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Year : 2020  |  Volume : 16  |  Issue : 71  |  Page : 592-599  

Profiling of antiviral and antioxidant phytochemicals of Pterocephalus frutescens hochist. using high-resolution ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer

1 Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University (Boys), Cairo 11884, Egypt
2 Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University (Girls), Cairo 11884, Egypt
3 Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University (Boys), Cairo 11884; Department of Pharmacognosy, Faculty of Pharmacy, Sinai University, Kantara 41636, Egypt

Date of Submission15-Dec-2019
Date of Decision05-Feb-2020
Date of Acceptance16-Apr-2020
Date of Web Publication20-Oct-2020

Correspondence Address:
Hatem S Abbass
Department of Pharmacognosy, Faculty of Pharmacy, Sinai University, Kantara
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/pm.pm_558_19

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Background: Medicinal plant extracts and herbal preparations are complex mixtures of active and ballast substances, which may contain in frequently up to several hundreds of different constituents, with no exactly defined structures at which chromatography undoubtedly is a fundamental tool to overcome the challenges of these phytoanalytics. Aim of the Study: To investigate Pterocephalus frutescens aerial part extracts biologically and chemically and the correlation of biologically active constituents with their corresponding activities. Materials and Methods: Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer with principal component statistical analysis was used for the identification of phytoconstituents, while spectrophotometer was used for quantitative analysis and both qualitative and quantitative antioxidant effects using stable radical diphenylpicrylhydrazyl, and the cytopathic effect inhibition assay was used for antiviral screening. Results and Conclusion: P. frutescens extracts exhibited antioxidant and antiviral activities along with tentative identifying of 46 compounds from which 38 compounds were correlated with either antiviral and/or antioxidant activities, besides the quantification of phenolic, flavonoid, phenylethanoid, and iridoid contents.

Keywords: Antioxidant, antiviral, mass spectrometry, principal component analysis, ultra-performance liquid chromatography

How to cite this article:
El-Hela AA, Hegazy MM, Abu Bakr MS, Abbass HS. Profiling of antiviral and antioxidant phytochemicals of Pterocephalus frutescens hochist. using high-resolution ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer. Phcog Mag 2020;16:592-9

How to cite this URL:
El-Hela AA, Hegazy MM, Abu Bakr MS, Abbass HS. Profiling of antiviral and antioxidant phytochemicals of Pterocephalus frutescens hochist. using high-resolution ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer. Phcog Mag [serial online] 2020 [cited 2022 Sep 30];16:592-9. Available from: http://www.phcog.com/text.asp?2020/16/71/592/298702


  • Biological investigation of Pterocephalus frutescens growing in Yemen showed both antiviral and antioxidant activities at which the responsible secondary metabolites for these activities were identified using rapid and accurate chromatographic analysis method as well. Quantification of the most important chemical classes was done for further confirmation and explaining of these activities.

Abbreviations used: BHT: Butylated hydroxy toluene; DPPH: Diphenylpicrylhydrazyl; ESI: Electrospray ionization; IC50value: Inhibition concentration of sample at 50% fall in absorbance; HAV: Hepatitis virus type A; HSV-I: Herpes simplex virus type I; HSV-II: Herpes simplex virus type II; PCA: Principal component analysis; RCMB: Regional Center for Mycology and Biotechnology; RT: Retention time; UPLC/QTOF-MS/MS: Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer.

   Introduction Top

Quadrupole time-of-flight mass spectrometer attached to ultra-performance liquid chromatography (UPLC/QTOF-MS/MS) is nearly standard equipment in a modern analytical laboratory and is indispensable for scientific research on a high level as screening and identifying compounds in different medicinal plants in a fast and accurate manner.[1] The honeysuckle family or Caprifoliaceae (Dipsacaceae) family is consisting of about 860 species and 42 genera, which distributed in Europe, Asia, and Africa. Some species of this family have been naturalized in other places.[2]Pterocephalus, comprising 25 species, ranges from the Mediterranean, to Central Asia, the Himalayas, Western China, and tropical Africa.[3] Plants of the genus Pterocephalus are widely used in folkloric medicines, around the entire world, as anti-inflammatory, analgesic, antihepatotoxic, antioxidant, antibacterial, spasmolytic, hemostatic, and astringent properties.[3],[4],[5],[6] Several active constituents have been isolated from genus Pterocephalus including iridoid, hydroxycinnamic acid esters, phenolic glycosides, lignans, triterpenoid saponins, and flavonoid C-glycosides.[7],[8],[9],[10] Surprisingly, Pterocephalus frutescens was not evaluated phytochemically and/or biologically, up to this date. In the present work, it is worthy to give an idea about the evaluation of antiviral and antioxidant activities and illustrating secondary metabolites responsible for both activities using reliable, fast, accurate, simple, and reproducible technique UPLC/QTOF-MS/MS in association with principal component analysis (PCA) as a qualitative technique. Quantitative measurements of polyphenolic content are of interest as a lot of findings have been reported earlier, suggesting a relationship between total polyphenols content and health effects,[11] mainly antioxidant activity which tested in our work.

