In vitro and in vivo anti-inflammatory properties of Annona muricata, Launaea taraxacifolia and Tridax procumbens leave extracts

doi:10.21203/rs.3.rs-4809894/v1

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In vitro and in vivo anti-inflammatory properties of Annona muricata, Launaea taraxacifolia and Tridax procumbens leave extracts

https://doi.org/10.21203/rs.3.rs-4809894/v1

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Background

In recent years, the role of inflammation in the development of several non-communicable diseases such as diabetes, cardiovascular diseases and cancer has been increasingly highlighted. Many medicinal plants contain bioactive substances with anti-inflammatory activities. The objective of this work was to study cytotoxicity and anti-inflammatory activity of hydroethanolic extracts of the leaves of Annona muricata, Launaea taraxacifolia and Tridax procumbens, three plants used in traditional medicine in Benin.

Method

Cytotoxicity and anti-inflammatory activity of the extracts was first assessed in vitro on the human monocyte THP-1 cells. In vitro, cytotoxicity was measured using the lactate dehydrogenase (LDH) assay. LPS/IFNγ-activated THP-1 cells were used to evaluate the anti-inflammatory effect of the three extracts by measuring gene expression level of pro-inflammatory (COX2, TLR8) and anti-inflammatory (IL-10-Receptor 1) markers by RT-qPCR, and by measuring by ELISA the production of pro- and anti-inflammatory cytokines (TNF, IL1β, IL-10) by THP-1 cells. Second, the model of acute Wistar rat hind paw edema induced by 1% formalin was used for the in vivo anti-inflammatory test.

Results

In vitro, the three plant extracts were nontoxic to THP-1 cells until 250 µg/mL. Expression of the pro-inflammatory markers COX2 and TLR8 were significantly lower for LPS/IFN_ϒ activated-cells treated with plant extracts at 50 and 100 µg/mL compared to untreated activated cells (P ˂ 0.05). IL-10-R1 expression was similar in untreated and plant-treated LPS-IFN_ϒ activated-cells, except for treatment with 50 µg/mL L. taraxacifolia leaf extract which resulted in lower expression of IL-10-R1. The production of TNF was completely abolished following the three plant treatment at 100 µg/mL of LPS/IFN_ϒ activated-cells, while IL-1β production was reduced (P < 0.001). For the anti-inflammatory cytokine IL-10, the production was higher by activated cells after treatment with 100 µg/mL L. taraxacifolia extract compared to untreated activated cells (P < 0.05). In vivo, per os administration of the three extracts at 300 mg/kg significantly prevented paw edema in rats (P < 0.05), similarly to acetylsalycilic acid.

Conclusion

The results suggest that the hydro-ethanolic extracts of the three plants have significant anti-inflammatory properties and deserve further studies for their use in the treatment of inflammatory pathologies.

