Anti-inflammatory activity of novel natural plant extracts composition—LevidorTM
, , , , und | 22. Mai 2023
Article Category: Original Study
Online veröffentlicht: 22. Mai 2023
Seitenbereich: 49 - 58
Eingereicht: 14. Okt. 2022
Akzeptiert: 21. März 2023
DOI: https://doi.org/10.2478/ahem-2023-0008
Schlüsselwörter
© 2023 Monika Kuczyńska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Inflammation is a complex response of body tissues to harmful factors, such as pathogens, irritants, or damaged cells. It plays a protective role involving the local immune and vascular system as well as molecular mediators [1]. The primary aim of inflammatory response is fast elimination of the initial cause of injury and initiation of tissue repair. However, if the inflammation is prolonged, a state called chronic inflammation occurs. Over time, chronic inflammation can the trigger immune system to attack healthy tissue, leading to their damage. When left untreated, prolonged chronic inflammation can increase the risk of diseases such as diabetes, heart disease, or even cancer [1]. Among various inflammatory mediators implicated in pathological processes, cyclooxygenase (COX) and its downstream effector molecules are of greater significance. Cyclooxygenase 2 (COX-2) is a major enzyme involved in the modulation of inflammation and acts by catalyzing the rate-limiting step, leading to the production of prostaglandins (PGs) from arachidonic acid [2]. COX-2 is expressed at low levels under physiological conditions; however, it is highly and transiently expressed in response to the aforementioned harmful stimuli. This leads to a burst of PGs production. It has been proven that prolonged COX-2 expression may override immunological balance of the body, resulting in pathological states [3]. In different examples of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, or diabetes, the hyperactivation of such subpopulations of immune cells has been shown to be directly dependent on the over-expression of COX-2 [4]. The hyperexpression of COX-2 is the result of cross-action between broad range of inflammatory markers, namely interleukins and cytokines (i.e., IL-1, IL-6 or TNF-
The other key cyclooxygenase is COX-1, an enzyme that is constitutively and stably expressed at low levels in many tissues. Whereas COX-2 is predominantly implicated in inflammation, COX-1 is a critical regulator of homeostatic functions [4]. Protein COX-1 ensures the constant production of PGs, which contribute to the maintenance of important physiological functions, such as platelet aggregation, renal water balance, and most importantly, gastric mucosal protection [5]. Its expression is also found in fetal and amniotic cells, uterine epithelium in early pregnancy, and the central nervous system. It is believed to exert complex integrative functions [6].
Consequently, strategies to decrease chronic inflammation in a broad range of disorders aimed at selective inhibition of COX-2 enzyme activity. Non-steroidal anti-inflammatory drugs (NSAIDs), which have their origin in the extracts of salicylate-containing plants, represent the first large class of inhibitors of COXs available on the market [7].
Clinically proven effectiveness of the aspirin led to the development of other classical NSAIDs. These pharmaceuticals belong to a group of carboxylic acids. Aside from aspirin, this group consists of naproxen, diclofenac, ketoprofen, flurbiprofen, ibuprofen, and indomethacin [8]. Eventually, these drugs hit the pharmaceutical markets. Unfortunately, the classical NSAIDs have varying selectivity towards COX-1. Thus, prolonged intake of NSAIDs was immediately recognized to cause serious side effects, such as damage to the gastric mucosa [9]. For this reason, patients taking NSAIDs for an extended period of time must use proton-pomp inhibitors to protect mucosa. The negative impact of NSAIDs on the gastrointestinal tract was related to co-inhibition of concomitant perturbation of COX-1 activity [10]. Also, preferential COX-2 inhibitors such as carboxamides (meloxicam), sulphonanilides (nimesulide) and naphthalenes (nabumetone), and selective COX-2 inhibitors called coxibs (celecoxib, etoricoxib, parecoxib, rofecoxib, valdecoxib, lumiracoxib) have been introduced into the market [8]. These drugs initially exhibited improved safety profiles regarding the health of the gastrointestinal tract as compared to the classical NSAIDs. Unfortunately, despite the early success of these drugs, many cardiovascular disorders have been reported when compared to classical NSAIDs. Rofecoxib and valdecoxib were withdrawn from the market because of severe side effects in the cardiovascular system. For the same reason, etoricoxib is not approved in several countries. Side effects affecting the liver caused the withdrawal from the market of drugs based on lumiracoxib. Currently, celecoxib is the safest NSAID and the only selective COX-2 inhibitor with market authorization both in the European Union (EU) and the United States. However, the labels of celecoxib-based drugs highlight the several potential adverse effects [11].
