HPLC-based cytotoxicity profiling and LC-ESIQTOF-MS/MS analysis of Helichrysum leucocephalum

Abstract

Introduction: Helichrysum leucocephalum Boiss. (Asteraceae) is an endemic plant to Iran. No reports have studied the cytotoxicity of the plant. The current study aimed to evaluate the cytotoxicity of H. leucocephalum collected from Fars province (Iran) against MCF-7 and HDF cell lines using HPLC-based activity profiling and to annotate the active constituents by LC-ESIQTOF-MS/MS.

Methods: H. leucocephalum was collected from three locations in Fars province. The dried flowers and leaves were separately extracted by percolation using methanol. The crude extracts were fractionated by liquid-liquid partitioning with dichloromethane (DCM) and aqueous methanol. The cytotoxicity of the fractions was evaluated against MCF-7 and HDF cells by Alamarblue assay. HPLC-based activity profiling was used to track the active constituents. LC-MS dereplication strategy was used for the annotation of the compounds in the active time window. LC-MS data were preprocessed by MZmine 3.3.0 and submitted to multivariate analysis to compare the differences and similarities in the metabolites of the samples.

Results: The DCM fractions showed a dose-dependent cytotoxicity against the cancerous cells (IC50s, 9.8-105.1 μg/ml). In general, the metabolites of the flowers and their cytotoxicity were higher than the leaves. LCESIMS/MS analyses revealed that prenylated and geranylated α,β-unsaturated spiroketal phloroglucinols were among the active constituents.

Conclusion: It can be concluded that H. leucocephalum is a rich source of phloroglucinol derivatives with cytotoxic activities. Further phytochemical analysis is needed to characterize the bioactive components.

Keywords: Asteraceae; Cytotoxicity; HPLC; Helichrysum leucocephalum; LC-ESIQTOF-MS/MS.

Fig. 1

Viability of HDF cells treated with different concentrations of DCM fractions after 48 h (p < 0.05*p < 0.01**p < 0.001***). HLED, HLND, and HLAD refer to the DCM fraction from leave of plant samples collected from Estahban, Neyriz, and Ashraf regions in Fars province. HLNFD and HLAFD refer to the DCM fraction of flower samples collected from Neyriz and Ashraf regions in Fars province (Iran).

Fig. 2

IC₅₀ values of the DCM samples against HDF cells after 48 h. HLED, HLND, and HLAD refer to the DCM fraction of leave samples collected from Estahban, Neyriz, and Ashraf regions in Fars province, respectively. HLNFD and HLAFD refer to the DCM fraction of flower samples collected from Neyriz and Ashraf regions in Fars province (Iran), respectively.

Fig. 3

Viability of MCF-7 cells treated with different concentrations of DCM fractions after 48 h (p < 0.05*p < 0.01**p < 0.001***). HLED, HLND, and HLAD refer to the DCM fraction of leave samples collected from Estahban, Neyriz, and Ashraf regions in Fars province. HLNFD and HLAFD refer to the DCM fraction of flower samples collected from Neyriz and Ashraf regions in Fars province (Iran).

Fig. 4

IC₅₀ values of the DCM fractions against MCF-7 cells after 48 h. HLED, HLND, and HLAD refer to the leave of plant samples collected from Estahban, Neyriz, and Ashraf regions in Fars province, respectively. HLNFD and HLAFD refer to the flowers of plant samples collected from Neyriz, and Ashraf regions in Fars province (Iran), respectively.

Fig. 5

Viability of MCF-7 cells treated with concentrations 2.5, 5, 20, and 100 μg/ml of HPLC microfractions (1–15) after 48 h (p < 0.05*p < 0.01**p < 0.001***).

Fig. 6

HPLC-based activity profiling of the dichloromethane extract of H. leucocephalum (HLNFD). The PDA chromatogram (285 nm) of a separation of 10 mg of extract on a semipreparative reverse phase HPLC column is shown. Activity of 5 min microfractions is indicated with colored columns for cytotoxic activity, expressed as IC₅₀.

Fig. 7

LC-MS chromatograms of the flower DCM fractions of H. leucocephalum collected from Neyriz (black) and Ashraf (pink) in positive ionization mode. ESIMS: base peak chromatogram (BPC); m/z 50–1000; column Purospher® STAR RP-18 encapped (250 × 4.6 mm, 5 μm; Merck). MeOH 0.05% TFA/aq. 0.05 TFA in 80 min, a flow rate of 0.7 ml/min. The following gradient mode was employed: 0.0−1.0 min: 40% B isocratic, 1.0−10.0 min: 40−55% B, 10.0−15.0 min: 55–75% B, 15.0–50.0 min: 75−90% B, 50–55 min: 90.0–100.0% B, and 55.0−75.0 min: 100% B. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

LC-MS chromatograms of the leave (pink) and flower (black) DCM fractions of H. leucocephalum collected from Neyriz in positive ionization mode. ESIMS: base peak chromatogram (BPC); m/z 50–1000; column Purospher® STAR RP-18 encapped (250 × 4.6 mm, 5 μm; Merck). MeOH 0.05% TFA/aq. 0.05 TFA in 80 min, a flow rate of 0.7 ml/min. The following gradient mode was employed: 0.0−1.0 min: 40% B isocratic, 1.0−10.0 min: 40−55% B, 10.0−15.0 min: 55–75% B, 15.0–50.0 min: 75−90% B, 50–55 min: 90.0–100.0% B, and 55.0−75.0 min: 100% B. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

The peak number of the detected compounds in the active time windows for the DCM fraction of Helichrysum leucocephalum flowers in positive ionization mode.

Scheme 1

The plausible MS/MS fragmentation pattern for compound 2 in positive ionization mode.

Fig. 10

MS¹ (A) and MS² (B) spectra of compound 2 (helipyrone C), [M+H]⁺ = 293.

Fig. 11

MS¹ (A) and MS² (B) spectra of compound 11 (helispiroketal C), [M−H]⁻ = 399.

Scheme 2

The plausible MS/MS fragmentation pattern for compound 11, a prenylated α,β-unsaturated spiroketal phloroglucinol derivative, in negative ionization mode.

Fig. 12

The plausible MSⁿ fragmentation pathway for the geranylated α,β-unsaturated spiroketal phloroglucinol derivatives.

Scheme 3

The plausible MS/MS fragmentation pattern for geranylated α,β-unsaturated spiroketal phloroglucinols in negative ionization mode.

Fig. 13

The compounds annotated in the active time windows.

Fig. 14

PCA score plot derived from multivariate statistical analysis of LC-ESIQTOF-MS profiling data acquired from Helichrysum leucocephalum samples. HLED and HLND refer to the DCM fraction of leave samples collected from Estahban and Neyriz regions in Fars province, respectively. HLNFD and HLAFD refer to the DCM extract of flower samples collected from Neyriz, and Ashraf regions in Fars province (Iran), respectively.

Fig. 15

Heat-map of the metabolites in the samples of Helichrysum leucocephalum. In this analysis, only LCESIMS/MS results in negative ionization mode were used. The colors represent the abundance of compounds in the samples; the most abundant compounds are red and the absent compounds are blue. The X-axis shows the detected ions in the time interval from 0 to 60 min. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)