Annona muricata

Launaea taraxacifolia

Tridax procumbens

Medicinal plants

Anti-inflammatory properties

THP-1 cells

Rat model

Gene expression

Cytokines

Inflammation is a set of protective mechanisms by which the body defends itself against various attacks (infection by a pathogenic organism, burns, allergy, etc.) and repairs damaged tissues [1]. However, uncontrolled inflammation that persists for a long duration can lead to chronic inflammation. The occurrence or progression of several non-communicable diseases such as neurodegenerative diseases, cancers, diabetes and cardiovascular diseases are directly or indirectly favored by inflammation [2, 3]. Activation of innate immune cells (macrophages, neutrophils, and dendritic cells) by pathogen-associated molecular patterns such as lipopolysaccharide (LPS) occurs through pattern recognition receptors, including Toll-like receptors (TLR), the stimulation of which affects each stage of inflammation development [4]. Among them, TLR8 is a key TLR, expressed by many immune cells (monocytes, myeloid dendritic cells and B lymphocytes), which activation has been implicated in many human autoimmune diseases, including rheumatoid arthritis [5, 6], and inflammatory bowel disease [7]. Macrophage activation is mediated by proinflammatory and prooxidant signaling pathways. In particular, NFkB pathway leads to pro-inflammatory cytokine production while activation of the COX-2 enzyme leads to the production of numerous inflammatory mediators, including prostaglandins [8, 9]. Activation of the NADPH-oxidase and NO-synthase pathways are involved in the prooxidant response through the production of reactive oxygen species (ROS) and nitric oxide (NO) [10]. Among pro-inflammatory cytokines, tumor necrosis factor (TNF), IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and their receptors are central markers of inflammation [11, 12]. Several studies have shown that inflammation plays an important role in the initiation and progression of atherosclerosis. A large number of pro-inflammatory cytokines are present in atherosclerotic plaque (TNF, IL1β, IL6) and anti-inflammatory cytokines that participate in the resolution of inflammation (IL-10) [13, 14]. Even diabetes-related hyperglycemia creates inflammation that underlies the complications of the disease [15, 16]. Plants from traditional medicine constitute a great source of medicines, including therapies against inflammation. Medicinal plants contain bioactive substances such as flavonoids and vitamin E presenting anti-inflammatory properties [17–19]. Our recent work has shown that the leaves of Annona muricata, Tridax procumbens and Launaea taraxacifolia contain flavonoids and vitamin E [20]. Therefore, in this work, we decided to evaluate the anti-inflammatory properties of the hydroethanolic extracts of the leaves of these three plants through in vitro experiments with the human monocyte THP-1 cell line and in vivo approach with Wistar rats.

Plant material

The leaves of A. muricata, L. taraxacifolia and T. procumbens were collected in Dassa-zoumé, a city in central Benin, from October to November 2019. The harvested plants were identified in the national herbarium of Benin at the University of Abomey-Calavi using the analytical flora of Benin [21]. A voucher specimen was deposited with number YH 776/HNB for A. muricata, YH 777/HNB for L. taraxacifolia and YH 778/HNB for T. procumbens. The leaf samples were cleaned with water and dried at room temperature during 14 days in the dark to better preserve heat- and light-sensitive molecules, and then crushed into powder.

Animals and ethical rules

Wistar rats were provided by and handled at the animal facility of the Laboratory of Pharmacology and Improved Traditional Medicines of the University of Abomey-Calavi. Wistar rats used were adult male and female rats aged 10 weeks with a body weight of 150 to 200 g. Animals were kept in constant laboratory conditions (25–28°C, relative humidity around 60%, and light/dark cycles, i.e., 12/12 h) with unlimited access to food and tap water. Animal welfare requirements were strictly followed during the experiments and the study was authorized with permit number 003-2023/EPAC/LARBA/URMAT/CE/R by the ethics committee of the polytechnic school of Abomey Calavi (EPAC).

Preparation of hydro-ethanolic plant extracts

Leaf powder (100 g) of each plant was macerated with 500 mL of 70% ethanol in distilled water for 72 hours under constant stirring. After filtration, the extract was evaporated to dryness at 40°C using a rotavapor (Heidolph Laborota 4000 efficient) coupled to a water cooler (Julabo FL 300) and the residue obtained was weighed to determine the yield according to the relationship: Extraction yield = m2/m1*100, where m1 = mass of dried matter used (100 g) and m2 = mass of final extract.

Cell culture

THP-1 cells, kindly provided by PHARMADEV research unit (IRD, Toulouse 3 university, France), were cultured with RPMI 1640 medium (Gibco, UK) supplemented with 2 mM L-glutamine (Gibco, UK), 100 units/mL Penicillin (Gibco, UK), 100 µg/mL streptomycin (Gibco), and 10% bovine calf serum (FCS, Sigma). Cultures were maintained at 37°C in humidified atmosphere at 5% CO2 with replacement of medium every 3 days. Cell concentration was maintained below 1 x106 cells/mL.

Cytotoxicity test

To assess the cytotoxicity of plant extracts, THP1 cells (1 × 106 viable cells/well) were seeded in 24-wells cell culture plates and incubated for 24 hours at 37°C, 5% CO2 with different concentrations of extracts (1000 µg/mL, 500 µg/mL, 250 µg/mL and 125 µg/mL). Each condition was tested in triplicate wells. The release of lactate dehydrogenase (LDH) in the culture supernatant, which correlates with the amount of cell death and membrane damage, has been quantified using the commercial LDH CyQUANT™ kit (Invitrogen, Eugene, Oregon, USA) following the manufacturer’s specifications. Background fluorescence (negative control) was determined by adding the LDH detection reagent to wells containing cells and culture medium without extract. A 100% cell lysis control (positive control) was done to determine the maximum amount of LDH present by adding to the wells containing cells 10 µl of a LDH Positive Control toxic to the cells. Absorbance was read at 492 nm using a microplate spectrophotometer (TECAN Infinite F200 pro) and results were expressed as percent of cytotoxicity [22].

