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Characterization and Discrimination of Different Pulicaria Species Using UHPLC-UV-MS QTOF (Quadrupole Time-of-Flight Mass Spectrometer).

Byline: Yan-Hong Wang, Adnan Jathlan Al-Rehaily, Muhammad Yousaf, Mohammad Shamim Ahmad and Ikhlas Ahmad Khan

Summary: A rapid and sensitive UHPLC-UV-MS/MS method was established for the screening and identification of polysaccharides, polyphenols, and flavonoids from methanolic extracts of P. schimperi, P. incisa, and P. glutinosa. This technique will facilitate qualitative analysis of these compounds as well as differentiation of Pulicaria species.

Keywords: Pulicaria, Traditional medicine, Phytochemical study, Chemical constituents.

Introduction

Pulicaria Gaertner is a genus of the Asteraceae, tribe Inulae, subtribe inulinae, comprised of about 80 species distributed in Europe, North Africa and Asia [1]. The genus is represented by twelve species in Saudi Arabia and distributed throughout the country [2]. These are Pulicaria arabica (L.) Cass, P. crispa (Forssk.) Benth. et Hook f., P. glutinosa (Boiss.) Jaub. and Spach, P. guestii Rech. f. and Rawi, P. incisa (Lam.) DC., P. inuloides (Poir) DC., P. jaubertii Gamal-Eldin, P. petiolaris Jaub. and Spach., P. schimperi DC., P. somalensis O. Hoffm., P. undulata (L.) Kostel., and P. vulgaris Gaertn. Most of these species have been reported as traditional medicines. Pulicaria arabica is reported for the treatment of digestive disorders [3], P. crispa is used to treat inflammation and as an insect repellent [4]. P. undulata is reported as hemostyptic and counterirritant [3]. In addition, P. incisa is used for the treatment of heart disease and as a hypoglycemic agent [5, 6].

Due to this traditional medicinal uses, various Pulicaria species have been investigated both phytochemically and biologically.

The phytochemical work suggested that the genus is not homogenous even though sesquiterpenes, sesquiterpene lactones and flavonoids are the most common metabolites present in the studied species [7-11]. Other species are also reported to produce diterpenes, coumarins and even alkaloids [12]. Systemic characterization and discrimination of different Pulicaria species are not yet reported. This work is designed to characterize and discriminate the different chemical constituents presented in three local Pulicaria species, namely, P. schimperi, P. incisa and P. glutinosa using UHPLC-UV-MS.

Experimental

Plant Material, Chemicals and Reagents

Plant samples Pulicaria schimperi (#15690 and 15719), P. incisa (#15083), and P. glutinosa (#15073) were collected in Saudi Arabia. The plant material was identified by Dr. Mohammed Yusuf, taxonomist, College of Pharmacy, King Saud University (KSU), Riyadh, Saudi Arabia. Specimens were deposited at the Repository of Botanicals, NCNPR, The University of Mississippi, University, Mississippi, USA.

The reference compounds chlorogenic acid, luteolin and quercetin were purchased from Sigma- Aldrich (St. Louis, MO, USA) with purity greater than 98%. Standard compounds 3,5-dicaffeoyl quinic acid (3,5-DCQA) and 4,5-dicaffeoyl quinic acid (4,5- DCQA) were isolated at the National Center for Natural Products Research (NCNPR), The University of Mississippi, University, MS, USA. The identity of 3,5-DCQA and 4,5-DCQA were determined by analysis of the spectral data ( H- and C-NMR and HR-ESIMS), and the purity was confirmed to be above 95% by chromatographic methods.

HPLC grade methanol, acetonitrile, and formic acid were purchased from Fisher Scientific (FairLawn, NJ, USA). Water for the HPLC mobile phase was purified using a Milli-Q system (Millipore).

Plant Sample Preparation

A dry powder of plant sample (0.5 g) was accurately weighed and sonicated in 2.5 mL methanol for 30 min, followed by centrifugation at 4000 rmp for 15 min. The supernatant was transferred to a 10 mL volumetric flask. The procedure was repeated three more times with 2.5 mL methanol and the respective supernatants were combined. The final volume was adjusted to 10 mL with methanol and mixed thoroughly. Prior to LC analysis, an adequate volume of sample was passed through a 0.45 m PTFE filter and collected in a LC sample vial.