   Materials and Methods Top

Plant materials

The aerial parts of P. frutescens Hochist. were collected in June 2017 at flowering stage from Jabal An-Nabi Shu'ahyb, Sanaa, Al Yemen. The plant was collected, identified, and authenticated by Dr. Abdo H. Marey, Professor of Botany and Plant Taxonomy, Faculty of Science, Al-Azhar University, Cairo, Egypt. A voucher sample was kept in the Herbarium of Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.

Extraction and isolation

Air-dried powdered aerial part plant material of P. frutescens (250 g) was extracted by Soxhlet apparatus with n-hexane (2 × 2 L) and then with methanol (3 × 3 L) to afford (7.5 and 35 g), respectively, after evaporation under vacuum by a rotatory evaporator (BUCHI Rotavapor® R-210/R-215, Germany). From methanol extract, 0.5 g was subjected to the quantitative determination of polyphenolics and UPLC/QTOF-MS/MS analysis; 1 g reserved for biological investigations and the remaining residue were dissolved in water and then partitioned successively with ethyl acetate (2 × 200 mL) and n-butanol saturated with water (3 × 200 mL) to afford light brown masses (8.5 and 11 g), respectively.

Quantitative determination of polyphenolics

Quantitative determination of phenolic, flavonoid, phenylethanoid, and iridoid contents using selective method for each phytochemical class[12] was done using Genesys Spectrophotometer (Milton Roy, INC., Rochester, NY, USA). The reagents used for each were Folin-Ciocalteu (Sigma Chemical Co., St. Louis, MO, USA) and gallic acid (Merck, Darmstadt, Germany) for phenolic content quantification; quercetin (Merck Co. Darmstadt, Germany) and aluminum chloride (Merck, Darmstadt, Germany) for flavonoid content quantification, Arnow's reagent and verbascoside (Sigma Chemical Co., St. Louis, MO, USA) for phenylethanoid content quantification; and Trim and Hill reagent and Herbagoside (Sigma Chemical Co., St. Louis, MO, USA) for iridoid content quantification.

Antiviral assay

Screening of antiviral activity was achieved using cytopathic effect (CPE) inhibition assay against hepatitis virus type A (HAV) and herpes simplex virus type I (HSV-I) and II (HSV-II) at the Regional Center for Mycology and Biotechnology, Al-Azhar University.[13] Incubation was done at 37°C for 5 days and observation was made every 24 h to record the CPE. Antiviral activity was determined by the inhibition of CPE compared with control. The results were expressed as:

–ve: No inhibition, +: 25% inhibition, ++: 50 inhibition, +++: 75% inhibition, and ++++: 100% inhibition of CPE.

Antioxidant potentials

Determination of the antioxidant effect of the different extracts of P. frutescens was done using stable radical diphenylpicrylhydrazyl (DPPH) (Sigma Chemical Co., St. Louis, MO, USA). Both qualitative and quantitative antioxidant effects were experimented.

TLC assay (qualitative method)

20 μL aliquot of each extract was spotted on silica gel plates and developed using butanol:acetic acid:water (4:1:5) as a mobile phase; after development, the dried TLC plates were sprayed with 0.2% DPPH solution (Sigma Chemical Co., St. Louis, MO, USA) in methanol and examined after 30 min, where active extracts as antioxidants appeared as yellow spots against purple background.[14]

Spectrophotometric assay (quantitative method)

The test was carried out by adding 160 μL (0.1 mM) DPPH solution in methanol to 40 μL of each sample solution in methanol. After 30 min, the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity according to recent published method.[15] Butylated hydroxy toluene (BHT) (Sigma Chemical Co., St. Louis, MO, USA) in methanol was used as standard. The capability of scavenging the DPPH radical was calculated using the following equation:

DPPH scavenging effect (%) = [(Acontrol− Asample)/Acontrol] – 100.

