Comparison of the Anti-Inflammatory and Immunomodulatory Mechanisms of Two Medicinal Herbs: Meadowsweet (Filipendula
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International Journal of Plant, Animal and Environmental Sciences DOI: 10.26502/ijpaes.002 Research Article Comparison of The Anti-Inflammatory and Immunomodulatory Mechanisms of Two Medicinal Herbs: Meadowsweet (Filipendula ulmaria) and Harpagophytum (Harpagophytum procumbens) Juliette Cholet1*, Caroline Decombat1†, Marjolaine Vareille-Delarbre1,† , Maël Gainche2, Alexandre Berry1, Clémence Ogéron2, Isabelle Ripoche2, Laetitia Delort1, Marion Vermerie1, Didier Fraisse1, Catherine Felgines1, Edwige Ranouille3, Jean-Yves Berthon3, Albert Tourette4, Yves Troin2, François Senejoux1, Pierre Chalard2, Florence Caldefie-Chezet1 1Université Clermont Auvergne, INRA, UNH, Unité de Nutrition Humaine, CRNH Auvergne, F-63000, Clermont-Ferrand, France 2Université Clermont Auvergne, CNRS, SIGMA Clermont, ICCF, F-63000 Clermont-Ferrand, France 3Greentech, Biopôle Clermont-Limagne, 63360 Saint-Beauzire, France 4AltoPhyto, 7 rue des gargailles, 63370 Lempdes, France †Equal contributions *Corresponding Author: Juliette Cholet, Université Clermont Auvergne, INRA, UNH, Unité de Nutrition Humaine, CRNH Auvergne, F-63000, Clermont-Ferrand, France, Tel: +33-4-73-17-79-72; E-mail: [email protected] Received: 24 June 2019; Accepted: 10 July 2019; Published: 22 July 2019 Abstract Background: Meadowsweet (Filipendula ulmaria) and Harpagophytum (H. procumbens) are two medicinal herbs traditionally used for their anti-inflammatory effect. Nonetheless, if the effects of the single compounds isolated from these plants have been well described, little is known about the molecular mechanisms behind whole extracts. Methods: We studied and compared the effects of methanolic extracts from the aerial parts of F. ulmaria (FUE) and from the roots of H. procumbens (HPE) on different markers of inflammation such as antioxidant capacity, leukocyte ROS production, COX-2/PGE2 pathway or cytokine secretions. Results: FUE proved to be better than HPE in terms of antioxidant capabilities. Even if their effect on COX-2/PGE2 were similar, we found that their immune-modulatory activities were quite different. In the basal state, the FUE favored Int J Plant Anim Environ Sci Vol. 9 No. 3 September 2019. [ISSN 2231-4490] 145 International Journal of Plant, Animal and Environmental Sciences DOI: 10.26502/ijpaes.002 cytokines associated with Th1 lymphocytes whereas the HPE decreased the secretion of IL-21 and IL-23, associated with Th17 cells. In PHA-stimulated cells, the HPE increased the characteristic cytokines of Th1 cells, whereas the effects of the FUE were more nuanced. Conclusion: Though both plants are known as anti-inflammatory herbs, these results suggested that, apart from their similar anti-inflammatory effect on COX-2/PGE2, both could improve neutrophil and monocyte recruitment, as well as monocytes/macrophages and Th1, and presumably Th17, activation. Therefore, their impact on immune response was more likely immunostimulant. Keywords: Harpagophytum procumbens; Filipendula ulmaria; Cytokines; Inflammation; Immune-modulation; Molecular mechanism; Natural products; Radical oxygen species; COX-2/PGE2 1. Introduction Medicinal plants are one of the oldest sources of treatment used for healthcare, and for several years there is a reviving interest in them, especially in the context of chronic ailments and inflammation. Most of the well-known traditional medicinal plants have been found through empirical observations, the trial and error process. Among them, Harpagophytum procumbens DC. (Pedaliaceae), Devil’s claw, and Filipendula ulmaria (L.) Maxim. (Rosaceae), also known as Meadowsweet, have been extensively used for their anti-inflammatory and antirheumatic properties [1,2]. Although the beneficial effects of both Devil's Claw and Meadowsweet have been observed on inflammation in vivo, little is known about their underlying mechanisms. Indeed, most studies have been focused on their capacities to reduce inflammation in vivo, in models such as the paw edema or hot plate, or, in particular, on their action on the arachidonic acid pathway in vitro [3-7]. Thus, their inhibitory effect on the generation of leukotrienes and prostaglandins, considered as their main mechanism of action, has been well documented. However, their broad mechanism, and eventual side effects at the cellular level, remains unclear. In particular, some authors have pointed out that these plants, or certain compounds (such as quercetin or harpagoside) isolated from them, might impact the secretion of some cytokines during inflammation [8-10]. Nevertheless, the inflammation process is highly complex as is the interplay between immune cells via cytokines, prostaglandins or