each sample was injected into the LC/MS instrument for analysis.

Animals and drug disposal

All carnal processs were conducted in conformity with the guidelines from the Review Committee of Animal Care and Use at the Shanghai Institute of Materia Medica ( Shanghai, China ) . 18 male Sprague-Dawley rats ( 200-220 g ; Shanghai SLAC Laboratory Animal Co. , Shanghai, China ) were kept in an environmentally controlled genteelness room for one hebdomad and fasted 12 H ( but with entree to H2O ) before get downing the experiments. These animate beings were indiscriminately divided into three groups: a dosed urine aggregation group ( group A, n=6 ) ; a dosed plasma aggregation group ( group B, n=6 ) and a control group ( group C, n=6 ) .

Sample Preparation

After the unwritten disposal, heparinized blood samples were collected at 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 H ( n=6 ) from the abdominal aorta of the rats, shaken up and so centrifuged at 12,000 g for 10 min to obtain the plasma. Urine samples were collected during the clip period 0-24h ( n=6 ) . All samples were stored at -80°C until analysis.

All the readying processs of plasma and urine samples were harmonizing to Liu et Al [ 19 ] and Chen et al [ 20 ] . Then, a 600 I?L aliquot of urine and plasma samples was loaded onto an SPE cartridge, which was preconditioned with 2 milliliters methyl alcohol and 2 mL H2O. Then the cartridge was washed with 1 milliliters of H2O and the analyte was eluted with 1 milliliters of methyl alcohol. The eluted solution was evaporated to dryness in a H2O bath at 37°C under a soft watercourse of N and the residue was reconstituted in 200 I?L methyl alcohol. The ensuing solution was centrifuged at 12,000 g for 10 min at 4°C and 10 I?L of supernatant was injected into the chromatographic system for LC-MS/MS analysis. Blank samples as controls were prepared with the same method as the drug-containing samples.

Consequences and treatment

Structure elucidation of the mention compounds by LC-MSn

In footings of the chemical diverseness of nature merchandises, the scheme we late proposed was used with minor alterations to place the compounds in Zhimu-Huangqi herb-pair by LC/MS. When a pure criterion was available, the compound was identified by comparing its HPLC keeping clip and mass spectra with those of the criterions. When no criterion was available, the constructions were proposed chiefly based on the mass spectra. Analysis of keeping clip and MS spectral informations of the mention compounds and decision of the regulations will be valuable for subsequent online elucidation of structurally related compounds. Therefore, the first measure of this work was to qualify the chromatographic and aggregate spectral belongingss of the mention compounds. These informations will supply a scientific footing for designation of other bioactive compounds in works infusions. A typical MS entire ion current chromatogram of 13 mention compounds studied in the experiment is in shown in Fig. 1.

Among the 13 mention compounds analyzed, there were 2 xanthones, 3 timosaponins, 4 astragaloside and 4 isoflavonoids. The MSn spectra for S1 ( m/z 583 ) , S3 ( m/z 491 ) , S4 ( m/z 919 ) and S10 ( m/z 871 ) in negative ion manner were shown in Fig. 2. Negative ESI analysis of S1 gave the [ M-H ] i?? ion at m/z 583. The MS2 experiment of the m/z 583 ion yielded three outstanding ions at m/z 565, 493 and 421 through impersonal losingss of H2O ( 18 Da ) , C3H6O3 ( 90 Da ) and C6H11O5 ( 162 Da ) , severally. The ion at m/z 421 was subjected to MS3 analysis to afford four ions at m/z 403, 331, 301 and 259 by losingss of H2O ( 18 Da ) , C3H6O3 ( 90 Da ) , C4H8O4 ( 120 Da ) and C6H11O5 ( 162 Da ) , severally. Based on these informations, the construction of S1 could be identified as neomangiferin. The mechanistic tract for fragments formed may be explained harmonizing to Fig. 3 ( A ) .