Due to the severe side effects of existing COX-inhibitors, there is an urgent need to find new natural or synthetic chemicals that could act as selective inhibitors of COX-2. The most valuable source of new bioactive compounds and potential drugs are plants. Historically, plant products with medicinal properties have been used to treat several inflammatory diseases [12]. These traditional anti-inflammatory remedies later become the basis for the production of aspirin, the first natural-product-derived synthetic anti-inflammatory drug [12]. The exploration of phytochemicals has been promising and resulted in the discovery of many plant secondary metabolites with therapeutic activities such as artemisinin, vinblastine, or pilocarpine [12]. Recently, attention has been paid to
The second plant exhibiting anti-inflammatory potential is
The studies showed that both plants have a strong analgesic and anti-inflammatory effect [13,16,17,18,19,20,21,22,23,24,25,26]. Recent investigations on bioactive phytochemicals showed that combinations of bioactive compounds such as different extracts may lead to the formation of a new preparation with modified biological properties due to interactions between phytochemicals [27]. Moreover, the extraction and fixation procedures have crucial impact on maintenance of bioactivity of isolated phytocomplex. The extraction method applied in the present study has been patented (P.412214, WO2016118027A1). For this reason, the present study aimed at the investigation of LevidorTM, the composition based on oil from the seeds of
The following chemicals were used for the study: lipopolysaccharide (LPS) from
Plant preparations of LevidorTM were dissolved in DMSO at a concentration 20 mg/mL. The stock solution was stored in dark at 4 °C. For every experiment, extracts were taken from the stock vial and dissolved as needed. The extraction method has been patented (P.412214, WO2016118027A1). The extract has been phytochemically characterized previously [20].
The cell line of human monocytes U937 (ECACC 85011440) and murine macrophages RAW 264.7 (CLS 400319) cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cell line derived from a rat pheochromocytoma PC-12 (CRL-1721) was cultured in RPMI-1640 medium. To make the complete growth medium, the following components to the base medium were added: heat-inactivated horse serum to a final concentration of 10% and FBS to a final concentration of 5%, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cell lines were maintained at 37 °C under 5% CO2 atmosphere in a cell incubator (Galaxy 170S, New Brunwick, Canada).
In the case of cytotoxicity assessment by MTT assay, the RAW264.37 and PC-12 cells were seeded at a density of 1 × 104 in 96-well plate. For determination of IL-6 and TNF-
In the selected experiments, the human U937 cell line was applied. This cell line under appropriate stimulation is commonly used as a functional model of macrophages. For differentiation into macrophage-like cells, PMA at concentration 10 ng/mL in culture medium was added and cells were allowed to differentiate for 48 h at 37 °C under 5% CO2 atmosphere in a cell incubator. Then the cells were incubated without treatment for 40 h. After that time, the cells were treated with extract and LPS stimulation was performed as described below.
In order to determine the influence of LevidorTM extract on the inflammatory response of RAW 264.7 or differentiated U937 cells, the extract at concentrations 1, 2.5, 5, 10, 25 and 50 μg/mL was added to cells for 4 h. Diclofenac was used as reference (50 or 100 μg/mL). After that time, the cells were stimulated with 1 μg/mL of LPS for 2, 4, 6, and 16 h when
In the case of immunofluorescence staining performed to verify the level of COX-1 and COX-2 proteins under LevidorTM treatment, the U937 cells after 24 h pre-treatment with PMA (10 ng/mL), followed by 24 h incubation without treatment, were given 4 h treatment with 10, 25, and 50 μg/mL of LevidorTM extract and TNF-
To determine the impact of LevidorTM extract on RAW264.37 and PC-12, MTT test was applied. The cells were treated for 72 h with concentrations ranging from 6.25 to 100 μg/mL of sample. After 72 h of incubation, 100 μL of MTT (1 mg/mL) were added to each well for 4 h. After that time, medium was aspirated from wells and formazan crystals were dissolved in 0.05 mL of DMSO. The absorption of the obtained solutions was measured at 540 nm with the aid of microplate reader SynergyTM HT, (Biotek Instruments, USA). The impact of investigated samples on cells growth was expressed as IC50.