Cell processing for evaluation of in vitro anti-inflammatory activity

THP1 cells (1×105 viable cells/well in 500 µL of culture medium) were seeded in 24-wells cell culture plates and stimulated with 5 µg/mL of LPS (eBioscience, Carlsbad, USA) and 100 ng/mL of IFN_ϒ (STEMCELL™ Technologies, Carlsbad, USA) for 6 hours at 37°C, 5% CO2. Plates were then washed three times with PBS. Crude extracts (50 and 100 µg/mL) were then added and plates were incubated at 37°C, 5% CO2 for 4 hours or 24 hours for evaluation of gene expression by RT-qPCR and cytokine production by ELISA, respectively. Positive and negative control wells contained THP-1 cells respectively stimulated or not with LPS/IFN_ϒ. Each condition was tested in triplicate wells.

Evaluation of gene expression by RT-qPCR

After 4 hours of incubation of THP-1 cells with plant extracts, plates were centrifugated and the supernatant was discarded. THP1 cells were lysed with 300 µL of RLT lysis buffer (PureLink TM, RNA Mini Kit, Invitrogen, Carlsbad, USA) and mRNA was prepared according to the manufacturer’s protocol. The concentration and quality of isolated RNA were assessed using a NanoDrop 8000 (Thermo Scientific). Synthesis of cDNA was performed according to the manufacturer’s recommendations (Themoscientific Verso cDNA Synthesis kit, Vilnius, Lithuania). qPCR was performed on a ViiA7 system using POWER SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) in the following PCR conditions: 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 10 s, and 72°C for 60 s, and a dissociation phase from 60°C to 90°C for 30 s. The primers used (IDT, France) were designed using Primer 3 software. βactin mRNA was used as the invariant control. The primers used are listed in Table 1.

Table 1

Sequences of primers used in RT-qPCR experiments
Gene	Sens	Antisens	Product size (Pb)
NOX 2	5’-TTAGTGGGAGCAGGGATTGG	5’-GGCATTGTTCCTTTCCTGCA	198
IL10-R1	5’-GTGCTGAAGAGAAGGCAACC	5’-CCTGAGGAAGCCAGAGTCAT	175
COX 2	5’-ATTCTTTGCCCAGCACTTCA	5’-ATCCTTGAAAAGGCGCAGT	151
TLR 8	5’-ACTCCAGCAGTTTCCTCGTCTC	5’-AAAGCCAGAGGGTAGGTGGGAA	145
Βactin	5’-GTGGCCGAGGACTTTGATT	5’-ATGCTATCACCTCCCCTGTG	164

Cytokine production

Enzyme-linked immunosorbent assays (ELISAs) were performed to measure the cytokine levels produced by THP-1 cells after LPS/IFN_ϒ activation and plant extract treatment. After 24 hours as described above, plates were centrifuged and the supernatant kept. The levels of pro-inflammatory cytokines TNF, IL-1β and the anti-inflammatory cytokine IL-10 were measured in duplicates in supernatants following instructions provided by the manufacturer (BioLegend, Inc. San Diego, CA, USA). Absorbance at 450 nm and 570 nm was read using a microplate spectrophotometer (EON, BioTeck, USA). Absorbance at 450 nm was normalized against absorbance at 570 nm. After subtraction of background values, the standards (OD and concentrations) were used to draw a linear regression line and cytokine concentrations were calculated.

