# Ribes nigrum leaf extract downregulates pro-inflammatory gene expression and regulates redox balance in microglial cells

Alvard Minasyan, Vivien Pires, Catherine Gondcaille, Mikayel Ginovyan, Marika Mróz, Stéphane Savary, Mustapha I Cherkaoui-Malki, Barbara Kusznierewicz, Agnieszka Bartoszek, Pierre Andreoletti, et al.

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## ► To cite this version:

Alvard Minasyan, Vivien Pires, Catherine Gondcaille, Mikayel Ginovyan, Marika Mróz, et al. Ribes nigrum leaf extract downregulates pro-inflammatory gene expression and regulates redox balance in microglial cells. BMC Complementary Medicine and Therapies, 2025, 25 (1), pp.49. 10.1186/s12906-025-04780-7. hal-04958552

HAL Id: hal-04958552

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# Ribes nigrum leaf extract downregulates pro-inflammatory gene expression and regulates redox balance in microglial cells

Alvard Minasyan¹, Vivien Pires², Catherine Gondcaille², Mikayel Ginovyan¹, Marika Mróz³, Stéphane Savary², Mustapha Cherkaoui-Malki², Barbara Kusznierewicz³, Agnieszka Bartoszek³, Pierre Andreoletti² and Naira Sahakyan¹*

## Abstract

**Background** This study focuses on the investigation of the antioxidant and anti-inflammatory activities of alcohol extracts from *Ribes nigrum* leaves on murine BV-2 microglial *Wt* and Acyl-CoA oxidase 1 deficient (Acox1⁻/⁻) cell line models, useful for the investigation of some neurodegenerative disorders.

**Methods** The extract chemical composition was analyzed via LC-Q-Orbitrap HRMS. Various assays, including DPPH, MTT, and H₂DCFDA, were used to assess the extract's antioxidant capacity, cell viability, and reactive oxygen species (ROS) production. Immunoblotting and RT-qPCR techniques were employed to measure protein expression and gene transcription in treated cells. Statistical analysis was conducted using GraphPad Prism, with significance determined at *p* < 0.05.

**Results** Investigations showed the presence of phenolic compounds in this extract, among which flavan-3-ols, flavonols, furanocoumarins, hydroxycinnamates were major components, which are known for their biological activity in various test systems. The MTT test revealed a concentration of 0.125 mg/mL of *R. nigrum* extract as the highest non-toxic. The investigated extract showed high antioxidant activity in chemical-based tests. The antioxidant potential of the *R. nigrum* leaf extract was furtherly explored using the BV-2 microglial cell line models. Moreover, the extract was found to alter the activity of the main antioxidant enzyme, catalase and fatty acid oxidation enzyme, Acyl-CoA oxidase 1 (ACOX1) as well as the expression of appropriate genes in *Wt* and Acox1⁻/⁻ BV-2 microglial cells such as *Cat*, *iNos*, *Il-1β*, *Tnf-a*, and *Abcd1*. In *Wt* cells, after the 24-hour treatment with *R. nigrum* leaf extract, ACOX1 activity was downregulated, meanwhile the catalase activity remains unchanged. Further treatment led to the downregulation of catalase and the upregulation of ACOX1 activity. However, in Acox1⁻/⁻ cells, which represent a model of oxidative stress, an increase in catalase activity was observed only after 48 h of treatment. It was also observed the reduced ROS and NO formation in cells, showing the pronounced antioxidant capacity of *R. nigrum* extract in the investigated cell-models.

**Conclusion** Our study demonstrated the protective effects of *R. nigrum* leaf extracts on BV-2 microglial cells by reducing oxidative and nitrosative stress, decreasing pro-inflammatory gene expression, and normalizing peroxisomal

Full list of author information is available at the end of the article

<footnote>*Correspondence:
Naira Sahakyan
sahakyannaira@ysu.am</footnote>
function, highlighting the potential of these extracts as therapeutic agents for managing oxidative stress and
inflammation.

**Keywords** Oxidative stress, Armenian flora, Reactive oxygen species, Polyphenols, Neurodegenerative disorders

**Introduction**

During aging process and various physiological changes, we observe oxidative misbalance in organisms, resulting in a range of deviations. There is well established link between the redox balance and the development and progression of several diseases. According to Loreto Palacio et al. (2022) [1] this imbalance may result in alterations to the redox system and thiol modifications. Oxidative stress typically results from the overproduction of free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are generated by internal metabolic processes and external environmental factors like ultraviolet radiation, chemical pollutants, and physical overexertion [2] Studies have shown a direct correlation between excess free radicals, DNA damage, cancer cell growth, type II diabetes mellitus induction and many other deviations [3, 4].

When analyzing the available data, it can appear that controlling any specific deviation from the norm by altering the cell's redox status should be quite simple because so many processes are taking place at the expense of proteins and other molecules undergoing redox transformations [5]. But in reality, it is not so clear-cut, as there are many interrelated factors at play.

First, the issue of bioavailability of active ingredients in organisms and the inability of extrapolating in vitro or even in situ experiment results to in vivo systems remain constant problems [6]. Additional possible causes of the bioavailability issues include dose, the degradation of components by GUT microbiome, interactions with various chemicals and ions among other factors [6, 7]. Further reason is the fact that active metabolites potentially can play as pro-oxidants depending on a collection of variables: the presence of reduced metals in the environment, flavonoids, vitamins that in addition to their stated antioxidant activity, can act also as pro-oxidants [8].

In addition, one more explanation would be the so-called “reductive stress” when a biological system’s reducing power increases, leading to a variety of pathological problems linked to an excessive buildup of reducing equivalents, ROS depletion, and disruption of the signaling pathways [1].

Anyway, multiple investigations showed that natural antioxidant agents such as plant-origin metabolites could alter some organismal metabolic processes by acting with enzymes included in antioxidant defense system, by regulating the enzyme expressions through translational control mechanisms [9–11] or by directly quenching ROS and RNS [12]. Plants and agro-waste can serve as almost

endless sources of metabolites possessing high biological activity, including those, which are strong antioxidants [13–15].

Over the last few decades, there has been a significant increase in the quantity and the quality of published articles related to the biological activity of plant origin metabolites. Several factors have contributed to this trend: the advancements in research techniques particularly in fields like genomics, metabolomics, and bioinformatics, which have allowed researchers to explore and analyze plant metabolites in more depth than ever before; the growing interest in natural species with the increasing awareness of the potential health benefits and therapeutic applications of these substances. Another reason is the continuous need in drug discovery, new approaches in cosmetic industry, agriculture and other fields [13, 16, 17].

*Ribes nigrum* L. or black currant (Grossulariaceae family) is extensively consumed cultivar throughout the world, which is applicable also for the dietary management of various diseases [18, 19]. This species' effectiveness in the traditional treatment of different diseases is linked to the presence of a wide range of biologically active metabolites, including potent antioxidants and reducing agents. For the prophylaxis and treatment of various deviations in human metabolism, the vegetal product of *R. nigrum* is frequently used in European traditional medicine [20, 21]. Infusions made from black currant leaves have been used to control kidney function and effectively remove toxins from the body. Its extracts are employed in the treatment of inflammatory diseases [21]. Lipophilic proanthocyanidins isolated from *R. nigrum* vegetal products have been shown to have excellent in vivo anti-inflammatory effects in rat models by inhibiting the production of cytokines [22].

Some literature sources mention the hydrophilic epigallocatechin and epigallocatechin-3-gallate as the main antioxidant components in blackcurrant fruit methanol extracts [23, 24]. In-depth research and descriptions of the phytochemical profile of *R. nigrum* fruit alcohol extract have been published by Paunović et al. [25]. It's noteworthy that leaf extracts contain significantly higher quantity of active phenolic metabolites than fruits do [21], which are primarily responsible for this plant's antioxidant and other biological activities. According to reports, the hydroalcohol leaf extract of *R. nigrum* exhibits less antimicrobial activity than fruit extract [25]. There is some gap in the scientific literature concerning the mode of action of the metabolites, isolated from this
plant. No published research on acute toxicity, genotoxicity, reproductive and developmental toxicity, or carcinogenicity of this extract. This fact is important by the means of introducing this particular plant as potentially valuable medicinal plant species.

