Metabolomic characterization of plant samples with LC-Q-Orbitrap HRMS analysis
According to our investigations, the main metabolic groups identified in the R. nigrum leaf extract (RN) were hydroxycinnamic acids and derivatives, dimeric glycosides, flavan-3-ols, condensed tannins and derivatives, and flavonols (Table 2, Fig. 1). The mentioned metabolites identified as major components.
Table 2
The major identified compounds in R. nigrum extract and the features of their possible biological activity.
Compound Group | Tentative Identification | Possible biological activities |
Hydroxycinnamic acids and derivatives | Caffeoylquinic acid | Antioxidant, antibacterial, antiparasitic, anti-inflammatory, anticancer, antiviral, etc. [41, 42] |
Coumaric acid derivative |
Dimeric glycosides | Musizin glucoside | Antioxidant, anticancer, anti-inflammatory, neuroprotective [43–45] |
Flavan-3-ols | (Epi)gallocatechin | Anticancer, antioxidant, neuroprotective, antivirus, antibiotic modulatory and antibacterial [46–50] |
(epi)Catechin | Antioxidant, antimicrobial, antibiotic modulatory, neuroprotective [48, 51–52] |
Epicatechin gallate | Antioxidant, cytotoxic, antimicrobial, anticancer, antivirus, neuroprotective [50, 53–56] |
Condensed tannins and derivatives | B-Procyanidin dimer | Antitumor, antioxidant, antiviral and anti-inflammatory, neuroprotective [49, 57] |
B-Procyanidin trimer |
A-typeprocyanidin trimer |
B-Procyanidin tetramer |
Flavonols | Quercetin O-hexoside | Antioxidant, anti-inflammatory, anti-cancer, antimicrobial, neuroprotective [4, 58] |
Quercetin O-glucoronide |
Quercetin O-pentoside |
Rhamnetin glucoside |
Quercetin O-deoxyhexoside |
Literature data suggest that all of the identified components possessed biological activity in different tests. This suggestion was confirmed also by our investigations according which R. nigrum leaf extract provided an IC50 value of 61.28 ± 2.34 µg/mL (R² = 0.9462) in DPPH test.
Effects of R. nigrum extract on cell viability
An MTT test was implemented to evaluate the effect of R. nigrum leaf extract on the mitochondrial function and cell viability of Wt and Acox1−/− BV-2 microglial cells. The effect of RN on the cell viability was assessed at a concentration range of 0.0625 to 1 mg/mL for 4, 24 or 48 h treatment period. According to obtained data all concentrations of RN exceeding the 0.25 mg/mL influenced as cytotoxic, regardless the cell genotype and the inhibition of cell viability reached 70% and higher when cells were treated at higher concentrations of extract (Fig. 2 (a) and (b)).
As the sub-cytotoxic the concentration of RN 0.125 mg/mL was chosen, under the influence of which there were not revealed any substantial deviation in cell viability, compared to control. The selected concentration of extract was applied in almost all further experiments.
Effect of R. nigrum extract on LPS-induced intracellular ROS accumulation
In this study, we investigated the effect of the sub-cytotoxic concentration of RN on intracellular ROS accumulation induced by LPS treatment (at 1 µg/mL). For this, we applied H2DCFDA as a ROS probe.
As shown in Fig. 2, in contrast to the Acox1−/− cells, LPS treatment induced oxidative stress with the significant intracellular ROS production in the Wt cells, exhibiting approximately 20% increase in LPS-treated cells compared to the untreated control (Fig. 3a). The observed LPS-induced increase of ROS production in BV2-Wt cells which was significantly overturned by the co-treatment with RN. Moreover, in the cells treated only with RN the ROS level decreased up to 20% compared to untreated control cells (Fig. 3a). LPS treatment did not stimulated the excessive production of ROS in Acox1−/− BV-2 cells, but the treatment with RN decreased ROS accumulation in these cells both in the absence or presence of LPS (Fig. 3b).
R. nigrum extract effects on LPS-induced nitric oxide generation, on the expression of genes encoding different inflammation markers or peroxisomal ABCD transporter protein-
Nitric oxide (NO) is a signaling molecule known for its significant impact on acute and chronic inflammation processes in the nervous system as well as on apoptosis. The inducible isoform iNOS in many cell types produces large amounts of NO as a defense mechanism in response to cytokines and different stress factors [29]. To evaluate the anti-inflammatory effects of RN, we measured the level of NO production and the expression of iNOS gene the in the absence or the presence of LPS (Fig. 4, Fig. 5).
