Anti-inflammatory effects of Brazilian ginseng (Pfaffia paniculata) on TNBS-induced intestinal inflammation: Experimental evidence

International Immunopharmacology 28 (2015) 459–469

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Anti-inflammatory effects of Brazilian ginseng (Pfaffia paniculata) on TNBS-induced intestinal inflammation: Experimental evidence C.A.R.A. Costa, A. Tanimoto, A.E.V. Quaglio, L.D. Almeida Jr., J.A. Severi, L.C. Di Stasi ⁎ Laboratory of Phytomedicines, Pharmacology and Biotechnology (PhytoPharmaTech), Department of Pharmacology, Institute of Biosciences, Unesp — Univ Estadual Paulista, P.O. Box 510, 18618-970 Botucatu, São Paulo, Brazil

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Article history: Received 13 March 2015 Received in revised form 19 June 2015 Accepted 4 July 2015 Available online xxxx Keywords: Inflammatory bowel disease Adaptogen Pfaffia paniculata Hebanthe eriantha Hebanthe paniculata Trinitrobenzenesulfonic acid

a b s t r a c t Inflammatory bowel disease (IBD) is a chronic, relapsing, idiopathic inflammation of the gastrointestinal tract. Clinical studies suggest that the initiation of IBD is multifactorial, involving genetics, the immune system and environmental factors, such as diet, drugs and stress. Pfaffia paniculata is an adaptogenic medicinal plant used in Brazilian folk medicine as an “anti-stress” agent. Thus, we hypothesised that the P. paniculata enhances the response of animals subjected to colonic inflammation. Our aim was to investigate the intestinal antiinflammatory activity of P. paniculata in rats before or after induction of intestinal inflammation using trinitrobenzenesulfonic acid (TNBS). The animals were divided into groups that received the vehicle, prednisolone or P. paniculata extract daily starting 14 days before or 7 days after TNBS induction. At the end of the procedure, the animals were killed and their colons were assessed for the macroscopic damage score (MDS), extent of the lesion (EL) and weight/length ratio, myeloperoxidase (MPO) activity and glutathione (GSH), cytokines and Creactive protein (CRP) levels. Histological evaluation and ultrastructural analysis of the colonic samples were performed. Treatment with the 200 mg/kg dose on the curative schedule was able to reduce the MDS and the EL. In addition, MPO activity was reduced, GSH levels were maintained, and the levels of pro-inflammatory cytokines and CRP were decreased. In conclusion, the protective effect of P. paniculata was related to reduced oxidative stress and CRP colonic levels, and due to immunomodulatory activity as evidenced by reduced levels of IL-1β, INF-γ, TNF-α and IL-6. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Inflammatory bowel disease (IBD) is a chronic and relapsing inflammation of the gastrointestinal tract [1]. Crohn's disease and ulcerative colitis, the main forms of IBD, exhibit aetiology and mechanisms of pathogenesis that are still unknown. However, clinical studies suggest that the initiation of IBD is multifactorial, involving genetics, the immune system and environmental factors, such as diet, drugs and mainly stress [1–3]. The intestinal inflammatory process related to the stress response involves the complex integration of interconnected brain regions and the hyperactivation of the brain–gut and hypothalamic– pituitary–adrenal (HPA) axis as well as the autonomic and enteric nervous system, which can interact directly with the endocrine and immune systems [1]. Contemporary IBD treatments are centred on the use of aminosalicylates, corticosteroids and immunomodulators. Though the drugs available have shown beneficial effects, they may pose risks to patients, and not all patients respond to these treatments [4]. Recently, IBD treatment has expanded to include biological agents, such as the anti-TNF ⁎ Corresponding author. E-mail address: [email protected] (L.C. Di Stasi).

http://dx.doi.org/10.1016/j.intimp.2015.07.002 1567-5769/© 2015 Elsevier B.V. All rights reserved.

drugs infliximab, adalimumab, and certolizumab, which has enabled a new look and a new hope for the treatment of disease [5–7]. However, monoclonal antibodies have numerous adverse effects related to their specific targets and carry the risk of an immune response, leading to a short serum half-life, a weakened immune response, and the possible generation of antibodies, acute anaphylaxis, and a potentially fatal allergic reaction [8,9]. Pfaffia paniculata (Martius) Kuntze (Suma or Brazilian ginseng) is a medicinal plant from Brazilian folk medicine that presents a wide range of uses as a tonic, including invigoration, reduction in stress, aphrodisiacal effects, and memory improvement [10,11]. P. paniculata, which is also called Hebanthe eriantha or Hebanthe paniculata, was recently renamed to its old botanical name [12] and belongs to the group of medicinal plants known as “ginseng” as well as a specific group of plants called adaptogens. While the pharmacological effects of other species known as “ginseng” (Eleutherococcus senticosus — Siberian ginseng, Panax quinquefolius — American ginseng, Panax ginseng — Korean ginseng, Withania somnifera — Indian ginseng) are widely reported and documented [13], studies on genera Pfaffia and P. paniculata in particular are scarce [11]. This is especially true considering the extent of the use of P. paniculata in Brazil and its importance as an export product to Asian countries [11,14].