Our previous investigations showed remarkable antioxidant potential of *R. nigrum* leaf alcohol extract in chemical-based tests [18, 24]. On this base, we decided to investigate the activity of the components of this extract in different cellular test-systems to be closer to processes taking place in living organisms. In the presented work we selected two microglial BV-2 cell lines (Acyl-CoA oxidase 1 deficient mutants (*Acox1*−/−) and *Wt* cells) to reveal how the plant origin secondary metabolites possessing antioxidant activity can act during the oxidative stress in these cells. ACOX1 is the rate-limiting enzyme of the peroxisomal very-long-chain fatty acid (VLCFA) β-oxidation. ACOX1 deficiency is a rare peroxisomal neurogenerative disorder characterized by the accumulation of VLCFA, leading to cellular oxidative damage [26]. Recently, it has been generated by CRISPR/Cas9 gene editing the abovementioned microglial *Acox1*−/− cell line model, which accumulates VLCFA and can be considered as a model of oxidative stress [27, 28]. Moreover, lipopolysaccharides (LPS) were applied to stimulate additional oxidative stress in both cell lines to evaluate the protective power of *R. nigrum* leaf extract (known to have antioxidant activity) against the formation and accumulation of ROS. In addition, the selection of these cell models was based on obligatory requirements for cell cultures under investigation as they are well-studied, easy-to cultivate and play central role in the formation of metabolic dysregulations. The ROS overproduction in microglial cells may by a cause of neurotoxicity, and therefore, may trigger neurodegenerative processes [10, 29, 30]. The role of enzymes enrolled in the antioxidant defense mechanisms, including superoxide dismutase (SOD), catalase and ACOX1 is very important there [31, 32]. So, the investigation of influence of *R. nigrum* extract on the activity of abovementioned enzymes is of interest. In addition to these pivotal enzymes, the investigation of other factors, which potentially are able to influence on the redox status of cells is also paramount. As such factors can play the ROS and cytokine levels in the cells, membrane-related processes, as well as the expression levels of the genes, responsible for these processes.

The aim of our research was to investigate the antioxidant and anti-inflammatory properties of *R. nigrum* (RN) leaf extract, focusing on its influence on oxidative stress, gene expression, and enzymatic activities in microglial cells. Additionally, the study evaluates the potential of RN to mitigate LPS-induced oxidative damage and inflammation, contributing to its potential therapeutic use in treating oxidative stress-associated disorders.

# Materials and methods

## Chemicals and reagents

Ethanol (POCH S. A., Lot no. 1156/11/21, Gliwice, Poland), DPPH (#STBB0828V, SigmaAldrich, Taufkirchen, Germany), MTT (#MKCR0748, Sigma-Aldrich, Taufkirchen, Germany), catechin-amino2-(hydroxymethyl)-1,3-propanediol (Trizma-Base) (Lot #BCBF0735V, Sigma-Aldrich, Taufkirchen, Germany), Triton X-100 (#STBH6272, Sigma-Aldrich, Taufkirchen, Germany), phosphate-buffered saline (PBS) (#SLCL7026, Sigma-Aldrich, Taufkirchen, Germany), dimethyl sulfoxide (DMSO) (#BCCJ0028, Sigma-Aldrich, Taufkirchen, Germany), ABTS (#SLCH6886, Sigma-Aldrich, Taufkirchen, Germany). The OxiSelect™ Cellular Antioxidant Assay Kit (Cell Biolabs, Inc., Cat. No. STA-349, San Diego, CA, USA), the mouse microglial BV-2 cell line was purchased from Banca-Biologica e Cell Factory (catalog no. ATL03001), LPS from *Escherichia coli* O55:B5 (Prod No. L4524, Sigma-Aldrich, Taufkirchen, Germany), DMEM (Lonza), RNeasy kit (Qiagen), Dionex Ultimate 3000 UHPLC system (Thermo Scientific™ TM, Dionex, San Jose, CA, USA), penicillin/streptomycin (Dutscher), heat-inactivated FBS (Corning), PBS, RIPA, Griess reagent, NaNO₂, loading buffer, 10% SDS-PAGE gel, PVDF membrane, anti-catalase, AF3398 from R&D Systems Noyal Châtillon sur Seiche, France, dilution 1/400; α-tubulin, A2228 from Sigma-Aldrich, Saint-Quentin-Fallavier, France, dilution 1/10,000), Supersignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, Illkirch-Graffenstaden, France), RNeasy Mini kit (Qiagen, Valencia, CA, USA), iScript cDNA Synthesis Kit (Bio-Rad), FG Power SYBR Green (Thermo Fischer Scientific, Illkirch-Graffenstaden, France), iCycler iQ Real-Time Detection System (Bio-Rad, Marnes-la-Coquette, France), MESA Green qPCR Mastermix (Eurogentec, Uppsala, Sweden), 2',7'-dichlorodihydrofluoresceindiacetate (H₂DCFDA) from Sigma-Aldrich GmbH (CAS No.: 4091-99-0, Taufkirchen, Germany). Following HPLC standards were purchased: chlorogenic acid, gallic acid, caffeic acid, isoorientin, quercitrin hydrate, kaempferol (Sigma Aldrich, St. Louis, MO, USA), as well as isovitexin and rutin (Extrasynthese, Genay, France).

## Plant material

The plant material (*R. nigrum* L.) was harvested from Lori province (Armenia, 1600–1650 m a.s.l., Latitude: 408322, Longitude: 442673) during the fruiting period (July 2021) as suggested previously [33, 34]. The sampling was conducted on private land, and the land owner gave permission for this. The identification of the plant was carried out by Dr. Narine Zakaryan at the Department of Botany and Mycology, Yerevan State University (YSU), Armenia. Plant samples were deposited at the herbarium of the same department. The voucher specimen number
was not provided as this plant is not wildly growing and leaves are considered as agri-bio-waste.

## Extract preparation

The collected leaves of *R. nigrum* were washed, dried in the shadow at room temperature and subsequently crushed to obtain the powder, which was stored in a dry and dark place until use. Plant material was extracted as described by Hovhannisyan et al., 2022 [24]. For this 1 g of plant leaves powder was weighed and suspended into 10–15 mL of 70–80% ethanol and stirred overnight at ~10 °C. The extract was subsequently centrifuged for 5 min at 5000 rpm, and the supernatant was isolated. The precipitate was extracted four times with the same procedure, and the combined supernatant fractions were evaporated at room temperature to obtain dried extracts which were then stored at 4°C until further use. The extract yield was 14.8 ± 0.1% o dry weight. In further research, solvents were used at non-toxic concentrations to cells.

## 2,2-Diphenyl-1-picrylhydrazyl free radical scavenging assay

The DPPH free radical scavenging assay was performed as described previously. For this, various concentrations of plant extracts (ranging from 31.25 to 500 µg mL-1) were prepared by dilution method. 0.0.1 mM DPPH was prepared in 96% ethanol. 200 µL of plant extracts of different concentrations were added to 1 mL of DPPH solution. The mixture was shaken vigorously and allowed to stand in dark condition at room temperature for 30 min. The absorbance was measured at a wavelength of 517 nm against a blank (96% ethanol) using a UV–Vis spectrophotometer (Genesys 10 S, Thermo Scientific, USA). Catechin was used as a positive control under the same assay condition. The radical scavenging activity was calculated employing the following equation:

$$ \text{Radical scavenging activity (\%)} = \frac{(\text{Abc} - \text{Abs})}{\text{Abc}} \times 100 $$

where Abc and Abs represented the absorbance of the control (DPPH solution alone) and the absorbance of the sample in the presence of an extract or standard, respectively. The results were expressed as IC50 values (µg/mL), which represented the concentration of sample required to inhibit 50% of the DPPH free radicals [24, 35]. 2.