The treatment with RN alone had no any influence on NO production in both BV-2 cell lines (Fig. 4a, b). After the incubation with LPS, the level of NO was significantly increased (up to 30% in Wt and up to 180% - in the Acox 1−/− cells) in comparison to the control cells (Fig. 5). In Wt cells the treatment with RN reduced the LPS-induced NO level (Fig. 4a). The more expressed LPS-induced NO level reduction was observed in the Acox 1−/− BV-2 cell line after the treatment with RN (Fig. 5b).
The expression level of iNOS mRNA was increased by LPS treatment concomitantly to the increase in NO production in both cell lines (Fig. 5a). In response to LPS treatment, the mRNA expression level of iNOS was increased by more than 8–10 folds, in both cell lines (Fig. 5a). The combined treatments of RN + LPS significantly attenuated the LPS-induced iNOS mRNA level increase (Fig. 5a).
The expression of other inflammatory marker encoding genes was also change accordingly (Fig. 5b and c). The treatment with RN kept the Tnf-α and Il-1β gene expression levels constant in both Wt and Acox1−/− cell lines (Fig. 5b, c). Expectedly, the treatment with LPS increases the Tnf-α and the Il-1β gene expression significantly. The co-treatments of RN + LPS limited the increased mRNA levels of both cytokine genes (Fig. 5b, c).
Another picture was observed in case of Abcd1 gene expression: treatment with the RN increases the Abcd1 gene expression level; meanwhile LPS treatment decreases the expression level of this Abcd1 mRNA encoding peroxisomal protein transporter. The treatment with RN + LPS combination elevated this gene expression level to the level of control (Fig. 5d).
Effects of R. nigrum extract on the activity of peroxisomal enzymes and the expression of these protein-encoding genes
The evaluation of activity of the main antioxidant enzymes in peroxisomes of BV-2 cells (ACOX1—the rate-limiting enzyme of β-oxidation process in cells; catalase—antioxidant enzyme, quenching hydrogen peroxide) under the treatment of RN is of interest in order to fully understand the antioxidant capacity of test-extract.
As it is presented in Fig. 6 (a) the catalase activity in Wt cells remained almost unchanged during the 24-hour treatment, meanwhile the further treatment lead to significant decrease of this enzyme activity (at up to 40% compared to untreated control). The long-term (72-hour) treatment of Wt cells with RN had no significant effect on catalase activity (Fig. 6a). In case of Acox1−/− BV-2 cells, during the first 24 hours of treatment with RN, a strict decrease in catalase activity was observed. During the further treatment, the activity of the enzyme almost equaled to its activity in control cells, and the long-term treatment did not have any effect (Fig. 6a).
The ACOX1 activity in Wt BV-2 microglial cells decreased by almost 50% during the first 24 hours of treatment with RN compared to the untreated control. The further 48-hour treatment resulted in an increase in the activity of this enzyme by almost 70%. In case of the long-term treatment (72-hour), the activity of ACOX1 decreased more than 3 times compared to the second stage of the treatment, reaching the indicator of 24-hour treatment (Fig. 6b).
To further analyze the change in catalase activity under the treatment of RN, the expression of its mRNA and protein levels have been investigated. For this, 24-hour treated cells were used. According to our investigation, the selected concentration of extract did not induce the oxidative stress in the investigated cell lines (Fig. 3; Fig. 4; Fi 5a). The next step of our experiment was the triggering the oxidative stress and ROS formation in both cell lines by LPS and further treatment with RN in order to observe the possible mitigating influence of this extract on the LPS-induced oxidative deviations in cell models (Fig. 7).
All values are presented as means ± SD of two independent experiments performed in triplicate (*p < 0.1; **p < 0.01; ***p < 0.01 and ns, not significant). Statistical significance was determined using one-way ANOVA followed by Tukey’s test for multiple comparisons.
According to our investigations the presence of 1 µg/mL LPS in the cultivation medium significantly trigger the catalase activity in both cell lines at up to 60% and more (Fig. 7a). The LPS treatment also increased the Cat mRNA level in both Wt and Acox1−/− cells (Fig. 7b). The co-treatments of RN + LPS decreased the LPS-induced catalase activity as well as the CAT gene expression in both cell lines (Fig. 7a, b). The immunoblotting assay shows that the CAT protein quantity change replicates the changes in catalase activity in both cell lines (Fig. 7c,d).