After treatment of the U937 cells, cells were washed twice with PBS, collected and the protein concentration in the cell free supernatants was determined by BCA assay. The assay was performed according to the manufacture's instruction. Obtained material was stored in −80 °C. IL-6 and TNF-
After treatment, total RNA was extracted using Total RNA Mini Plus kit according to the supplier's protocol. The extracted RNA was quantified, and input amounts were optimized for each amplicon. Then, the RNA samples were reverse-transcribed with the aid of Transcriba kit, and the resulting cDNA samples were used as templates for real-time PCR analysis of selected gene expression using SYBR Green PCR reaction mix namely SensiFAST SYBR No-ROX and gene specific primers (Table 1).
Forward and reverse sequences of applied primers
| Gene | Forward | Reverse |
|---|---|---|
| 5′-AACGACCCCTTCATT-GAC-3′ | 5′-TCCACGACATACTCAG-CA-3′ | |
| 5′-TCTCAGGCTA-CACCCTAGACCA-3′ | 5′-ATCGGGGTAGTCCGAG-TAACGT-3′ | |
| 5′-CAGCACTTCACGCAT-CAGTT-3′ | 5′-CGCAGTTTACGCTGTC-TAGC-3′ |
Reactions were conducted on an Miniopticon system (Biorad) with following stages: 95 °C for 3 min., 39 cycles of denaturation 95 °C for 10 sec. and a melting curve stage of 60 °C to 90 °C for 5 sec. Gene expression levels were determined by the ΔΔCt method. RT-PCR data was expressed as fold changes relative to control group. No-reverse transcribed mRNA served as a negative control. GAPDH was used as the housekeeping gene for normalization of the results.
After treatment, the cells were lysed in RIPA buffer combined with homogenization. The protein concentration was evaluated by BCA assay. The lysates (30 μg of proteins) were fractionated by 7.5% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies: anti-COX-1 (1:1000), anti-COX-2 (1:1000), anti-
Fluorescent immunocytochemistry was performed on U937 cells using monoclonal antibodies against COX-1 and COX-2. The cells were pre-treated with PMA (10 ng/mL). This step was followed by 24 h of incubation without treatment, 4 h treatment with LevidorTM at 10, 25, and 50 μg/mL as well as TNF-
All values are expressed as means ± SD of three independent experiments unless stated otherwise. The statistical significance of changes between samples and control was evaluated by nonparametric Mann-Whitney one-tailed statistical test. The level of statistical significance was set at p ≤ 0.05. All statistical analyses were performed using Prism 4.0 software package (GraphPad Software, Inc., USA).
The impact of studied extract on RAW264.37 and PC-12 was assessed by MTT test. The cells were treated with tested extract at concentrations ranging from 0.1 to 200 μg/mL. Table 2 presents IC50 determined after 72 h of exposure. The extract did not significantly influence the RAW264.37 cell growth at any of the investigated concentrations. In the case of PC-12 cell line, the IC50 at 193.1 μg/mL was observed. The obtained results indicate that LevidorTM extract does not exhibit cytotoxic activity.
Values of IC50 obtained by MTT test
| Cell line | IC50 [μg/mL] |
|---|---|
| RAW264.37 | not cytotoxic at concentration range tested |
| PC-12 | 193.1 |
The RAW 264.7 cells were treated with tested extract at concentrations ranging from 1 to 50 μg/mL.