Compound S3 exhibited a [ M+Cl-H ] i?? and [ M+HCOOH-H ] i?? ion at m/z 481 and 491. Its MS/MS spectra produced a outstanding ion at m/z 283, arising from loss of branch glycoside concatenation ( 162 Da ) . In the MS3 of m/z 283, fragments of m/z 268 was generated having to loss of one H2O molecule. Therefore compound S4 was tentatively identified as Calycosin-7-O-I?-glucoside. The proposed atomization tract for compound S3 is shown in Fig. 3 ( B ) .

Compound S4 displayed a typical [ M-H ] i?? at m/z 919. In add-on, three ions at m/z 757 [ M-H-Glu ] i?? , 577 [ M-H-Glu-Gal ] i?? and 433 [ M-H-2Glu-Gal ] i?? could be detected in the MS/MS spectrum of the ion at m/z 919, arising from loss of branch glycoside concatenation ( 162 Da ) . The above atomizations were consistent with those of Timosaponin B-II. The mechanistic tract for fragments formed may be explained harmonizing to Fig. 3 ( C ) .

Compound S10 showed a typical [ M-H+HCOOH ] i?? and [ M-H ] i?? at m/z 871 and 825. Furthermore, three ions at m/z 765 [ M-H-Ac-H2O ] i?? , 633 [ M-H-H2O-Acetylated Xyl ] i?? and 489 [ M-H-Glu-Acetylated Xyl ] i?? could be detected in the MS/MS spectrum of the ion at m/z 825. The above atomizations were consistent with those of Astragaloside II. The proposed atomization tract for compound S10 is shown in Fig. 3 ( D ) .

Compound S8 and compound S9 displayed the same quasi-molecular ion at m/z 829. Additionally, the characteristic fragment ions in compounds S8 and S9 are the same, i.e. , m/z 651 [ M-H-Xyl ] i?? , 621 [ M-H-Glu ] i?? and 489 [ M-H-Xyl-Glu ] i?? . Similar to Astragaloside II, these characteristic fragment ions are yielded by the loss of branch glycoside concatenation. However, compounds S8 and S9 can be differentiated by their keeping times since Astragaloside IV was eluted earlier than the compound Astragaloside III on different chromatographic columns. Therefore, compounds S8 and S9 were identified as Astragaloside IV and Astragaloside III, severally.

Taking the same designation method as for S1, S3, S4 and S10 described above, the other 7 mention compounds, S2, S5, S6, S7, S11, S12 and S13, were unequivocally identified as Mangiferin, Ononin, Calycosin, Timosaponin B-III, Formononetin, Astragaloside I and Timaosaponin A-III. The keeping clip, ESI-MSn informations and atomizations of 13 standard compounds are summarized in Table 1. The ESI-MSn information of these compounds shared some common characteristics, such as the impersonal loss of H2O ( 18 Da ) , branch glycoside concatenation ( 162 Da ) in timosaponins and astragalosides or C3H6O3 ( 90 Da ) and C4H8O4 ( 120 Da ) in xanthones. These specific atomization regulations discussed above can be faithfully and efficaciously used for rapid showing and designation of other bioactive xanthones, timosaponins, isoflavonoids and astragalosides in Zhimu-Huangqi herb-pair infusion and their metabolites in vivo in the undermentioned subdivisions.

Designation of the major bioactivity components in Zhimu-Huangqi herb-pair infusion

In the entire ion current ( TIC ) chromatograms of Zhimu-Huangqi herb-pair infusion, a sum of 30 compounds were identified on the footing of their keeping clip and mass spectroscopy atomization forms. The LC-MS entire ion chromatograms for Zhimu-Huangqi herb-pair infusion are shown in Fig. 4 and their chemical constructions are presented in Fig. 5. Among them, peaks 1, 3, 6, 10, 11, 16, 17, 20, 21, 24, 27, 28 and 31 could be unequivocally identified as Neomangiferin, Mangiferin, Calycosin-7-O-I?-glycoside, Timosaponin B-II, Ononin, Calycosin, Timosaponin B-III, Astragaloside IV, Astragaloside III, Astragaloside III, Formononetin, Astragaloside I and Timaosaponin A-III by comparing their keeping times, molecular weights and fragment ions with those of criterions S1-S13. From the ESI-MSn surveies on criterions S1-S3, we can reason that the characteristic atomization tracts for the bioactivity components of Zhimu-Huangqi herb-pair infusion were the characteristic impersonal losingss of 18 Da ( H2O ) , 90 Da ( C3H6O3 ) , 120 Da ( C4H8O4 ) and the subdivision glycoside concatenation ( 162 Da ) , which could be used for theorizing as to the being of xanthones and saponins. These characteristic impersonal losingss and the characteristic cleavage of subdivision glycoside concatenation provided a sound footing for elucidation of other 17 compounds in Zhimu-Huangqi herb-pair infusion.