## Metabolomic characterization of R. nigrum extract
Qualitative analysis with LC-Q-Orbitrap HRMS

The identification of *R. nigrum* extract components was performed using a Dionex Ultimate 3000 UHPLC system (Thermo Scientific TM, Dionex, San Jose, CA, USA) equipped with Synergi TM Hydro-RP A (150 × 4.5 mm,

4 µm, Phenomenex) column, held at a temperature of 30 °C as described [15, 24]. The mobile phase consisted of water (A) and acetonitrile (B); both components contained 0.1% v/v formic acid. The flow rate was set at 0.3 mL/min and the injection volume of all samples was 2 µL. The program of gradient elution was as follows: 0 min, 15% B; 15 min, 40% B; 16 min, 100% B; 24 min, 100% B and finally, the initial conditions were held for 7 min as a re-equilibration step.

The chromatographic system was coupled to a PDA detector and Q ExactiveTM Focus quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a heated electrospray ionization source (HESI II). Detection was performed using a Q-Exactive mass spectrometer. The HESI parameters in negative polarity were as follows: sheath gas flow rate, 35 arb; auxiliary gas flow rate, 15 arb; sweep gas flow rate, 3 arb; spray voltage, 2.5 kV; capillary temperature, 350 °C; S-lens RF level, 50; heater temperature, 300 °C. The parameters of the full-scan analysis were as follows: resolution, 70,000; AGC target, 1e6; max IT, auto; scan range, 120–1200 m/z. The parameters of the data-dependent MS2 were as follows: resolution, 17,500; isolation window, 3.0 m/z; normalized collision energy, 30; AGC target, 1e6; max IT, auto.

Raw data from high resolution mass spectrometry were elaborated with Compound Discoverer (v. 2.1, Thermo, Waltham, USA). Identification of major metabolites was based on accurate mass and mass fragmentation pattern spectra against MS-MS spectra of compounds available on the customized database of different classes of phytochemicals created on the basis of literature data and implemented in the software.

## Quantitative analysis of major phenolic compounds by UHPLC-PDA

To prepare the standard solutions, 1 mg of the respective compounds were dissolved in 1 mL of 70% ethanol. The calibration curve was generated by the integration of the areas of absorption peaks at 265–325 nm determined during UPLC-PDA analysis of serial dilutions of authentic standards. The limit of detection (LOD) was defined as the absolute amount of analyte that maintained a signal-to-noise ratio (peak height) of 3:1, while the limit of quantification (LOQ) was defined as the absolute amount of analyte that produced a signal-to-noise ratio of 10:1. Noise was the magnitude of background response, which was determined by analyzing blank samples (70% ethanol). A linear regression method was used to determine the coefficient of determination (r2) and the linear equation. The major caffeoylquinic acids were quantified as chlorogenic acid equivalents (325 nm), hydroxybenzoic acids as gallic acid equivalents (265 nm), hydroxycinnamic acids as caffeic acid equivalents (325 nm), luteolin
glycosides as isoorientin equivalents (265 nm), quercetin glycosides as rutin equivalents (265 nm), apigenin glycosides as isovitexin equivalents (265 nm) and kaempferol glycosides as kaempferol equivalents (265 nm). The validation parameters such as linear range, standard curve, coefficient of determination, LOD and LOQ parameters determined for standards used were as follows: gallic acid, 20–500 µg/mL, y = 20299x + 69,832, r² = 0.999, 0.08 µg/mL, 0.29 µg/mL; chlorogenic acid, 25–250 µg/mL, y = 31342x + 80,516, r² = 0.999, 0.07 µg/mL, 0.25 µg/mL; caffeic acid, 40–400 µg/mL, y = 32239x + 43,733, r² = 0.992, 0.14 µg/mL, 0.46 µg/mL; isoorientin, 10–400 µg/mL, y = 24171x – 29,618; r² = 0.999, 0.20 µg/mL, 0.67 µg/mL; isovitexin, 45–450 µg/mL, y = 14857x – 25,520, r² = 0.997, 0.23 µg/mL, 0.78 µg/mL; rutin, 43–430 µg/mL, y = 13656x – 29,627, r² = 0.997, 0.20 µg/mL, 0.67 µg/mL; quercitrin hydrate, 27–270 µg/mL, y = 16417x – 5606, r² = 0.997, 1.07 µg/mL, 3.56 µg/mL; kaempferol, 35–350 µg/mL, y = 21807x – 67,453, r² = 0.997, 0.09 µg/mL, 0.29 µg/mL. The chromatographic system, column and conditions of separation were the same as in the case of LC-Q-Orbitrap HRMS.

### Cell cultures used

Investigated murine BV-2 cell lines, Acox1⁻/⁻ and Wt cells, were described by Raas et al. 2019 [27]. BV-2 cells were from female C57BL/6J mice purchased from Banca-Biologica e Cell Factory (catalog no. ATL03001) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Dutscher). Cultures were maintained at 37 °C in a humidified atmosphere with 5% of CO₂ [27]. BV-2 is derived from C57/BL6 murine. They are immortalized by v-raf/v-myc carrying J2 retrovirus. BV-2 express nuclear v-myc and the cytoplasmic v-raf oncogene products as well as the env gp70 antigen at the surface level. BV-2 microglia cell line retains microglia morphological and functional characteristics. In accordance with v-raf/v-myc expressed characters, the metabolic and proliferation rate of in vitro BV-2 greatly exceeds that of other microglia [27, 36].

BV-2 cells were seeded on 96-well microplates at 2 × 10⁴ cells per well for viability and 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay while 6-well microplates at 5 × 10⁵ cells per well for enzymatic activity investigations, immunoblotting and quantitative Reverse-Transcription-PCR analysis [10, 36].

### MTT assay

Cell proliferation and mitochondrial activity was evaluated using 3-(4,5-dimethyltrazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells plated in 96-wells plates were treated for 4–48 h with various concentrations of extract (0.0625–1 mg/mL) and were incubated for 2–3 h with 100 µL MTT (5 mg/mL) followed by the

absorbance (Abs) measurement at the 570 nm. The formazan crystals were dissolved in acidic isopropanol. The sub-cytotoxic concentration of extract was applied in further experiments. For all assays, the extract was dissolved in 1% dimethyl sulfoxide (DMSO). The MTT test was carried out to clarify the effect of R. nigrum extract on microglial cell viability and mitochondrial activity [10]. The untreated cells have been considered as control in all experiments.

### BV-2 cell lysate preparation

After the treatment of BV-2 microglia cells with plant extract, cells were washed with Phosphate-buffered saline (PBS) and were lysed in 50 µL of radioimmunoprecipitation (RIPA) buffer: 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecylsulfate, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 50 mM NaF. Lysate was cleared by centrifugation at 20,000 × g for 20 min (at 4 °C) as described previously [36]. Protein content was measured according to Smith et al. [37].