In vivo evaluation of the anti-inflammatory activity of the extracts

The model used was the induction of edema described by Sen and colleagues [23]. The rats were divided into 5 batches of 3 rats: positive control, negative control, T. procumbens, L. taraxacifolia, and A. muricata. Rats were fasted for 12 h before the experiment. The initial volume (V0) of the left hind paw of all rats was measured before injection of formalin and any treatment. Then, a volume of 0.1 ml of 1% formalin was injected into the aponeurosis of the soles of the paws of the rats. Paw volume were evaluated every hour from 1 to 4 h after formalin injection. All rats were treated orally 30 minutes before formalin injection, as follows: the negative control received 0.9% NaCl isotonic saline solution at 10 ml/kg; the positive control was treated with acetylsalicylic acid at 100 mg/kg, the reference anti-inflammatory product; the three-test received 300 mg/kg of hydroethanolic extracts of the leaves of T. procumbens, L. taraxacifolia, or A. muricata. The experimental device used to assess the rats paw volume was that proposed by Bhatt and collaborators [24]. The paw volume was determined by immersion in a tube containing distilled water. The introduction of the paw causes an increase in the water level which is brought back to its initial level using 2 small syringes combined with the tube containing distilled water. The volume of the paw which corresponds to the quantity of water displaced was directly read on the 1 ml syringes. For each rat, the differences between the paw volume at 1, 2, 3 and 4 hours after formalin injection (Vt) and the initial paw volume (V0) were calculated. Then, the percentage of edema increase (PEInc), and the percentage of edema inhibition (PEInh) were calculated for each rat from the following formulas:

PEInc = [(Vt-V0)/V0]×100, where V0 = volume of the right hind paw at time 0 (before injection of formalin and any treatment) and Vt = volume of the right hind paw at time t (after injection and treatment).

PEInh = [(PEinc) negative control – (PEinc) treated rat) / (PEinc) negative control] × 100.

Statistical analysis

Graph Pad Prism software (version 8.0) was used to produce the graphs and statistical tests. For in vitro anti-inflammatory activities, differences between control and other conditions were tested by unpaired t-test. For the anti-inflammatory activity in vivo, comparisons were made using a two-way analysis of variance (ANOVA) with the Geisser-Greenhouse correction and Sidak's multiple comparison test. The differences were considered significant if P < 0.05.

Yield of extractions

The yields of the hydroethanolic extraction of the three plants were 8.7% for T. procumbens, 9.6% for A. muricata and 9.7% for L. taraxacifolia.

In vitro cytotoxicity of plant leaf extracts

Cytotoxicity of the three plant extracts was evaluated in vitro on THP-1 cells by LDH quantification. As shown on Fig. 1, concentrations of A. muricata and L. taraxacifolia leaf extracts until 250 µg/mL had no effect on cell viability. Concentrations of T. procumbens leaf extracts from 1 µg/mL to 250 µg/mL decreased cell viability by less than 1%. Concentrations of the extracts of the three plants from 250 to 500 µg/mL decreased cell viability by less than 10% (Fig. 1). At 1000 µg/mL, plants presented cytotoxicity of 8%, 20% and 30%, for T. procumbens, L. taraxacifolia, and A. muricata, respectively. We can deduce that concentrations of plant leaf extracts until 250 µg/mL were non-toxic for THP-1 cells. Concentrations of extracts above 250 µg/mL progressively decreased cell viability indicating that high concentrations of extracts can be toxic (Fig. 1).

In vitro anti-inflammatory activity of the plant extracts

To evaluate the anti-inflammatory activity of the three plant extracts, we first evaluated the in vitro capacity of the plant extracts to limit gene expression of inflammatory markers in activated THP-1 cells. Plants extracts were tested at two concentrations, 50 and 100 µg/mL. The expression level of TLR8 mRNA significantly decreased after treatment of LPS/IFN_ϒ activated-THP-1 cells with 50 and 100 µg/mL of each of the three plant extracts compared to untreated activated cells (Fig. 2A). In addition, plant-treated cells had a similar expression level to that of untreated and non-inflammatory cells (Fig. 2A). For COX-2 mRNA, the expression level was also significantly decreased after treatment of LPS/IFN_ϒ activated-THP-1 cells with 50 µg/mL and 100 µg/mL of each of the three plant extracts, except for A. muricata extract at 50 µg/mL. For IL10 receptor (IL-10-R1), an anti-inflammatory marker, no difference of expression was observed between LPS/IFN_ϒ activated-THP-1 cells with or without plant treatment, except for L. taraxacifolia at the concentration of 50 µg/mL whose treatment slightly reduced the expression level of IL-10-R1 (Fig. 2C).