As shown in Fig. 1, control cells produced low levels of both tested proteins. The addition of LPS strongly induced production of both IL-6 and TNF-
Fig. 1
The effect of LevidorTM extract on pro-inflammatory markers (IL-6, TNF-α) induced by LPS in the RAW267.7 cells. All results are given as means ± SD of three independent determinations. Negative control (C−) refers to cells treated with solvent, while positive control (C+) relates to cells treated with inflammatory inducer only. Significantly different values are marked as (*) p ≤ 0.05
The presented study aimed to elucidate the impact of LevidorTM on
Fig. 2
Changes in COX-1 (A) and COX-2 (B) gene expression in the U937 cells after 48 h pre-treatment with PMA (10 ng/mL), followed by 40 h of incubation without treatment, 4 h treatment with 1, 2,5, 5, 10, 25, and 50 μg/mL of LevidorTM as well as LPS addition (1 μg/mL) for 2, 4, 6, and 16 h. Results are presented as fold changes in the expression of investigated genes. Negative control (C−) refers to cells treated with PMA, while positive control (C+) relates to cells treated also with inflammatory inducer. Significantly different values are marked as (*) p ≤ 0.05. The results are calculated based on three independent experiments
Subsequent to gene expression analysis, the determination of COX-1 and COX-2 protein level was performed (Fig. 3). The non- and differentiated U937 cells were treated with tested extract at concentrations ranging from 1 to 50 μg/mL. Western blot examination provided the confirmation of hypothesis that LevidorTM does not affect COX-1, while abundance of COX-2 protein decreases in a concentration dependent manner. Importantly, COX-1 level was not affected both in monocytes and macrophages. In turn, COX-2 level determined in macrophages treated with 50 μg/ml reduced protein level in comparison to negative control level. The concentrations 10 and 25 μg/ml of LevidorTM caused a similar effect on COX-2 as non-steroidal anti-inflammatory drug, namely diclofenac at 100 μg/mL. The lowest concentrations of the LevidorTM extract did not impact COX-2 protein level notably.
Fig. 3
Western blot analysis of the modulation of COX-1 and COX-2 protein abundance in the U937 after 48 h pre-treatment with PMA (10 ng/mL), followed by 40 h of incubation without treatment, 4 h treatment with 1, 2.5, 5, 10, 25, and 50 μg/mL of LevidorTM as well as LPS addition (1 μg/mL) for 24 h. Negative control (C−) refers to cells treated with PMA, while positive control (C+) relates to cells treated also with inflammatory inducer (LPS)
The two forms of COX-1 exist—glycosylated with a molecular weight of 70 kDa and deglycosylated with a molecular weight of 65 kDa. For this reason, two protein bands have been observed. The three protein bands for COX-2 derived from fully N-glycosylated COX-2 with a molecular weight of 72 and 74 kDa and the aglycosylated form (70 kDa) [29].
Immunofluorescence staining confirmed that LevidorTM extract did not cause drop in COX-1 protein level (Fig. 4). Conversely, it was observed that the extract potentiated fluorescence derived from COX-1. This effect was noted especially at 25 μg/mL. In the case of COX-2, the protein level was significantly decreased when compared to positive control. Such reduction exhibited dose dependent character similarly as it was observed in Western blot analysis.
Fig. 4
Visualisation of COX-1 and COX-2 protein in the U937 cells after 24 h pre-treatment with PMA (10 ng/mL), followed by 24 h of incubation without treatment, 4 h treatment with 10, 25, and 50 μg/mL of LevidorTM as well as TNF-α addition (100 ng/mL) for 20 h. Negative control (C−) refers to cells treated with PMA, while positive control (C+) relates to cells treated also with inflammatory inducer (LPS)
Over the last few decades, NSAIDs have been the drugs of choice for treating numerous inflammatory diseases. Unfortunately, the prolonged use of NSAIDs has been associated with severe and even life-threatening side effects. NSAIDs are inhibitors of cyclooxygenase enzymes (COX), which catalyze the conversion of arachidonic acid to inflammatory PGs. Unfortunately, their use is associated with side effects such as gastrointestinal and renal toxicity due to the inhibition of COX-1. Thus, the need arises to find more specific substances, in which therapeutic anti-inflammatory action results from COX-2 inhibition efficiency with intact COX-1 pathway activity.
Due to the fact that plants are rich source of diverse bioactive compounds, it makes them a potential source of novel compounds with anti-inflammatory activity. Such examples may be
The first series of experiments concentrated on evaluation of cytotoxicity of the investigated preparation. The results of a MTT cell viability test performed in two employed cellular models revealed no substantial impact of LevidorTM on cell growth in a broad range of investigated concentrations. These determinations provided the first information about the safety of the new preparation. Another interesting finding was the desirable effect of treatment with LevidorTM on pro-inflammatory cytokine profiles. For instance, TNF-
The observed strong anti-inflammatory activity may be related to relatively high concentrations of bioactive compounds from both plants due to the applied extraction technology. Our study is in line with available scientific literature [30,31]. It has been shown that extract of
In conclusion, LevidorTM exhibits strong anti-inflammatory properties in