### Enzymatic activity measurement

The activity of catalase was measured according to Cherkaoui-Malki et al. [38] and was expressed as % of untreated control. Catalase activity was evaluated following a photometric measurement of the H₂O₂ decomposition by catalase contained in the cell extract at 240 nm. The reaction was carried out in a special 96-well UV microplate (Greiner 655801 type, Dutscher, Bernolsheim, France France). A reaction mixture of H₂O₂ solution (30%) and Tris-HCl buffer (1 M, pH7.4) was added to the cell extract, and the reaction was carried out for 2 min. One unit of the enzyme is defined as 1 µmol of H₂O₂ consumed per minute, and the specific activity is calculated as units per milligram of protein according to the following formula:

$$ \text{Catalase specific activity} = \frac{\Delta\text{Abs}_{240\text{nm}} \text{ min}^{-1}}{43.2 \times 10^6} \times \frac{200}{10 \times 1} \text{ [protein] mg l}^{-1} $$

ACOX1 activity measurement was carried out, as described by Oaxaca-Castillo D. et al. [39] using a fluorometry-based assay and fresh cell lysate and was expressed as % of untreated control. The reaction was carried out in a 96-well plate using 200 µL reaction mixture containing Tris-buffer (50 mM, pH 8.3); horseradish peroxidase (20 mg/mL); homovanillic acid (0.75 mM); and acyl-CoA substrate (palmitoyl-CoA at a final concentration of 50 mM). The reaction was initiated at 30 °C by the addition of 10 µL enzymatic solution. The kinetics of the appearance of fluorescence was measured at 420 nm every 30 s for 120 cycles (total kinetic of 60 min) using a fluorimeter (Fluorimeter/luminometer Infinite M200
pro, TECAN). The initial rate of the reaction was determined from the kinetic curves, and the calculation of the specific activity of ACOX1 is expressed in units of H₂O₂ produced per minute per milligram of protein (1 µM d'H₂O₂ corresponds to 115.75 RFU) according to the following formula:

$$ \begin{aligned} \text{ACOX1 specific activity} &= \frac{\Delta \text{RFU min}^{-1}}{\text{RFU (correspond of 1 µM dH}_2\text{O}_2\text{ min}^{-1}}} \\ &= \frac{\text{Fd} \times 200}{10 \times 1} \text{ [protein] mg l}^{-1} \end{aligned} $$

## Evaluation of the ROS production in cells

Intracellular ROS production levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) assay [28]. Cells were seeded at a density of 2 × 10⁴ cells/well in a 96-well plate in DMEM phenol-red-free medium for 24 h with the treatments with R. nigrum leaf extract (RN) of the 0.125 mg/mL concentration and with or without LPS stimulation. LPS (at 1 µg/mL) was used to induce the additional oxidative stress in cells. α-tocopherol (0.4 mM) was applied as positive control. Then, the treatment medium was discarded; cells were washed with PBS and incubated with 5 µM H₂DCFDA for 70 min at 37 °C in 5% CO₂. Cell fluorescence emissions were measured using a fluorescence spectrophotometer (Tecan Infinite Pro M200, Tecan, Lyon, France). Emission was 535 nm and excitation – 535 nm. Hoechst staining was used to estimate cell density and calculate ROS production per cell using the formula below.

$$ \text{For H}_2\text{DCFDA: R} = \frac{(\text{mean H}_2\text{DCFDA} - \text{background})}{(\text{mean Hoechst} - \text{background})} $$

Data were expressed as % of untreated control.

### Griess test

Nitric oxide production in the culture medium was measured using the Griess reagent test [28]. After 24 h incubation at 5 × 10⁴ cells/well with different treatments, activation was performed with LPS (1 µg/mL). The supernatant was removed and placed in a 96-well plate; an equal volume of Griess reagent (1% sulphanilamide and 0.1% naphthyl-ethylene-diamine in 5% H₃PO₄) was added after 30 min incubation in the dark at room temperature; and the absorbance was measured at 540 nm. For the calibration curve, 0.39–100 µM NaNO₂ dilutions were used. The results were expressed as µM of released NO.

### Immunoblotting

Cell protein lysate was prepared as described above. 50 µg of protein was diluted (v/v) in the loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 14% mercaptoethanol, and 0.003% Bromophenol blue), and

the complete denaturation of the protein was achieved by incubation at 100 °C for 5 min. Samples were then separated on a 10% SDS-PAGE gel and transferred onto a PVDF membrane. The non-specific binding sites were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 8) for 1 h at room temperature. The membrane was incubated with the primary antibody diluted in 1% milk TBST overnight at 4 °C (anticatalase, AF3398 from R&D Systems Noyal Châtillon sur Seiche, France, dilution 1/400; α-tubulin, A2228 from Sigma-Aldrich, Saint-Quentin-Fallavier, France, dilution 1/10,000). Following three washes for 10 min in PBST, the membranes were immersed for 1 h at room temperature with an appropriate secondary antibody (dilution 1/5000 in 1% milk TBST) conjugated to horseradish peroxidase. Membranes were washed three times in TPBS for 10 min, and the Supersignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, Illkirch-Graffenstaden, France) was used to reveal the immunoreactivity through enhanced chemiluminescence and a Chemidoc XRS+ device (Bio-Rad, Marnes-la-Coquette, France). Image processing and quantification were performed using Image Lab software (Bio-Rad, Marnes-la-Coquette, France).

### Quantitative reverse-transcription-PCR

We used RT-qPCR to determine the mRNA expression in the *Wt* and *Acox1*⁻⁻ BV-2 microglia cell lines after the treatments with R. nigrum extract. Cells were collected by trypsinization (trypsin-EDTA, 2 mM solution) and then washed twice with PBS after centrifugation for 5 min at 300 g. Cell pellets were used for total RNA extraction and purification using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. The purity of nucleic acids was controlled by the ratio of absorbance at 260 nm to 280 nm, accepting a ratio between 1.8 and 2.2. cDNA was generated by reverse-transcription using an iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions. The quantitative PCR of cDNA was realized using FG Power SYBR Green (Thermo Fischer Scientific, Illkirch-Graffenstaden, France) and an iCycler iQ Real-Time Detection System (Bio-Rad, Marnes-la-Coquette, France). The primer sequences are described in Table 1. PCR reactions were carried out in triplicate at a final volume of 20 µL, containing 10 µL MESA Green qPCR Mastermix (Eurogentec, Uppsala, Sweden) 4 µL of cDNA, 0.06 µL forward, 0.06 µL reverse primers (final concentration 300 nM) and MQ H₂O 5.88 µL. Thermal cycling conditions were achieved by the activation of DNA polymerase at 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 10 s, 60 °C for 1 min. Melting curve analysis was performed to control the absence of non-specific products. For each transcript, the amplification
<table><caption>**Table 1** Sequences of the primers used for qPCR</caption><thead><tr><th>Gene Name</th><th>Accession Number</th><th>Primer Sequences</th></tr></thead><tbody><tr><td>Cat-F</td><td>NM_009804.2</td><td>5'AGCGACCAGATGAAGCAGTG3'</td></tr><tr><td>Cat-R</td><td></td><td>5'TCCGCTCTCTGTCAAAGTGTG3'</td></tr><tr><td>36b4-F</td><td>NM_007475.5</td><td>5'CGACCTGGAAGTCCAACTAC3'</td></tr><tr><td>36b4-R</td><td></td><td>5'ATCTGCTGCATCTGCTTG3'</td></tr><tr><td>Tnf- α-F</td><td>NM_013693.3</td><td>5'GACGTGGAAGTGGCAGAAGAG3'</td></tr><tr><td>Tnf- α-R</td><td></td><td>5'TGCCACAAGCAGGAATGAGA3'</td></tr><tr><td>Il-1β-F</td><td>NM_008361.4</td><td>5'GAGATTGAGCTGTCTGCTCA3'</td></tr><tr><td>Il-1β-R</td><td></td><td>5'AAGGAGAACCAAGCAACGAC 3'</td></tr><tr><td>iNos-F</td><td>NM_010927.4</td><td>5'CCTAGTCAACTGCAAGAGAA3'</td></tr><tr><td>iNos-R</td><td></td><td>5'TTTCAGGTCACTTTGGTAGG3'</td></tr><tr><td>Abcd1-F</td><td>NM_007435.2</td><td>5'GCCAAGTTGTGGATGTGGAG3'</td></tr><tr><td>Abcd1-R</td><td></td><td>5'TTCCGCAGAGTCGGGATAGA3'</td></tr></tbody></table>

efficiency was determined by the slope of the standard curve generated from two-fold serial dilutions of cDNA. The 2⁻ΔΔCt method was used to determine the relative gene expression. The results are presented as graphs of relative expression data (fold induction).