The the inhibition concentration of sample at 50% fall in absorbance of DPPH (IC50) value was used to compare the DPPH scavenging activity of the highest free radical scavenging activity extract with two standard controls, BHT and quercetin using various concentrations in methanol according to previously discussed method.[14]

Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer analysis

The lyophilized plant extracted sample injected in the positive mode at which separation of small molecules was carried out on a Axion AC system (Kyoto, Japan) connected with an auto sampler system, an In-Line filter disks precolumn (0.5 μm × 3.0 mm, Phenomenex, USA), and a Xbridge C18(3.5 μm, 2.1 mm × 50 mm) column (Waters Corporation, Milford, MA, USA) maintained at 40°C and a flow rate of 300 μL/min. The mobile phase composition and the gradient elution were performed according to the method discussed in Fayek et al.[16] LC-MS grade acetonitrile and gradient solvents including isopropanol, methanol, dichloromethane, and ethyl acetate were provided by Thermo-Fisher (Thermo Fisher Scientific, USA). Formic acid 98%, ammonium hydroxide, ammonium formate, and ammonium acetate were purchased from Sigma-Aldrich (Sigma-Aldrich Co., Louis St., MO, USA). MS was performed on a TripleTOF™ 5600 System QTOF/MS with a Duo-Spray™ source operating in the electrospray ionization mode (AB SCIEX, Concord, Canada). The sprayer capillary and declustering potential voltages were 4500 and 80 V. The source temperature, collision energy, ion tolerance, and Triple TOF 5600 operation parameters were reported in Fayek et al.[16] The method consisted of high-resolution survey spectra from 50 to 1100 m/z, and the mass spectrometer was operated in a pattern where a 50-ms survey scan was detected. Subsequently, the top 15 intense ions were selected for acquiring MS/MS fragmentation spectra after each scan.[16]

Data were processed using MS-DIAL 3.96 software for nontargeting small molecule comprehensive analysis of the sample. According to the acquisition mode, ReSpect Positive (2737 records), MassBank Positive (8068 records), and GNPS Positive (8782 records) databases were used as reference databases. The search parameters were set according to Tsugawa et al.[17] The MS-DIAL output was used to make manual annotation and confirmation of the identified peaks using PCA, which is an unsupervised clustering method requiring little prior knowledge of the data set.[18] PCA scores' plot analysis was performed in Excel using a macro written by Tsugawa et al.[17] and is available for free.

   Results and Discussion Top

Quantitative determination of polyphenolics

The results of this study provide evidence that the methanol extract has phenolic, flavonoid, phenylethanoid, and iridoid compounds in relatively high amounts [Table 1]; therefore, this species may have a great relevance in the prevention and therapies of diseases in which oxidants or free radicals are implicated.
Table 1: Quantitative determination of total phenolic, flavonoid, phenylethanoid, and iridoid contents of the methanol extract of Pterocephalus frutescens

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Antiviral assay

The results of antiviral screening of different extracts of P. frutescens [Table 2] showed that the observed activity of both hexane and ethyl acetate extracts on (HAV, HSV-I, and HSV-II) was attributed to flavonoids identified in the plant extract by UPLC/QTOF-MS/MS, due to inhibitory structure–function relationship between flavonoids and various enzymes associated with the life cycle of viruses.[19] The chromatographic UPLC/QTOF-MS/MS method revealed the presence of 30 compounds with antiviral potentials which were reported previously as shown in [Table 6]. For example, amentoflavone has activity against HSV-I and HSV-II with EC50 values of 17.9 and 48.0 mg/ml, respectively, as well apigenin which reported to have the highest inhibition against HSV-I with the widest therapeutic range (0.4–1.6 μg/mL) and loliolide which has both anti-HSV and anti-hepatitis virus type C (HCV).[30],[53],[57] Phenolic and iridoid compounds also play an important role as antiviral agents; the presence of 98 mg% of phenolic compounds along with 40 μg% of iridoid compounds explains both hexane and ethyl acetate antiviral activities.[31]
Table 2: Antiviral screening of different extracts of Pterocephalus frutescens