UHPLC Chromatographic Conditions

The UHPLC analyses were performed on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) including binary solvent manager, sampler manager, column compartment and a PDA detector (Waters Acquity model code UPD). The separation was carried out on a Waters Acquity UPLC BEH C18 column (100 mm A- 2.1 mm i.d., 1.7 m). The column was equipped with a guard column (Vanguard 2.1 A- 5 mm, Waters Corp., Milford, MA, USA). The sample temperature and column temperature were maintained at 10 C and 35 C, respectively. The mobile phase consisted of water containing 0.05% formic acid (v/v) (A) and acetonitrile with 0.05% formic acid (B). The analysis was performed using the following gradient elution at a flow rate of 0.25 mL/min: 0-7 min, 5% B to 30% B; 7-12 min, 30% B to 50% B; 12-15 min, 50% B to 75% B. Each run was followed by a 5 min wash with 100% B and an equilibration period of 3.5 min with the initial conditions.

Strong needle wash (90/10; acetonitrile/water, v/v) and weak needle wash solution (10/90; acetonitrile/water, v/v) were used. The total run time for analysis was 20 min.

ESI/TOF/MS

QTOF, the high resolution ESI-MS experiments were carried on Waters ACQUITY XEVO QTOF mass spectrometer (Waters Corporation, Manchester, UK) that was connected to the UHPLC system via an ESI interface. The ESI source was operated in the positive ionization mode with the capillary voltage at 3.0 kV. The source and desolvation temperatures were set at 150 and 350C, respectively. The cone and desolvation gas flows were 50 and 900 L/h, respectively. The cone voltage was set at 35 V. All data collected in centroid mode were acquired using MassLynx NT 4.1 software (Waters Corp., Milford, MA, USA). Leucine- enkephalin was used for the lock mass at a concentration of 5 ng/mL and flow rate of 5 L/min. Ions [M+H]+ (m/z 556.2771 Da) and fragment ion (m/z 278.1141 Da) of leucine-enkephalin were employed to ensure mass accuracy during the MS analysis. The lock spray interval was set at 30 s, and the data were averaged over three scans.

The mass spectrometer was programmed to step between low (20 eV) and elevated (2050 eV) collision energies on the gas cell, using a scan time of 1.0 s per function over a mass range of 501500 m/z. When data were acquired with MSE, two interleaved scan functions were used. The first scan function acquired a wide mass range using low collision energy. This scan function collected precursor ion information for the sample. The second scan function acquired data over the same mass range; however, the collision energy was ramped from low to high (2050 eV). This scan function allowed for the collection of a full-scan accurate mass fragment with precursor ion information.

Results and Discussion

Compounds Characterization

Characterization of various class of compounds in Pulicaria species were based on the following approaches: (1) unambiguous identification, by comparing with reference standards in terms of the retention time, accurate mass, and MS-MS fragment ions, and (2) tentative characterization of compounds without reference by analyzing accurate mass, MS-MS product ions, and surveying of the literature.

As a result, a total of nineteen compounds (Table-1) were identified from three species of Pulicaria. These compounds included seven polysaccharides, four polyphenols and eight flavonoids. Structural information including the retention time, precursor ions ([M+H]+/[M+Na]+), MS-MS product ions, and whether existing in each Pulicaria specie is summarized in Table-1. The Base Peak Ions (BPI) chromatograms of extracts of P. shimperi, P. incisa, and P. glutinosa are shown in Fig. 1.

From the methanolic extracts of P. shimperi, P. incisa, and P. glutinosa, polysaccharides and disaccharide were characterized between 0.95 min and 1.50 min at m/z 1337.4213, 1175.3649, 1013.3181, 851.2645, 689.2114, 527.1580, and 365.1047 that corresponded to sodium adducted ions of octa-hexose (1), hepta-hexose (2), hexa-hexose (3), penta-hexose (4), tetra-hexose (5), tri-hexose (6), and di-hexose (7), respectively. The successively loss of 162 Da from the beginning at m/z 1337.42 to the end at m/z 365.10 suggested that the difference between each closely related polysaccharide, such as ions at m/z 1337.4 and 1175.3, 1175.3 and 1013.3, 1013.3 and 851.2, and so on, was a hexose. Ions at m/z 203.04 corresponded to sodium adducted ions of hexose.

