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. 2021 Jul 2;11(7):975.
doi: 10.3390/biom11070975.

Phytochemical Analysis and Anti-Inflammatory Activity of Different Ethanolic Phyto-Extracts of Artemisia annua L

Giulia Abate  1 , Leilei Zhang  2 , Mariachiara Pucci  1 , Giulia Morbini  1 , Eileen Mac Sweeney  1 , Giuseppina Maccarinelli  1 , Giovanni Ribaudo  1 , Alessandra Gianoncelli  1 , Daniela Uberti  1 , Maurizio Memo  1 , Luigi Lucini  2 , Andrea Mastinu  1
Affiliations

Phytochemical Analysis and Anti-Inflammatory Activity of Different Ethanolic Phyto-Extracts of Artemisia annua L

Giulia Abate et al. Biomolecules. .

Abstract

Artemisia annua L. (AA) has shown for many centuries important therapeutic virtues associated with the presence of artemisinin (ART). The aim of this study was to identify and quantify ART and other secondary metabolites in ethanolic extracts of AA and evaluate the biological activity in the presence of an inflammatory stimulus. In this work, after the extraction of the aerial parts of AA with different concentrations of ethanol, ART was quantified by HPLC and HPLC-MS. In addition, anthocyanins, flavanols, flavanones, flavonols, lignans, low-molecular-weight phenolics, phenolic acids, stilbenes, and terpenes were identified and semi-quantitatively determined by UHPLC-QTOF-MS untargeted metabolomics. Finally, the viability of human neuroblastoma cells (SH-SY5Y) was evaluated in the presence of the different ethanolic extracts and in the presence of lipopolysaccharide (LPS). The results show that ART is more concentrated in AA samples extracted with 90% ethanol. Regarding the other metabolites, only the anthocyanins are more concentrated in the samples extracted with 90% ethanol. Finally, ART and all AA samples showed a protective action towards the pro-inflammatory stimulus of LPS. In particular, the anti-inflammatory effect of the leaf extract of AA with 90% ethanol was also confirmed at the molecular level since a reduction in TNF-α mRNA gene expression was observed in SH-SY5Y treated with LPS.

Keywords: Artemisia annua L.; TNF-α; artemisinin; phenolic compounds; terpenoids.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Crops of Artemisia annua.
Figure 2
Figure 2
Chromatograms of the phyto-extracts of Artemisia annua and of the standard artemisinin. (A) Chemical structure of artemisinin; (B) details of the elution of STD artemisinin (pink) and (C) the ART in the AA phyto-extract samples; and (D) superimposed chromatograms of the ART standard and AA phyto-extracts at different concentrations of ethanol.
Figure 3
Figure 3
ESI-MS spectra of the sample manually collected from HPLC-DAD of the hydroalcoholic phyto-extract in 90% ethanol. (A) Full ESI-MS spectrum. (B) Fragmentation spectrum resulting from the CID experiment carried out on the isolated signal at m/z = 283.12 (normalized collision energy = 13).
Figure 4
Figure 4
ESI-MS spectra of the sample manually collected from HPLC-DAD of the ART. (A) Full ESI-MS spectrum. (B) Fragmentation spectrum resulting from the CID experiment carried out on the isolated signal at m/z = 283.12 (normalized collision energy = 12).
Figure 5
Figure 5
Orthogonal projection to latent structures discriminant analysis (OPLS-DA) on different concentrations of hydroalcoholic phyto-extracts of Artemisia annua, i.e., AA ethanol 0%, AA ethanol 25%, AA ethanol 50%, and AA ethanol 90%. The parameters: goodness of fit R2Y: 0.997; goodness of prediction Q2Y: 0.898; cross-validation ANOVA p < 0.01.
Figure 6
Figure 6
Effects of ethanolic extracts of Artemisia annua L. on SH-SY5Y cell viability. The bars indicate the percent cell viability of cells treated with (A) artemisinin standard; (B) phyto-extracts of AA in 25% ethanol; (C) phyto-extracts of AA in 50% ethanol; and (D) phyto-extracts of AA in 90% ethanol with the indicated concentrations (ranging from 25 μg/mL to 5 ng/mL) for 48 h and subjected to an MTT assay. Phyto-extract treatments were performed so that the ART content within the phyto-extracts was at the same concentration of STD Artemisinin. This estimation was calculated based on the phyto-extract ART quantification by HPLC. Data are representative of three replicates and shown as mean ± standard deviation; *** p < 0.001 vs. control group; ** p < 0.005 vs. control group.
Figure 7
Figure 7
Protective effect of ethanolic extracts of Artemisia annua L. in SH-SY5Y cells after LPS pro-inflammatory insult. Cell viability of SH-SY5Y cells subjected to 24 h of co-treatment with LPS and Artemisinin standard (A), AA hydroalcoholic phyto-extract in ethanol 25% (B), 50% (C), and 90% (D). Phyto-extract treatments were performed so that the ART content within the phyto-extracts was at the same concentration of STD Artemisinin. This estimation was calculated based on the phyto-extract ART quantification by HPLC. Cell viability was measured with the MTT assay and is expressed as a percentage of the control (white column) that did not receive any treatment. Data are expressed as mean ± standard error of the mean: *** p ≤ 0.001, ** p ≤ 0.01 and * p < 0.05 versus untreated cells and ### p ≤ 0.001, ## p ≤ 0.01 and # p ≤ 0.05 versus LPS.
Figure 8
Figure 8
Ethanolic extracts of Artemisia annua and ART are able to act as a TNF-α inhibitor in the presence or absence of LPS. SH-SY5Y cells were treated for 24 h with STD Artemisinin and AA Ethanol 90% (A) or pre-treated for 2 h with the same treatments and then subjected to a LPS stimulus for 24 h (B). Cells were then processed in order to measure TNF-α m RNA levels by real-time PCR. GAPDH was used to normalize the results. Data are shown as mean ± SEM. Statistically significant differences are represented as follows: **** p ≤ 0.0001, *** p ≤ 0.001, and ** p ≤ 0.01 versus untreated cells and #### p < 0.0001, ### p ≤ 0.001, and # p ≤ 0.05 versus LPS.
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