Secondly, we measured cytokine production by LPS/IFN_ϒ activated-THP-1 cells treated or not with plant extracts, at three different concentrations, 10, 50, and 100 µg/mL (Fig. 3). For TNF, plant treatment at 100 µg/mL completely abolished TNF production by inflammatory THP-1 cells (Fig. 3A). At 10 µg/mL, only T. procumbens was able to reduce of around 3 times TNF production, whereas L. taraxacifolia and A. muricata had no effect. At 50 µg/mL, both T. procumbens and A. muricata reduced TNF production, respectively by 3 and 2 times approximatively (Fig. 3A). For IL-1β production, a slight significant decrease was observed for plant-treated cells with the three plant extracts at 100 µg/mL, compared to untreated ones (Fig. 3B). For IL-10, only L. taraxacifolia treatment of inflammatory cells had an effect, as IL-10 production was slightly increased with cells treated at 100 µg/mL of this plant extract (Fig. 3C).

In vivo anti-inflammatory activity of the plant extracts

To study anti-inflammatory properties of the three plant extracts in vivo, Wistar rats were treated with plant extracts before the induction of paw edema by formalin injection. The size of the edema was measured regularly every hour during 4 hours after formalin injection (Table 2). One and two hours after the induction of inflammation, there was no significant difference between the increase of the paw observed in the negative control rats compared to the treated rats. Three hours after inflammation, the edema was significantly inhibited by acetylsalicylic acid and T. procumbens treatments (P < 0.05 for both), with respective percentage of inhibition of 93% and 98.8% (Table 2). Both treatments led to 100% inhibition after four hours. For A. muricata and L. taraxacifolia treatments, their anti-inflammatory effects on edema development was significant 4 hours after formalin injection, with respective percentage of inhibition of 99% and 100% (Table 2).

Table 2

Effect of *T. procumbens*, *A. muricata* and *L. taraxacifolia* treatment on the volume of formalin induced-edema in rats. Paw edema was assessed before (T0) and every hour after formalin injection (T1 to T4) in Wistar rats treated or not by plants. Values are means ± SE of the differences between the volume of the paw at time T (V1 to V4) and the initial volume (V0). The percentage inhibition of edema ± SE is shown in brackets.
	Change in paw edema in mL ± SE (% inhibition of paw edema)
Group	T1	T2	T3	T4
Negative cont.^§	0.16 ± 0.01	0.20 ± 0.02	0.22 ± 0.02	0.42 ± 0.03
Positive cont.^§	0.11 ± 0.04 (40.6% ± 20.5)	0.09 ± 0.05 (54.5% ± 27.6)	0.02 ± 0.009* (93.0% ± 3.5)	0* (100% ± 0.0)
T. procumbens	0.09 ± 0.03 (50.6% ±13.8)	0.02 ± 0.003 (91.5% ± 2.6)	0.003 ± 0.003* (98.8% ± 1.2)	0* (100% ± 0.0)
A. muricata	0.10 ± 0.05 (54.8% ± 22.0)	0.25 ± 0.09 (24.2% ± 17.9)	0.12 ± 0.03 (62.6% ± 8.1)	0.007 ± 0.007** (99.0 ± 1.0)
L. taraxacifolia	0.26 ± 0.09 (16.7% ± 12.0)	0.14 ± 0.04 (55.2% ± 14.5)	0.06 ± 0.09 (72.0% ± 19.5)	0** (100% ± 0.0)
^§ Negative control received NaCl isotonic saline solution; Positive control received acetylsalicylic acid. Data were compared to the corresponding negative control values by a two-way ANOVA with the Geisser-Greenhouse correction and Sidak's multiple comparison test, * P < 0.05, P < 0.01, * P < 0.001, **** P < 0.0001.

This work aimed to evaluate the anti-inflammatory properties of the hydroethanolic extracts of the leaves of A. muricata, L. taraxacifolia and T. procumbens, three medicinal plants traditionally used in West Africa for numerous diseases, such as arterial hypertension, cardiovascular diseases, diabetes, cancer, liver diseases, malaria, bronchitis, conjunctivitis, fever and diarrhea [20]. Our approach combined in vitro experiments to evaluate plant extracts cytotoxicity and identify a nontoxic work concentration, with in vitro and in vivo models of inflammation to evaluate their anti-inflammatory potential.