Table-1 Characterized compounds from three Pulicaria species

####Compound Name###Structure###RT###[M+H]+ or###UV###MS and key####15690####15719####15083####15073

###(min)###[M+Na]+###Fragments P. schimperi P. schimperi###P. incisa###P. glutinosa

1###octa-hexose###0.95-1.50###C48H82O41Na+###1337.4213###+###+###+###+

2###hepta-hexose###0.95-1.50###C42H72O36Na+###1175.3649###+###+###+###+

3###hexa-hexose###0.95-1.50###C36H62O31Na+###1013.3181###+###+###+###+

4###penta-hexose###0.95-1.50###C30H62O26Na+###851.2645###+###+###+###+

5###tetra-hexose###0.95-1.50###C24H42O21Na+###689.2114###+###+###+###+

6###tri-hexose###0.95-1.50###C18H32O16Na+###527.1580###+###+###+###+

7###di-hexose###0.95-1.50###C12H22O11Na+###365.1047###+###+###+###+

###217/244/###355.1024,

8###chlorogenic acid###3.57###C16H19O9+###+###+###+###+

###298/326###163.0388

###465.1022,

###303.0502,

###quercetin 3-###+###204/255/

9###5.63###C21H21O12###285.0392,###+###+###+###-

###galactoside###353

###257.0446,

###163.0411

###449.1088,

###kaempferol 3-###204/255/###287.0541,

10###6.15###C21H21O11+###+###+###+###-

###galactoside###353###259.0574,

###163.0396

###517.1329,

###499.1224,

###3,4-dicaffeoylquinic###218/242/###355.1030,

11###6.17###C25H25O12+###+###+###+###+

###acid###298/327###337.0905,

###319.0813,

###163.0394

###517.1334,

###499.1237,

###3,5-dicaffeoylquinic###218/242/###355.1030,

12###6.35###C25H25O12+###+###+###+###+

###acid###298/327###337.0917,

###319.0809,

###163.0395

###517.1334,

###499.1228,

###4,5-dicaffeoylquinic###218/242/###355.1015,

13###6.73###C25H25O12+###+###+###+###+

###acid###298/327###337.0916,

###319.0819,

###163.0391

###303.0499,28

###204/255/###5.0333,

14###quercetin###8.37###C15H11O7+###+###+###+###-

###353###257.0418,16

###3.0397

###217/280/###287.0559,

15###luteolin###8.55###C15H11O6+###+###+###+###-

###327###259.0588

###287.0557,

16###isomer of luteolin###10.33###C15H11O6+###220/288###259.0935,###+###+###+###-

###167.0367

###317.0655,

###quercetin-3-methyl###220/255/###301.0726,

17###11.17###C16H13O7+###+###+###+###-

###ether###353###271.0549,

###243.0699

###287.2215,

###202.1246,

18###unknown-1###11.32###C16H31O4+###+###+###-###-

###189.1246,

###175.1134

###331.0814,

###quercetin-3,3'-

###316.0566,

###dimethyl ether

###199/255/###301.0687,

19###or###11.66###C17H15O7+###+###+###+###-

###355###287.0551,

###quercetin-3,7-

###273.1138,

###dimethyl ether

###167.0339

###301.2376,

###216.1501,

20###unknown-2###12.20###C17H33O4+###+###+###-###-

###189.1240,

###175.1137

###331.0803,

###quercetin-3,3'-

###316.0567,

###dimethyl ether

###197/220/###301.0595,

21###or###12.82###C17H15O7+###+###+###+###-

###291/360###287.0595,

###quercetin-3,7-

###273.0826,

###dimethyl ether

###167.0327

+ = detected, - = not detected

Chlorogenic acid (8, tR3.57 min), 3,5- dicaffeoyl quinic acid (3,5-DCQA, 12, tR6.35 min), and 4,5-dicaffeoyl quinic acid (4,5-DCQA, 13, tR6.73 min) were unambiguously identified after comparing with reference standards. Ions at m/z 377.08 (C16H18O9Na+) and 163.03 (C9H7O +) of chlorogenic acid corresponded to sodium adducted ions [M+Na]+ and fragments ions [M-quinic acid]+, respectively.

Key fragments of two dicaffeoylquinic acid were at m/z 499.12, 337.09, 319.08, and 163.03 which corresponded to fragments [M-H2O]+, [M-C9H8O4]+, [337- H2O]+, and C9H7O +, respectively. Peak at 6.17 min had very similar MS and MS-MS spectra as that of 3,5-DCQA (12) and 4,5-DCQA (13). This compound was tentatively characterized as 3,4- dicaffeoyl quinic acid (3,4-DCQA, 11). In three Pulicaria species (P. shimperi, P. incisa, and P. glutinosa), 3,5-DCQA (12) is a major component, contents of 3,4-DCQA (11) and 4,5-DCQA (13) are less than that of 12.

A group of flavonoids and flavonoid glycosides were characterized from three Pulicaria species. As flavonoids usually show UV absorption at 220, 255-280 and 320-355 nm, peaks having these characteristics were extracted and further identified. Quercetin (14, tR 8.37 min) and luteolin (15, tR 8.55 min) were unambiguously identified after comparing with reference standards. Elemental analysis of quercetin was determined as C15H11O7+ ([M+H]+, found 303.0499, calc. 303.0499).