## Statistical analysis

The results represent mean ± standard error or standard deviations of at least three independent experiments performed in triplicate. All statistical analyses were carried out using GraphPad Prism 8 software (GraphPad Software, Inc., USA). A *p*-value of less than 0.05 was considered significant.

## Results

### 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, musizin dimeric glycoside, flavan-3-ols, condensed tannins and derivatives, and flavonols (Table 2; Fig. 1). The mentioned metabolites identified as major components.

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) (the IC50 value of positive control – catechin was 12.62 ± 0.8 µg/mL(R2 = 0.9841) 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 viability of *Wt* and *Acox1*⁻⁻ BV-2 microglial cells. The effect of RN on cell viability was assessed at a concentration range of 0.0625 to 1 mg/mL for 4, 24–48 h treatment period. According to obtained data all concentrations of RN exceeding the 0.25 mg/mL considered 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. 2a and b).

For our investigations the concentration of 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. 3, 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 stimulate 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-

For the evaluation of anti-inflammatory effects of RN [28], we measured the level of NO production and the expression of iNOS gene the in the absence or the presence of LPS (Figs. 4 and 5).

The treatment with RN alone did not have 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 *Acox1*⁻⁻ 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 pronounced LPS-induced NO level reduction was observed in the *Acox1*⁻⁻ 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
<table><caption>**Table 2** Major identified compounds in *R. nigrum* (RN) leaf extracts in negative polarity with retention time (RT), quantity and relevant MS and MS/MS information</caption><thead><tr><th>Peak Nº</th><th>RT</th><th>Name</th><th>Class of compounds</th><th>Proposed formula</th><th>Exper. MW</th><th>Content (mg/g DW)</th><th>Δm (nm)</th><th>Experimental m/z</th><th>Adduct</th><th>Δm (ppm)</th><th>Main Product fragments (m/z)</th><th>Reported</th></tr></thead><tbody><tr><td>3</td><td>2.12</td><td>Quinic acid</td><td>Alcohols and polyols</td><td>C<sub>7</sub>H<sub>12</sub>O<sub>6</sub></td><td>192.0627</td><td>nd</td><td>221, 197, 272</td><td>191.01913</td><td>M-H</td><td>3.59</td><td>85.02811; 93.0332; 87.00736; 59.01253; 71.01244; 81.03316; 109.02834; 69.0332; 67.01757; 191.05505</td><td>[40] HMDB</td></tr><tr><td>6</td><td>2.27</td><td>Citric acid</td><td>Tricarboxylic acids and derivatives</td><td>C<sub>6</sub>H<sub>8</sub>O<sub>7</sub></td><td>192.02638</td><td>nd</td><td></td><td>191.01907</td><td>M-H</td><td>3.25</td><td>87.00738; 111.00748; 57.03323; 85.02808; 67.01755; 73.02805; 59.01251; 72.99168; Software</td><td>HMDB, Software</td></tr><tr><td>7</td><td>2.30</td><td>Gallic acid hexoside (2)</td><td>Phenolic acids and derivatives</td><td>C<sub>13</sub>H<sub>16</sub>O<sub>10</sub></td><td>332.07464</td><td>0.423±0.012</td><td></td><td>331.06735</td><td>M-H</td><td>-0.87</td><td>125.02318; 151.00266; 168.00543; 169.01332; 83.01243; 149.99474; 175.00298; 126.02671; 124.01508; 152.00616</td><td>[41]</td></tr><tr><td>26</td><td>3.58</td><td>5-Caffeoylquinic acid (Neochlorogenic acid) (3)</td><td>Phenolic acids and derivatives</td><td>C<sub>16</sub>H<sub>18</sub>O<sub>9</sub></td><td>354.09535</td><td>0.481±0.058</td><td>206, 324</td><td>353.08807</td><td>M-H</td><td>-0.75</td><td>191.05537; 135.04401; 179.03415; 192.05872; 136.04732; 180.03778; 161.02328; 85.02808; HMDB</td><td>[24]</td></tr><tr><td>40</td><td>4.47</td><td>Caffeoylthreonic acid</td><td>Phenolic acids and derivatives</td><td>C<sub>13</sub>H<sub>14</sub>O<sub>8</sub></td><td>298.06907</td><td>nd</td><td>205, 326</td><td>297.06177</td><td>M-H</td><td>-0.67</td><td>135.02876; 75.00735; 89.02299; 135.04362; 59.01246; 161.02325; 72.99171; 136.03209; 117.01809; 133.02815</td><td>[41]</td></tr><tr><td>42</td><td>4.80</td><td>5-p-Coumaroylquinic acid</td><td>Phenolic acids and derivatives</td><td>C<sub>16</sub>H<sub>18</sub>O<sub>8</sub></td><td>338.10052</td><td>nd</td><td>206, 298</td><td>337.09326</td><td>M-H</td><td>-1.04</td><td>119.04896; 163.03906; 191.05534; 120.05228; 164.04272; 111.0438; 192.05904; 155.03383; 93.03313; 173.04491</td><td>HMDB; HMDB; [42, 43]</td></tr><tr><td>44</td><td>5.06</td><td>Chlorogenic acid (3-O-Caffeoylquinic acid) (1)</td><td>Phenolic acids and derivatives</td><td>C<sub>16</sub>H<sub>18</sub>O<sub>9</sub></td><td>354.09536</td><td>0.364±0.085</td><td>216, 325</td><td>353.08813</td><td>M-H</td><td>-0.78</td><td>191.05545; 192.05898; 85.02813; 173.04477; 161.02335; 135.04393; 93.03318; 127.03879; 87.00759; 179.03423</td><td>HMDB; [42, 43]</td></tr><tr><td>43</td><td>5.49</td><td>(+)-Catechin(+)-Epicatechin</td><td>Flavonoids and derivatives</td><td>C<sub>15</sub>H<sub>14</sub>O<sub>6</sub></td><td>290.07927</td><td>nd</td><td></td><td>289.07196</td><td>M-H</td><td>-0.79</td><td>109.02821; 123.04391; 125.0232; 151.03908; 97.02811; 137.02333; 203.07109; 95.04882; 149.02325; 139.03926</td><td>[24, 43]</td></tr><tr><td>86</td><td>8.47</td><td>Gossypin (6)</td><td>Flavonoids and derivatives</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>13</sub></td><td>480.09072</td><td>0.390±0.027</td><td></td><td>479.08337</td><td>M-H</td><td>-0.68</td><td>316.02255; 317.02768; 479.0831; 318.03506; 178.99773; 271.02423; 480.08646; 287.02014; 151.00299; 270.01569</td><td>PubChem</td></tr><tr><td>90</td><td>9.35</td><td>Rutin (quercetin 3-O-rutinoside) (1)</td><td>Flavonoids and derivatives</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>16</sub></td><td>610.1538</td><td>1.133±0.118</td><td>207, 256, 353</td><td>609.14655</td><td>M-H</td><td>-0.67</td><td>609.14655; 300.02771; 301.03552; 178.99805; 343.04678; 151.00302; 299.02228; 597.16272; 255.06398; 591.13818</td><td>Pubchem, HMDB, [43]</td></tr><tr><td>92</td><td>9.66</td><td>Quercetin 3-6*-malonylneohesperidoside (7)</td><td>Flavonoids and derivatives</td><td>C<sub>30</sub>H<sub>32</sub>O<sub>19</sub></td><td>696.15431</td><td>0.549±0.041</td><td>207, 265, 348</td><td>695.14703</td><td>M-H</td><td>-0.75</td><td>651.15704; 300.02759; 652.16077; 301.03229; 609.14661; 178.99817; 610.14929; 489.10934; 487.08621; 302.03989</td><td>PubChem</td></tr><tr><td>99</td><td>10.14</td><td>Quercitrin (1)</td><td>Flavonoids and derivatives</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>12</sub></td><td>464.09589</td><td>1.242±0.073</td><td>205, 255, 354</td><td>463.08868</td><td>M-H</td><td>-0.88</td><td>300.02771; 301.03415; 302.03894; 271.02478; 463.08914; 151.00249; 178.99756; 255.02997; 464.0918; 272.0304</td><td>Pubchem, HMDB, [24, 44]</td></tr><tr><td>104</td><td>10.69</td><td>Kaempferol-3-O-rutinoside</td><td>Flavonoids and derivatives</td><td>C<sub>27</sub>H<sub>30</sub>O<sub>15</sub></td><td>594.15901</td><td>0.698±0.102</td><td>206, 265, 341</td><td>593.15167</td><td>M-H</td><td>-0.90</td><td>285.04037; 593.15192; 284.03265; 594.1554; 286.04395; 327.05127; 151.0024; 257.0452; 178.99739; 481.38022</td><td>[41, 43, 45]</td></tr></tbody></table>