The concentrations of the leaf extracts of the three plants from 250 µg/mL to 500 µg/mL decreased cell viability by less than 5%. The concentrations of T. procumbens, L. taraxacifolia and A. muricata leaf extracts from 500 µg/mL to 1000 µg/mL decreased cell viability by 8%, 20% and 30%, respectively. These results indicates that the plants were toxic only at very high concentrations. These results were consistent with our previous work on the toxicity of leaf extracts of L. taraxacifolia and T. procumbens on HepG2 cells of hepatic carcinoma [25, 26] and with other works which have studied the cytotoxicity of aqueous extracts of A. muricata on murine macrophages at the same concentrations [27]. Interestingly, Syed et al. reported high cytotoxicity of the methaolic extract of T. procumbens at 250 mg/mL against human lung cancer cell and breast cancer cell lines [28]. These differences may be related to the solvents used, the type of extract, or may reveal a specific cytotoxicity against the cancer cell types tested. Further toxicity evaluation on normal cells, primary cells or acute toxicity in murine model is needed to conclude this point.

T. procumbens, L. taraxacifolia and A. muricata extracts showed similar capacity to reduce the gene expression of TLR8 and COX2, two actors of the proinflammatory response. During inflammation, innate immune cells express Toll-like receptors (TLR) [4]. TLRs are a family of transmembrane receptors that appear to play an important role in maintaining tissue homeostasis by regulating inflammatory and tissue repair processes upon injury. TLR8 is expressed in monocytes and myeloid dendritic cells and is involved in the production of pro-inflammatory cytokines such as TNF. Monocytes then produce pro-inflammatory cytokines in response to TLR8 activation [29, 30], following the induction of inflammation in THP1 monocytes. COX2 are part of a family of enzymes inductive by multiple pro-inflammatory factors (cytokines and interferons). COX2 is a key enzyme involved in the production of prostaglandins during the inflammatory response [31]. However, the three extracts were not able to increase IL-10R1 expression in activated THP-1 cells, an anti-inflammatory marker, and L. taraxacifolia even limited its expression when compared to the untreated activated cells.

However, the impact of the plant extracts on cytokine production, which constitutes a highly relevant indicator of activity as we're talking about protein expression, not gene expression, was also conclusive. All three extracts were able to limit TNF production by activated THP-1 cells until a complete production inhibition at 100 µg/mL, a dose previously shown nontoxic for these cells. For IL-1β, the reduction of production was more modest, although a reduction of nearly 40% was observed with L. taraxacifolia. L. taraxacifolia extract was also able to increase IL-10 production by around 20%, confirming its anti-inflammatory activity in vitro. Pro-inflammatory cytokines are produced by macrophages and monocytes in response to aggression and infection to produce the inflammatory reaction characteristic of the innate immune response [3, 32]. Several studies have shown a decrease in pro-inflammatory cytokines and inflammation markers after induction of inflammation and treatment with anti-inflammatories such as quercetin [11, 33]. In addition, previous studies had shown that the extracts of T. procumbens could decrease the expression of TNF and COX2 genes in mice after induction of inflammation [34, 35].

The edema induced by the injection of 1% formalin is an animal model widely used to assess the anti-inflammatory activity of natural substances. The injection of formalin causes the release of several chemical mediators which are responsible for the inflammatory process. This inflammatory response is biphasic. The initial phase, which lasts about an hour, is due to the release of histamine and serotonin. Bradykinin is released during the second phase and prostaglandin biosynthesis occurs beyond the third hour [17]. The extracts of T. procumbens, L. taraxacifolia and A. muricata respectively showed an inhibition percentage of edema similar to that of acetylsalicylic acid in the second phase of the inflammatory process. However, this anti-edematous effect was weaker in the initial phase and not significant, similarly to acetylsalicylic acid. Several studies on acetylsalicylic acid have demonstrated that it acts in the second phase of inflammation by inhibiting the release of prostaglandins via COX2 [36, 37]. The inhibitory action of the plant extracts could therefore be due to the inhibition of cyclooxygenases, a hypothesis reinforced by the lower expression of COX-2 by the inflammatory THP-1 cells coincubated with the plant extracts.