Peak 2 exhibited a [ M+HCOOH-H ] i?? ion at m/z 521. The outstanding ions at m/z 313 resulted from the loss of branch glycoside concatenation C6H11O5 ( 162 Da ) , which was similar to Calycosin-7-O-I?-glycoside and Ononin. Compared with the known compound reported in the literature [ 21, 22 ] , peak 2 was tentatively identified as Odoratin-7-O-I?-glycoside. Similarly, peaks 13, 14 and 15 were identified as 9,10-dimethoxypteracarpan-3-O-I?-D-glycoside, Afrormosin-7-O-I?-glycoside and 2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-D-glycoside as reported in the literatures [ 22-24 ] .

Peak 4 gave a [ M-H ] i?? ion at m/z 421, bespeaking that it was the isomer of Mangiferin. Its MS2 spectrum yielded a typical atomization ion at m/z 283, matching to the loss of branchglycoside concatenation ( 162 Da ) . The ion represented a similar tract to Mangiferin. By seeking the known compound reported in the literature [ 25 ] , peak 4 was tentatively identified as Isomangiferin.

Peak 5 displayed a [ M-H ] i?? ion at m/z 901, proposing that it was a brace of isomer with Timosaponin B-III. The outstanding ions at m/z 739, 577, 415 resulted from the loss of subdivision glycoside ironss, which was the similar to Timosaponin B-III. These characteristic ions of extremum 5 manifested the same tract as peak 17. The above atomizations were consistent with known Timosaponin C, therefore, peak 5 was tentatively identified as Timosaponin C. Similarly, peaks 9, 12, 22, 23 and 26 were Timosaponin D, Timosaponin B-I, Timosaponin F, Anemarrhenasaponin I and Timosaponin G as reported in the literatures [ 16, 25-27 ] .

Peak 7 and 8 in the MSn spectra gave a quasi-molecular ion at m/z 931, including that these two compounds were isomers. Furthermore, five ions at m/z 773 [ M-H-Glu ] i?? , 755 [ M-H-H2O-Glu ] i?? , 611 [ M-H-Glu-Gal ] i?? and 449 [ M-H-2Glu-Gal ] i?? , 593 [ M-H-H2O-Glu-Gal ] i?? could be detected in MSn spectrum of the ion at m/z 935. Based on these informations, the construction of peak 7 and 8 could be identified as timosaponin N or timosaponin E1. Owning to miss of mention compounds and more characteristic signals in ESI-MSn, we could n’t separate them.

Peaks 18-21, 24, 25, 28, 29 displayed common characteristic ions of m/z 143, 401, 419, 437, 455, and 473, which were the same as the Astragaloside IV. All the major extremums in the MSn spectra of these compounds were in conformity with [ M+HCOOH-H ] i??ions showed that all of these 8 saponins, because of the inevitable presence of formic acid during the sample separation procedure, MS/MS spectra of these [ M-H+HCOOH ] – were 9,19-cyclolanostane aglycone ( m/z 489 ) and some sugar medieties. Based on the comparing of the keeping clip and MS/MS informations with the criterion, peak 20 was identified as Astragaloside IV and top out 21 was identified as Astragaloside III. Peak 19 gave the same MS and MS/MS informations as those of standard, and was therefore ascribed to isoastragaloside IV.