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Antioxidant potentials

Qualitative TLC-DPPH assay of the tested extracts showed that they are active as DPPH scavengers appearing as zones with different Rf values in the chromatogram; then, quantitative estimation of their antioxidant potentials at dose level of 0.1 mg/ml revealed that they possess significant DPPH free radical scavenging activity which is proven comparable to the reference synthetic antioxidant (BHT). This activity may be explained by high polyphenolic compound contents[11] [Table 1] and further confirmed by tentatively identification of 38 antioxidant compounds [Table 6], which are distributed in different plant extracts. For example, flavonoid aglycons (apigenin, kaempferol, and quercetin) concentrated in hexane extract;[64] flavonoid glycosides and other phenolic compounds concentrated in both ethyl acetate and n-butanol extracts. The most promising extract was ethyl acetate with 89.93% DPPH scavenging activity [Table 3] and an IC50 of 0.05 mg/mL nearly similar to that of BHT [Table 4].
Table 3: Calculated percent free radical scavenging activity of the different extracts of Pterocephalus frutescens and butylated hydroxy toluene

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Table 4: Diphenylpicrylhydrazyl free radical scavenging activity IC50 (mg/ml) of Pterocephalus frutescens ethyl acetate extract, butylated hydroxy toluene, and quercetin

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Interpretation of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer results

A total of 46 compounds were tentatively identified in [Table 5]; whereas the data were obtained from PCA scores' plot analysis from MS-DIAL exhibited a pseudomolecular ion peaks with different adducts. The compounds include amino acids (3), coumarins (4), simple phenolic compounds (9), and flavonoids of different subclasses (23) and of other different chemical classes (7) [Table 6].
Table 5: Metabolites identified from methanol extract of Pterocephalus frutescens using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometer analysis in positive ionization mode

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Table 6: The suspected biological active metabolites and their chemical class

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Compound identification based on the data of MS-DIAL includes assigning of the compounds with the highest total score above 70%, dissociating the link between precursors and their fragment ions, compromising the molecular identification process, and expressing the fragment presence by %. These two criteria were achieved by DIA methods; reducing false-negative results in contrast to traditional data-dependent MS/MS acquisitions.[17] The further confirmation of the compounds was done by comparison with METLIN (http://metlin.scripps.edu), a freely accessible web-based.[65] In addition to traditional methods of confirmation utilizing retention time, comparison of fragmentation pattern of each chemical class with literature reviews and high-resolution mass error values calculations using the following formula:

Error = (Experimental mass − Calculated mass)/Experimental mass.[66]

To improve mass accuracy, no error exceeds 14 ppm.[18] For comparison with literatures of amino acids, the characteristic m/z was due to the loss of 18 and 46 amu appears to be prevalent so that the [(M + H)-H2O]+ and [(M + H)-CO2H2]+, respectively.[67] The losses of hydroxyl, methyl, or carboxylic groups were helpful in the identification of the specific phenolic compounds to form fragments at m/z [M + H-16]+, [M + H-14]+, or [M + H-44]+, respectively.[68] Coumarin derivatives common fragment ions attributed to loss of CO from the pyrone ring to form a benzofuran ring at m/z [M + H-28]+ which is further fragmented by the loss of H2O moiety to form a fragment at m/z [M + H-18]+. Another fragment ions have been attributed to generate a ketene ion at m/z [M + H-72]+. The hydroxy-substituted coumarins showed fragmentation process with the loss of C2H2O to generate a signal at m/z [M + H-42]+.[69] The fragmentation patterns of flavonoids were very diverse according to subclass (flavones, flavonols, flavanones, and/or isoflavones) and according to glycosylation pattern which previously published in details.[70],[71],[72]

   Conclusion Top

Our results showed that P. frutescens has a high interesting value for pharmaceutical industries as it revealed antiviral and antioxidant activities. Tentative profiling of different secondary metabolites of P. frutescens aerial parts was done in a fast and accurate manner without tedious routine experiments, which could be applicable for chemical investigation of different plant materials.

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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