The anti-inflammatory activity of plants is often attributed to polyphenols [17, 18] and vitamin E [19]. Our previous work had highlighted the presence of polyphenols and vitamin E in the three plants [20]. Studies have previously shown the anti-inflammatory activities of quercetin, a polyphenol compound, and extracts of T. procumbens [33, 34]. The infiltration of macrophages and lymphocytes in the atherosclerotic lesion maintain a chronic inflammatory reaction. Moreover, a large number of pro-inflammatory and anti-inflammatory cytokines are present in atherosclerotic plaque, such as TNF, IL-1β, IL-6, IL-8, IL-10 and IL-12. Among the anti-inflammatory cytokines, IL-10 is one of the most interesting in the context of atherosclerosis. IL-10 is mainly produced by macrophages and is involved in the direct control of TNF production. Furthermore, IL-10 inhibits the expression by macrophages of the metalloproteinases, MMP-1 and MMP-9, and stimulates the expression of the endogenous inhibitor of MMPs, TIMP-1 [13, 14, 38]. It inhibits the activation of the transcription factor NF-κB, as well as the expression of TNF by activated monocytes. IL-10 is thus an antiatherogenic and antithrombotic cytokine. The reduced production of TNF and IL-1β in inflammatory THP-1 cells treated with T. procumbens, A. muricata and L. taraxacifolia extracts suggest that these plants may be used for the prevention or treatment of atherosclerosis. In addition, L. taraxacifolia treatment led to increased production of IL-10 compared to untreated inflammatory cells, highlighting the need for further research on this plant. The chronic hyperglycemia that characterizes type 2 diabetes leads to oxidative stress and chronic inflammation through the expression of pro-inflammatory enzymes and pro-inflammatory cytokines [15, 16]. Long-term inflammation leads to vascular complications including atherosclerosis and cardiovascular diseases, nephropathy and retinopathy. T. procumbens, A. muricata and L. taraxacifolia are therefore of interest in the field of research into anti-inflammatory treatments.

The hydroethanolic extracts of A. muricata, L. taraxacifolia and T. procumbens were cytotoxic only at very high concentrations on THP-1 cells, suggesting little risk if consumed. The hydro-ethanolic extracts of the three plants presented significant anti-inflammatory properties as they reduced the expression of pro-inflammatory markers at both gene and protein level in inflammatory THP-1 cells. L. taraxacifolia was also able to increase IL-10 production by these cells. In vivo, the three plant extracts prevented the onset of edema in rats, which confirms that these plants deserve further studies for their use in the treatment of inflammatory pathologies

COX2

cyclooxygenase-2

ELISA

enzyme-linked immunosorbent assay

IFNγ

interferon-γ

interleukine

LDH

lactate deshydrogenase

LPS

lipopolysaccharide

TLR

Toll-like receptor

Ethics approval

The study was authorized with permit number 003-2023/EPAC/LARBA/URMAT/CE/R by the ethics committee of the polytechnic school of Abomey Calavi (EPAC).

Consent for publication

Not applicable.

Availability of data and materials

All datasets generated in this study are available from the corresponding author on request.

Competing interests

The authors declare no competing interests.

Funding

There was no specific funding for this study.

Authors' contributions

OK and AA designed and planned the study, funded the research, supervised the experiments and wrote the publication. MB and JBA carried out in vivo experiments with the help of SS. MB and JBA helped BV who carried out most of the in vitro experiments. All authors read and approved the final manuscript.

Acknowledgements

We thank Pr. Achille Massougbodji for his support and welcome in the research laboratories of the Clinical Research Institute of Benin (IRCB) in Abomey Calavi.

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No competing interests reported.

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In vitro and in vivo anti-inflammatory properties of Annona muricata, Launaea taraxacifolia and Tridax procumbens leave extracts

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Abstract

Figures

Introduction

Material and methods

Plant material

Animals and ethical rules

Preparation of hydro-ethanolic plant extracts

Cell culture

Cytotoxicity test

Evaluation of gene expression by RT-qPCR

Cytokine production

Statistical analysis

Results

Yield of extractions

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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