chemokines. Therefore, the current study was conducted to investigate and compare the potential antioxidant, anti-inflammatory and immunomodulatory effects of a methanol extract of Harpagophytum procumbens root (Harpagophytum procumbens extract – HPE) and of a methanol extract of the aerial parts from Filipendula ulmaria (Filipendula ulmaria extract – FUE). We studied the extracts on peripheral blood mononuclear cells (PBMCs) and human leukemic monocytic cells THP-1, through different markers of inflammation: leukocyte radical oxygen species (ROS) production, monocyte differentiation, cytokine secretion, cyclooxygenase-2 (COX-2) expression and PGE2). Int J Plant Anim Environ Sci Vol. 9 No. 3 September 2019. [ISSN 2231-4490] 146 International Journal of Plant, Animal and Environmental Sciences DOI: 10.26502/ijpaes.002 2. Materials and Methods 2.1 Plant material and preparation of the extract The dried roots of Harpagophytum procumbens and the dried aerial parts of Filipendula ulmaria were obtained in a pharmaceutical dispensary (Pharmacie Fontgiève, Clermont-Ferrand, France) and were powdered and macerated 3 times in methanol for 24h. After each filtration, the solvent was removed in a rotary evaporator under reduced pressure. The yield obtained was found to be 41.2% and 29.2% for Harpagophytum procumbens extract (HPE) and Filipendula ulmaria extract (FUE), respectively. 2.2 Identification of the major compounds of the methanolic extracts The major constituents of the FUE and the HPE were determined using LC-MS (UHPLC Ultimate 3000 RSLC chain) and an Orbitrap Q-Exactive (Thermo Scientific) with an Uptisphere C18-3 (250 x 4.6 mm, 5 µm) column from Interchim, by comparison with analytical standard (Extrasynthèse, France) or literature data. 2.3 Phenolic content and antioxidant activity The total phenolic content was estimated using the Folin-Ciocalteu method as previously described by Dubost et al. This same method was modified (precipitation of tannins involving skin powder) in order to estimate tannin content [11]. The total flavonoid content was assessed with the Dowd method [12,13]. An oxygen radical absorbance capacity (ORAC) assay was done in 96-well plates with a final volume of 200 µL as described by Gillespie et al. [14]. Fluorescein was used as the fluorescent probe and 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) as the peroxyl radical generator. The decrease in fluorescence was measured every minute for 1 h (excitation/emission: 485/530 nm) using a microplate reader (TECAN infinite F200 PRO, Männedorf, Switzerland). The ORAC value of the FUE or the HPE were calculated using the regression equation between Trolox equivalent (TE) and the net area under the curve. The results were expressed as μmol TE per gram of dry extract. The DPPH scavenging activity was evaluated according to the method of Meda et al. [15] with slight modifications. Briefly, 10 µL of a solution of the HPE or the FUE (1 mg/mL in methanol) was mixed with 2.5 mL of fresh DPPH solution (25 µg/mL in methanol). After 30 min of incubation, DPPH absorbance was recorded at 515 nm using a spectrophotometer (JASCO V-630). A standard curve of Trolox, in the range of 100 µM to 3000 µM, was plotted. The results were expressed as µmol of Trolox equivalents (µmol TE) per gram of dry extract. The nitric oxide scavenging activity was performed according to a modified method of Balakrishnan et al. [11]. Briefly, sodium nitroprusside solution (SNP) (10 mM) in phosphate buffer saline (PBS 50 mM) was mixed with different concentrations of the extract (1-250 µg/mL for FUE and 100-600 µg/mL for HPE) dissolved in methanol and water and incubated at room temperature for 2 hours. Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine dichloride and 5% phosphoric acid) in PBS (50mM) was added to these preparations. The absorbance of the chromophores formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthyl ethylenediamine dichloride Int J Plant Anim Environ Sci Vol. 9 No. 3 September 2019. [ISSN 2231-4490] 147 International Journal of Plant, Animal and Environmental Sciences DOI: 10.26502/ijpaes.002 was read at 540 nm (TECAN infinite M200 PRO) against two blanks (one with SNP and PBS and the other with SNP, extract and PBS) in 96-well plates (same as above). The iron (II)-chelating activity of the extracts was measured according to Wang et al. [16]. A volume of 100 µL of the HPE or the FUE (5 mg/mL) was blended with 135 µL of distilled water and 5 µL of FeCl2 (2 mM) in microplate wells. After 10 min of incubation, the reaction was initiated by the addition of 10 µL of ferrozine (5 mM). The decrease of the purple ferrozine-Fe2+ complex was estimated 10 min later by reading the absorbance at 562 nm (TECAN infinite M200 PRO). Distilled water (100 µL) was used as a control and 10 µL of distilled water was used