<table><caption>Table 2 (continued)</caption><thead><tr><th>Peak Nº</th><th>RT</th><th>Name</th><th>Class of compounds</th><th>Proposed formula</th><th>Exper. MW</th><th>Content (mg/g DW)</th><th>Δmax (nm)</th><th>Experimental m/z</th><th>Adduct</th><th>Δm (ppm)</th><th>Main Product fragments (m/z)</th><th>Reported</th></tr></thead><tbody><tr><td>107</td><td>10.95</td><td>Quercetin 3-(6'-malonyl-glucoside) (7)</td><td>Flavonoids and derivatives</td><td>C<sub>24</sub>H<sub>22</sub>O<sub>15</sub></td><td>550.09635</td><td>1.759±0.365</td><td>205, 256, 354</td><td>549.08911</td><td>M-H</td><td>-0.86</td><td>300.02768; 301.03433; 505.09897; 302.0386; 506.10306; 463.08685; 178.99779; 151.00232; 464.09103; 271.02499</td><td>HMDB, [24]</td></tr><tr><td>109</td><td>11.48</td><td>Astragalin (Kaempferol 3-O-glucoside)</td><td>Flavonoids and derivatives</td><td>C<sub>21</sub>H<sub>20</sub>O<sub>11</sub></td><td>448.10087</td><td>1.242±0.277</td><td>197, 265, 347</td><td>447.09360</td><td>M-H</td><td>-0.68</td><td>284.03281; 285.03848; 255.02975; 447.09363; 227.03436; 256.0365; 286.04431; 448.09772; 151.00278; 327.05014</td><td>Pubchem, HMDB, [46]</td></tr><tr><td>114</td><td>11.82</td><td>Naringenin-7-O-glucoside isomer</td><td>Flavonoids and derivatives</td><td>C<sub>21</sub>H<sub>22</sub>O<sub>10</sub></td><td>434.12166</td><td>nd</td><td></td><td>433.11438</td><td>M-H</td><td>-0.83</td><td>271.06137; 151.00262; 272.06476; 119.04897; 177.01852; 93.03313; 145.02844; 169.01331; 152.00598; 61.98705</td><td>[43]</td></tr><tr><td>118</td><td>12.60</td><td>Kaempferol-O-acetylhexoside</td><td>Flavonoids and derivatives</td><td>C<sub>23</sub>H<sub>22</sub>O<sub>12</sub></td><td>490.11128</td><td>nd</td><td></td><td>489.10400</td><td>M-H</td><td>-0.31</td><td>284.03271; 285.03986; 286.04349; 255.02994; 489.10696; 257.0452; 256.03632; 490.10843; 227.03433; 229.04962</td><td>[47, 48]</td></tr><tr><td>119</td><td>12.60</td><td>Luteolin O-malonyl hexoside (5)</td><td>Flavonoids and derivatives</td><td>C<sub>24</sub>H<sub>22</sub>O<sub>14</sub></td><td>534.10116</td><td>1.251±0.236</td><td>197, 265, 348</td><td>533.09399</td><td>M-H</td><td>-0.37</td><td>285.04007; 284.03278; 286.04385; 489.10422; 490.10849; 257.0455; 255.03085; 327.0513; 151.00197; 83.60106</td><td>Sowtware; HMDB</td></tr><tr><td>120</td><td>13.61</td><td>Nuciferoside</td><td>Terpenes</td><td>C<sub>22</sub>H<sub>38</sub>O<sub>11</sub></td><td>478.24163</td><td>nd</td><td></td><td>477.23419</td><td>M-H</td><td>-0.45</td><td>101.02307; 71.01244; 89.02299; 59.01247; 113.02313; 161.04451; 119.03371; 221.06619; 85.02805; 73.02808</td><td>HMDB</td></tr><tr><td>129</td><td>16.34</td><td>Quercetin</td><td>Flavonoids and derivatives</td><td>C<sub>15</sub>H<sub>10</sub>O<sub>7</sub></td><td>302.0429</td><td>nd</td><td></td><td>301.03555</td><td>M-H</td><td>-0.81</td><td>151.00261; 107.01254; 121.02821; 65.00188; 83.01245; 178.99779; 169.01323; 63.02259; 152.00581; 149.02336</td><td>HMDB [41, 43, 49]</td></tr><tr><td>134</td><td>19.41</td><td>Carnosic acid derivative</td><td>Terpenes</td><td>C<sub>21</sub>H<sub>32</sub>O<sub>7</sub></td><td>396.21483</td><td>nd</td><td></td><td>395.20755</td><td>M-H</td><td>-0.06</td><td>331.19165; 287.20169; 332.19495; 259.20682; 110.03603; 303.19672; 288.20526; 243.17519; 97.02814; 269.19089</td><td>HMDB</td></tr><tr><td>136</td><td>19.87</td><td>Hydroxycarnosic acid</td><td>Terpenes</td><td>C<sub>20</sub>H<sub>28</sub>O<sub>5</sub></td><td>348.19378</td><td>nd</td><td></td><td>347.18646</td><td>M-H</td><td>-0.30</td><td>303.19656; 243.17529; 69.03315; 233.15446; 259.20651; 304.19879; 83.04893; 244.17877; 287.1662; 85.06448</td><td>HMDB</td></tr><tr><td>141</td><td>20.19</td><td>Carnosic acid</td><td>Terpenes</td><td>C<sub>20</sub>H<sub>28</sub>O<sub>4</sub></td><td>332.19887</td><td>nd</td><td></td><td>331.19159</td><td>M-H</td><td>-0.33</td><td>331.19168; 287.20197; 110.03613; 332.19455; 285.18597; 259.20731; 97.02821; 288.20459; 269.19009; 69.03304</td><td>HMDB</td></tr><tr><td>144</td><td>20.96</td><td>Rosmaridiphenol isomer</td><td>Terpenes</td><td>C<sub>20</sub>H<sub>28</sub>O<sub>3</sub></td><td>316.20394</td><td>nd</td><td></td><td>315.19662</td><td>M-H</td><td>-0.30</td><td>315.19669; 316.19983; 201.18394; 112.08009; 315.19156; 584.74454; 51.7416; 54.01874;</td><td>HMDB</td></tr></tbody></table>

Concentration was calculated: (1) based on authentic standards; (2) as gallic acid equivalent; (3) as chlorogenic acid equivalent; (4) as caffeic acid equivalent; (5) as isoorientin equivalent; (6) as isovitexin equivalent; (7) as rutin equivalent; (8) as kaempferol equivalent. The data are expressed as mean values of three independent experiments ± standard deviation. nd - not determined
<figure><img src="image_4.png" /><figcaption>**Fig. 1** The LC chromatographic profile of *R. nigrum* extract monitored either by Quadrupole-Orbitrap High resolution MS (black HRMS chromatograms) or UV light (orange UV chromatograms). (The UV chromatogram of extract at 265–325 nm. The peak numbers refer to those implemented in Table 2.)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Effect of *R. nigrum* leaf extract on the cell proliferation and viability of the *Wt* (a) and the *Acox1<sup>-/-</sup>* BV-2 (b) microglial cells for 4-, 24- or 48-hours treatment period (MTT test). The untreated cells were considered as control. The results are means ± SD of two independent experiments performed in triplicate (*p* ≤ 0.05)</figcaption></figure>

significantly attenuated the LPS-induced iNOS mRNA level increase (Fig. 5a).