After applying collision energy to ions at m/z 303.04, fragments were yielded at m/z 285.0333 and 163.0397 corresponding to the losses of oxygen atom and C6H4O4 from protonated molecular ions. Peak at 5.63 min had similar UV absorption as quercetin (14), but protonated molecule was 162 Da heavier than quercetin. Elemental analysis of this peak gave chemical formula as C21H21O + ([M+H]+, found 465.1022, calc. 465.1028).

In the MS/MS spectra of peak 5.63, key fragment ions at m/z 303.0502(C15H11O +) corresponded to the loss of hexose from protonated ions [M+H]+. This compound was tentatively identified as quercetin 3-galactoside (9) that has been found from P. incisa [5]. Protonated molecular ions of peak at 4.17 min were found at m/z 449.1088 (C21H21O11 calc. 449.1078). After applying collision energy to ions 449.10, fragment ions at m/z 287.0541 were produced corresponding to the loss of 162 Da, a hexose unit from protonated molecules. This compound was tentatively characterized as kaempferol 3-galactoside (10) that had been reported in the literature [5].

Compound at 11.17 min was characterized as C16H13O7+ ([M+H]+, found 317.0655, calc. 317.0656) which was 14 Da heavier than that of quercetin, indicating an additional methylene moiety in quercetin. The MS/MS key fragments of this compound presented at m/z 301.0726, 271.0549, and 243.0699 corresponding to C16H13O6+, C15H11O5+, and C14H11O4+, respectively. The MS/MS pattern followed the similar fragmentation pathway of quercetin. Thus, compound at 11.17 min was tentatively characterized as quercetin-3-methyl ether (17), a compound that was previously isolated from P. incisa [5, 13].

Two compounds at 11.66 and 12.82 min gave same protonated molecular ions at m/z 331.0814 and 331.0803, respectively. Through elemental analysis, two compounds had same chemical formula as C17H15O + ([M+H]+, calc. 331.0812) which was 28 Da heavier than that of quercetin, indicating two additional methylene moieties. Two compounds showed identical MS/MS spectra of quercetin. Key fragments at m/z 316.0567(C16H12O7+), 301.0595 (C16H13O6+), 287.0595 (C15H11O +), and 167.0327 (C8H7O4+) further proved that two compounds contained the core skeleton of quercetin. Therefore, two compounds were tentatively identified as quercetin-3,3'-dimethyl ether (19 or 21) and quercetin-3,7-dimethyl ether (19 or 21) that had been identified from P. incisa [5, 13].

Compound at 8.55 min was determined as C15H11O6+ ([M+H]+, found 287.0559, calc. 287.0550), indicating one oxygen atom less than that of quercetin molecule. This compound was eluted at the same retention time as that of luteolin and showed identical MS and MS/MS spectra to that of luteolin. Therefore, compound at 8.55 min was unambiguously identified as luteolin (15). Meanwhile, peak at 10.33 min had same MS and MS-MS spectra as that of luteolin. However, UV spectra of compound 16 had absorption at 220 and 288 nm which was different from that of luteolin. Compound 16 was tentatively characterized as an isomer of luteolin.

Discrimination of Pulicaria Species

Chromatograms of UV maximum absorption of three Pulicaria species are shown in Fig. 2. The peaks retention time at 1.04, 1.08, 2.41, 3.83, 4.21, 4.35, and 4.59 min, respectively, had very similar UV spectra in three Pulicaria species. The results from further discovering of mass and tandem mass spectra proved that all three species (P. schimperi, P. incisa, and P. glutinosa) contained seven polysaccharides (1- 7), chlorogenic acid (8), 3,4-dicaffeoyl quinic acid (11), 3,5-dicaffeoyl quinic acid (12), and 4,5- dicaffeoyl quinic acid (13).

In order to differentiate these species of Pulicaria, further investigating upon mass and tandem mass spectra along with UV spectra of those compounds in Table 1 was conducted. The results showed that the distribution of flavonoid and flavonoid glycosides among species P. schimperi, P. incisa, and P. glutinosa could discriminate three Pulicaria species. In P. glutinosa, none of those characterized flavonoids and flavonoid glycosides was identified.

Species P. shimperi and P. incisa, both contained quercetin and luteolin type flavonoids as well as kaemperfol 3-galactoside (10). However, only two compounds (peaks 18 and 20) were detected from P. shimperi. Compounds 18 and 20 had identical fragments. Chemical formula of compounds 18 and 20 were characterized as C16H31O4+ and C17H33O4+, respectively, in which compound 20 was 14 Da heavier than that of compound 18, indicating an additional methylene group in compound 20.

Acknowledgements

This work was partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University.

References

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