Extremums 24 and 25 are a brace of isomers. Both of them gave an adduct ion [ M-H+HCOOH ] i?? at m/z 871 and important [ M-H ] i?? signal at m/z 825, and m/z 871 produced similar fragment ions to those of standard, except for the ions incorporating an acetoxy group. Additional atomization extremums in the MSn spectrum at m/z 489 [ M-H- ( Ac-H2O ) -Glu-Xyl ] i?? , 603 [ M-H- ( Ac-H2O ) -180 ] i?? and 633 [ M-H- ( Ac-H2O ) -180 ] i?? , indicated that the ethanoyl group group was linked to the glucose in the sugar part. By analyzing the known Astragalosides in AM, it was found that there were two saponins, named Astragaloside II and isoastragaloside II, consisted with the above information. Harmonizing to the content difference and the keeping behaviour in old studies [ 28, 29 ] , peaks 24 and 25 were tentatively assigned to Astragaloside II and isoastragaloside II, repectively. Similarly, extremums 28 and 29 incorporating [ M+HCOOH-H ] i?? ion ( m/z 913 ) and [ M-H ] i?? ion ( m/z 867 ) were tentatively assigned as Astragaloside I and isoastragaloside I, severally. Additionally, peak 18 was ascribed to acetylastragaloside I or its isomer based on their MS and MS/MS spectra and by comparing with the literature informations [ 29 ] .

These 30 identified compounds represented the major constituents of the Zhimu-Huangqi herb-pair infusion. The bulk of them had been reported as the chief bioactive components of the single herbs.

Designation of the metamorphosis compounds in rat serum and piss samples

By comparing the accurate multitudes of extremums looking in the chromatograms of drug-containing serum and piss with those antecedently identified in Zhimu-Huangqi herb-pair infusion ( Fig. 6 ) , 3 extremums and 7 extremums were detected as prototype constituents of Zhimu-Huangqi herb-pair in serum and piss, severally. Beyond the paradigm compounds, more than 23 extremums were tentatively predicted to be metabolites of Zhimu-Huangqi herb-pair and could be by and large divided into two groups: isoflavonoid-related and xanthone-related metabolites. As shown in Table 3, most of the metabolites were identified severally by comparing of their keeping times and MSn informations with those of the paradigms and the literatures reported before [ 19, 20, 24 ] .

Based on these metabolites, the possible metabolic tracts of Mangiferin, Calycosin and Formononetin in Zhimu-Huangqi herb-pair were proposed and summarized in Fig. 7. The consequences revealed that glucuronidation and sulfation were the chief metabolic tracts of isoflavonoids in Zhimu-Huangqi herb-pair. In add-on, it showed that some Phase I reactions, such as hydrolysis, demethylation, methylation and hydroxylation, besides occurred in the metamorphosis of isoflavonoids in Zhimu-Huangqi herb-pair.

Saponins besides play an of import function in Zhimu-Huangqi herb-pair. However, merely really low sums of Astragaloside II ( P4 ) , Astragaloside IV ( P5 ) , Isoastragaloside II ( P6 ) and Timosaponin A-III ( P8 ) were detected in the drug-containing serum and piss. This can be perchance attributed to the fact that saponins were ill absorbed straight, and could be chiefly excreted through fecal matters or gall.


In the present survey, a LC-ESI-MSn method was developed for the analysis of the major components of the Zhimu-Huangqi herb-pair infusion and their metabolites in rat serum and piss. A sum of 30 compounds were identified or tentatively characterized in the decoction based on their mass spectra determined in negative ion manner. The Zhimu-Huangqi herb-pair was actively metabolized in rats, including 3 paradigms and 9 metabolites in serum, while 7 paradigms and 23 metabolites in piss. The developed method was simple, dependable and sensitive, which revealed it to be appropriate for rapid showing and structural word picture of the major components and their metabolites of Zhimu-Huangqi herb-pair. These consequences could ease the elucidation of the metabolic procedure of the Zhimu-Huangqi herb-pair in vivo and better understand its action mechanism.


We would wish to appreciate the State Key Program of National Natural Science Foundation of China ( no. 81030065 ) and the National Science & A ; Technology Major Project “ Key New Drug Creation and Manufacturing Program ” , China ( no. 2012ZX09301001-001 and 2013ZX09102027 ) for i¬?nancial support of this work.


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