The expression of other inflammatory marker encoding genes was also changed 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<sup>-/-</sup>* cell lines (Fig. 5b, c). Expectedly, the treatment with LPS increases the *Tnf-α* and the *Il-1β* gene expression significantly, but 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 up 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. 6a the catalase activity in *Wt* cells remained almost unchanged during the 24-hour treatment, meanwhile the further treatment led to significant decrease of this enzyme activity (at up to 40% compared to untreated control). In case of *Acox1<sup>-/-</sup>* BV-2 cells, during the first 24 h 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).
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Effects of *R. nigrum* leaf extract on ROS production induced by LPS in the *Wt* (a) and *Acox1*⁻/⁻ (b) BV-2 microglial cells assessed by the H₂ DCFDA (5 µM) dye test. Cells were incubated for 24 h with RN (0.125 mg/mL) in the absence or presence of LPS (1 µg/mL). All values are presented as means ± SD of two independent experiments performed in triplicate (*p* < 0.1; and **p** < 0.01). Statistical significance was determined using one-way ANOVA followed by Tukey's test. The α-tocopherol did not show any statistically significant effect</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4** Effect of *R. nigrum* extract on the NO production induced by LPS in the culture media of the *Wt* (a) and *Acox1*⁻/⁻ (b) BV-2 microglial cells assessed by the Griess test. Cells were incubated for 24 h with RN (0.125 mg/mL) in the absence or the presence of LPS (1 µg/mL). All values are presented as means ± SD of two independent experiments performed in triplicate (*p* < 0.1; **p** < 0.01; ***p** < 0.001; ****p** < 0.0001 and ns, not significant). Statistical significance was determined using one-way ANOVA followed by Tukey's test</figcaption></figure>

The ACOX1 activity in *Wt* BV-2 microglial cells decreased by almost 50% during the first 24 h 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%.

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 (Figs. 3, 4
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Effect of *R. nigrum* leaf extract on the gene expression of *iNos* (a), *Tnf-α* (b), *Il-1β* (c) and *Abcd1* (d) with or without the LPS influence. All values are presented as means ± SD of two independent experiments performed in triplicate (*p* < 0.1; **p** < 0.01; ***p** < 0.001; ****p < 0.0001 and ns, not significant). Statistical significance was determined using two-way ANOVA followed by Tukey's test for multiple comparisons</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 6** Effect of *R. nigrum* extract on catalase activity in the *Wt* and *Acox1*⁻/⁻ BV-2 microglial cells (a), and on *ACOX1* activity (b) in *Wt* BV-2 microglial cells. All values are presented as means ± SD of two independent experiments performed in triplicate (*p* < 0.1; **p** < 0.01; ***p** < 0.001 and ns, not significant). Statistical significance was determined using two-way ANOVA followed by Tukey's test for multiple comparisons (a) and one-way ANOVA followed by Tukey's test (b). The α-tocopherol did not show any statistically significant effect</figcaption></figure>

and 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).

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
<figure><img src="image_10.png" /><figcaption>**Fig. 7** Effect of *R. nigrum* extract on the level of catalase activity (a) and on the expression of the peroxisomal *Cat* mRNA (b) in the *Wt* and in *Acox1*⁻/⁻ BV-2 microglial cells. Panels (c) and (d) show the results of immunoblotting assay of RN effect on the expression of the peroxisomal protein *CAT* in *Wt* (c) and in *Acox1*⁻/⁻ (d) BV-2 microglial cells. Cells were incubated for 24 h with RN (0.125 mg/mL) in the absence or presence of LPS (1 µg/mL). Cell lysates were analyzed by PAGE-SDS electrophoresis and subjected to immunoblotting. Band intensities were analyzed by densitometry and standardized to α-tubulin expression level. Table values represent standardized densitometric analysis obtained after the signal intensity quantification of different proteins. 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</figcaption></figure>
the changes in catalase activity in both cell lines (Fig. 7c,
d).

Discussion

R. nigrum considered as an excellent source of biologi-
cally active substances [24]. While its fruits are well-
known for their nutritional value and health benefits,
the leaves of this plant are often considered as bio-waste.
However, recently published literature suggests them
as a good source of active metabolites. R. nigrum vege-
tal products contain a rich array of polyphenolics, such
as flavonoids (e.g., quercetin, rutin), anthocyanins, and
phenolic acids, which all possess remarkable antioxidant
capacity [24]. They contain various vitamins, minerals
like potassium, magnesium, and calcium [25]. Several
components found in R. nigrum leaves, such as quercetin
and anthocyanins, have anti-inflammatory, antioxidant,
antiviral, antimicrobial, antibiotic-modulatory and other
effects [21]. Some authors mention the neuroprotective
effect of R. nigrum extracts [50].

According to our previous investigations total pheno-
lic and flavonoid contents of extracts of R. nigrum vegetal
products harvested from high altitude Armenian land-
scape were 84.1 ± 1.6 mg gallic acid equivalent (GAE)/g
DW) and 35.42 ± 1.52 mg quercetin equivalent (QE)/g
DW), respectively [24]. The further HPLC analysis
showed that there are around 30 main components pres-
ent, the majority of which are flavan-3-ols, dimeric gly-
cosides, hydroxycinnamates, tannins, flavonols, flavonols
and flavonol derivatives (Table 2; Fig. 1).

All the identified components are known to have
expressed biological activity in different test-systems.
Moreover, these substances can interact with each other
as well as other metabolites influencing by synergism or
antagonism [15, 51]. So, it is very important to investigate
the activity of individual components, but more impor-
tant - the combination of these active metabolites.

The most studied feature of *R. nigrum* leaf extract is
antioxidant capacity. Our previous investigations showed
the high antioxidant activity in chemical-based tests.
Moreover, we reported also the ability to alter the activity
of SOD in *Wt* and *Acox1*⁻/⁻ BV-2 microglial cells treated
by *R. nigrum* extract [5]. Microglia play a central role in
the regulation of inflammation in nervous system [28,
52, 53]. In response to the stressors, such as accumula-
tion of the elevated concentration of ROS, microglia pro-
duces proinflammatory cytokines (TNF-α, IL-1β, IL-18).
This can induce the activation of iNOS and NO release
in cells [28, 54]. NO is a signaling molecule known for
its significant impact on acute and chronic inflammation
processes in the nervous system as well as on apopto-
sis. The inducible isoform iNOS in many cell types pro-
duces large amounts of NO as a defense mechanism in
response to cytokines and different stress factors [28].

NO in its turn is considered a free radical, which is toxic
for all cells and at the elevated concentrations it reacts
non-specifically with proteins, lipids, and nucleic acids
and, therefore, amplify the inflammation and neurode-
generative processes [55]. In recent years, plant origin
metabolites have been of interest as a potential source for
the development of new medicinal preparations in the
treatment of different neurological disorders. In the pres-
ent work, we continued the investigations on the ability
of RN to act as a redox regulator in BV-2 cell line models
and to attenuate and mitigate the influence of different
stressors. We showed that the activity of all investigated
enzymes embedded in the antioxidant defense system of
these microglial cells was altered by the treatment of the
selected concentration of RN. Peroxisomal proteins such
as catalase, SOD, ACOX1 are known to keep a significant
role in the development of tolerance to oxidative stress
in cells, particularly when there is decreased activity of
glutathione peroxidases or elevated H₂O₂ levels [56]. In
*Wt* cells, some decrease of catalase and ACOX1 activities
during the first period of treatment can be the evidence
of the fact that RN extract acts as antioxidant [18, 57, 58].
Additional arguments are shown by the decrease of the
intracellular concentration of ROS under both the RN
treatment alone and RN + LPS combination (Figs. 3 and
4). In *Acox1*⁻/⁻ cells the “picture” can be different as this
type of cells a priori can be considered as a model of oxi-
dative stress. In this case, we have some increase in cata-
lase activity only after the 48-hour treatment, meanwhile
our previous investigations showed the activation of SOD
activity in 24-hour treatment [18]. However, in both cell
lines we observed of ROS formation in H₂DCFDA test
(Fig. 3) which speaks about the expressed antioxidant
activity of RN.

The decreased expression rate of *Tnf-α* and *Il-1β* gene expression in RN + LPS treated cells, which is in correlation with iNOS gene-expression and NO release under the same conditions are also able to confirm the redox regulating power and anti-inflammatory effects of RN on LPS-stimulated BV2 microglial cells (Figs. 5b and c and 8).

The activation of *Abcd1* gene-expression in both RN and RN + LPS treated cells (Fig. 4d) evidences the activation of VLCFA transport into the peroxisomes and the β-oxidation of these fatty acids in both cell lines, reducing the oxidative stress in these particular cells (Fig. 8).

In addition, the expression of another peroxisomal pro-
tein CAT gene was also in line with the activity change of
catalase, which confirmed by both RT-qPCR and immu-
noblotting assays (Fig. 7).

All of these data in a complex with no expressed geno-
toxicity of RN [35] pointed out that this plant can serve
as an excellent source of valuable biologically active
<figure><img src="image_11.png" /><figcaption>**Fig. 8** The schematic representation of influence of the phenolic components of RN extract on microglial BV-2 Wt cells</figcaption></figure>

metabolites useful in the treatment of oxidative stress-associated neurodegenerative disorders.

Limitations of study: Despite these promising results, some limitations must be acknowledged. The examined extract has a complex composition and it is possible that the components included in its composition may demonstrate synergistic or antagonistic activity in test systems. Therefore, the study of individual components is inappropriate in this case.

## Conclusions

Our research focused on the phenolic and flavonoid contents of *R. nigrum* (RN) leaf extracts, sourced from the high-altitude Armenian landscape. We identified multiple bioactive components, which led us to further explore the antioxidant and anti-inflammatory properties of RN in BV-2 microglial cell models.

Our findings underscore the potential health benefits of RN leaf extracts, particularly their ability to modulate antioxidant enzyme activity, decrease oxidative stress level, and influence gene expression related to inflammation and peroxisomal function. These results are fundamental for advancing knowledge of RN's therapeutic potential, especially in addressing oxidative stress and inflammation-related disorders.

Importantly, our study highlights the extract's protective effects against LPS-induced oxidative stress, inflammation, and peroxisomal dysfunction in microglia. RN treatment reduced both oxidative and nitrosative stress, downregulated pro-inflammatory gene expression, and normalized the expression of peroxisomal catalase while restoring fatty acid β-oxidation functions.

These findings have both practical and scientific value. They contribute to the development of new antioxidant therapies and broaden our understanding of natural metabolites' role in promoting health. Future research should aim to elucidate the specific contributions of individual components or their synergistic effects to better comprehend their diverse biological impacts.

## Abbreviations

<table><tr><td>ABCD</td><td>Transporter Peroxisomal ATP-binding cassette transporter protein</td></tr><tr><td>ACOX1</td><td>Acyl-CoA oxidase 1</td></tr><tr><td>CAT</td><td>Peroxisomal protein catalase</td></tr><tr><td>DMEM</td><td>Dulbecco's modified Eagle medium</td></tr><tr><td>DMSO</td><td>Dimethyl sulfoxide</td></tr><tr><td>DPPH</td><td>2,2-diphenyl-1-picrylhydrazyl</td></tr><tr><td>DW</td><td>Dry weight</td></tr><tr><td>EDTA</td><td>Ethylenediaminetetraacetic acid</td></tr><tr><td>FBS</td><td>Fetal bovine serum</td></tr><tr><td>GAE</td><td>Gallic acid equivalent</td></tr><tr><td>H<sub>2</sub>DCFDA</td><td>2,7'-dichlorodihydrofluorescein diacetate</td></tr><tr><td>HPLC</td><td>High-performance liquid chromatography</td></tr><tr><td>II-1β</td><td>Interleukin-1 beta</td></tr><tr><td>iNOS</td><td>Nitric oxide synthase (inducible form)</td></tr><tr><td>LPS</td><td>Lipopolysaccharides</td></tr><tr><td>MTT</td><td>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</td></tr><tr><td>NO</td><td>Nitric oxide</td></tr><tr><td>PBS</td><td>Phosphate-buffered saline</td></tr><tr><td>ROS</td><td>Reactive oxygen species</td></tr><tr><td>PVDF</td><td>Polyvinylidene difluoride</td></tr><tr><td>QE</td><td>Quercetin equivalent</td></tr><tr><td>RIPA</td><td>Radioimmunoprecipitation (a buffer solution)</td></tr><tr><td>RN</td><td>Ribes nigrum extract</td></tr><tr><td>RT-qPCR</td><td>Quantitative reverse transcription polymerase chain reaction</td></tr><tr><td>SOD</td><td>Superoxide dismutase</td></tr><tr><td>TBS</td><td>Tris-buffered saline (a buffer solution)</td></tr><tr><td>TBST</td><td>Tris-buffered saline (TBS) with Tween 20</td></tr><tr><td>Tnf-α</td><td>Tumour necrosis factor alpha</td></tr><tr><td>VLCFA</td><td>Very-long-chain fatty acids</td></tr><tr><td>Wt</td><td>Wild type</td></tr></table>
**Supplementary Information**

The online version contains supplementary material available at https://doi.org/
g/10.1186/s12906-025-04780-7.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Supplementary Material 4

**Acknowledgements**

Not applicable.

**Author contributions**

All authors contributed to the study's conception and design. AM, VP, CG, MG, MM, BK, and NS carried out the investigations and analyzed the outcomes. NA wrote the manuscript. NA, SS, MCM, AB, and PA directed the experiments, corrected, and edited the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript and have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors provided approval of the final submitted manuscript.

**Funding**

The research was supported by the Science Committee of MESCS RA, in the frames of the research projects № 21AG-4D027.

**Data availability**

The datasets used and/or analyzed during the current study available from the
corresponding author on reasonable request.

**Declarations**

**Ethics approval and consent to participate**

There is no need to obtain the licenses for the plant sampling as the selected plant is not wild-growing and the leaves are considered as bio-waste.

**Consent for publication**

Not applicable.

**Plant ethics**

The *R. nigrum* plant was cultivated at the Lori province (Armenia, 1600–1650 m a.s.l.) and harvested during the fruiting period (July 2019) as suggested in literature [33]. The cultivation was carried out without the treatment with any fertilizer or pesticide. The identification of the plant was carried out at the Department of Botany and Mycology, Yerevan State University (YSU), Armenia. The plant samples are available at the Department of Microbiology & Plants and Microbes Biotechnology, Biology Faculty, Yerevan State University, Yerevan, Armenia. The voucher specimen number was not provided for the cultivated plant species. There is no permissions or licenses needed for harvesting of the cultivated plant samples.

**Competing interests**

The authors declare no competing interests.

**Author details**

Received: 29 April 2024 / Accepted: 23 January 2025

Published online: 12 February 2025

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