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Adaptogens were recently defined as herbal preparations that increased attention and endurance against fatigue and are capable of increasing an organism's non-specific resistance to different stressors. The activities of adaptogens are related to the HPA axis, which in turn contributes to the normal functions of the nervous, cardiovascular, immune, endocrine and gastrointestinal systems [15]. Based on this, we hypothesised that adaptogen products can be useful in the control and prevention of diseases in which stress is an aetiological factor, such as IBD. Therefore, the aim of the present work was to investigate the intestinal anti-inflammatory activity of P. paniculata crude extract in rats before or after the induction of colitis using trinitrobenzenesulfonic acid (TNBS). 2. Material and methods 2.1. Reagents All reagents and chemicals used were of analytical grade and were purchased from Sigma-Aldrich, Co., St. Louis, unless otherwise stated. The test substances were dissolved in 0.9% saline and prepared immediately before administration to the animals. 2.2. Animals Male Wistar rats (weighing 180–220 g) were obtained from ANILAB, Paulínia, SP, Brazil, and housed under standard environmental conditions (21 ± 2 °C) with 12-h light/dark cycles and air filtration. Animals had free access to water and food. All of the experiments were conducted in accordance with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Biosciences Institute — Ethics Committee for Animal Research (CEEA), Protocol number 042/04-CEEA. 2.3. Plant material and extract preparation P. paniculata roots were collected from the Chemical, Biological and Agricultural Pluridisciplinary Research Centre (CPQBA) in Paulínia/SP in September 2011, and a voucher specimen (CPQBA 0241) of the plant was kept in the herbarium. After collection, the roots were crushed with the aid of an industrial blender and dried in an aircirculating oven (45 °C for 4 days). After drying, the roots were ground in a Wiley mill, and the resulting powder was placed in appropriate containers and stored away from light and humidity. The dried powder was extracted by cold maceration with 70% methanol for 48 h (1:4, kg/L). The resulting material was filtered through filter paper under vacuum, and the maceration process was repeated three times in 24 hour cycles. The resulting solution was concentrated at 45 °C under reduced pressure, lyophilized and properly stored away from light and humidity. 2.4. Phytochemical analysis The crude extract was subjected to qualitative phytochemical screening aimed at detecting the presence or absence of various classes of active chemical constituents, including phenolics, flavonoids, anthocyanins, tannins, quinones, coumarins, terpenoids, cardiac glycosides, alkaloids, saponins and cyanogenic glycosides as described by [16] and modified from [17]. The extract was also characterised using thin layer chromatography (TLC) and HPLC. For TLC, approximately 15 mg of each sample was solubilised in 1 mL of 70% MeOH and applied with the aid of a capillary in aluminium foil coated with silica gel 60 (250 μm thickness, F254). The plate was visualised using UV–vis observation, and the spots were visualised using nebulisation anisaldehyde/H2SO4 with subsequent heating.

HPLC chromatography was performed with equipment from Jasco, Japan consisting of a PU-2089 plus quaternary pump, degasser and a photo diode array detector (Jasco MD-2010 Plus). A Phenomenex Synergi Hydro column (C18, 250 × 4.6 mm d. i., 4 μm) was connected to a Phenomenex pre-column (C18, 4 × 3 mm d. i.). Data were recorded and evaluated using the EZChrom software. 10 mg aliquots of the samples were solubilised in 400 μL of methanol (MeOH/H2O 90:10) using ultrasound and were subjected to the clean-up step on a Phenomenex Strata C18 cartridge (250 mg) using 1600 mL of MeOH/H2O 90:10 as the eluent. The resulting solutions were filtered through a 0.22 μL polytetrafluoroethylene (PTFE) microfilter and were analysed using HPLC–UV–PAD. The system was optimized for the gradient elution mode in which the solvents were acetonitrile (Solvent B) and water (Solvent A), which were both acidified with 0.1% trifluoroacetic acid (TFA). The separation was monitored by scanning the spectral range of 200 to 400 ηm and the final recordings of the chromatograms were made at 214 ηm to display the largest number of peaks. Total phenolic content was determined by reaction with Folin– Ciocalteu reagent adapted to a 96-well plate [18]. Solutions of P. paniculata extract in absolute methanol were prepared to concentrations ranging from 0 to 10,000 mg/mL. The reaction with diluted Folin– Ciocalteu reagent (2N, Imbralab) was analysed using a UV–vis spectrophotometer (Power wave 340, Bio-TEK) at 750 ηm. The results were expressed as mg of gallic acid per g of P. paniculata extract (GAE/g of extract) and compared to the control sample of gallic acid (0–3 μg/mL). All samples were pipetted in triplicate. 2.5. Determining the antioxidant and scavenger activities The antioxidant activity of P. paniculata extract (1–1000 μM) was determined by lipid peroxidation assay in rat brain membranes modified from the original protocol described by [19]. The flavonoid quercetin was used as a reference and was tested using the same assay system. Scavenger activity was performed using 2,2,-diphenyl-1-picrylhydrazil (DPPH) radical (Sigma-Aldrich) as previously described by [20,21] and adapted for use in a 96-well plate. Aqueous solutions of P. paniculata were prepared to final concentrations in the range of 0 to 900 μg/mL. Gallic acid was used as a standard antioxidant substance and was compared to the samples. 2.6. TNBS model of intestinal inflammation The intestinal inflammation process was induced using the method originally described by [22]. Briefly, animals were fasted overnight and then anaesthetized. Under anaesthesia, 10 mg of trinitrobenzenesulphonic acid (TNBS) dissolved in 0.25 mL of 50% ethanol (v/v) was administered through a Teflon cannula inserted 8 cm into the anus. During and after TNBS administration, the rats remained in a head-down position until they recovered from the anaesthesia. Rats from the non-colitic (normal) group received 0.25 mL of saline instead of TNBS. After the induction, which was performed using two different protocols explained below, the animals were killed and an incision in the abdominal linea alba was made, and the occurrence of adhesions to the adjacent organs was noted. The colon was withdrawn, cleaned of fat and mesentery and weighed, and its length was measured under a standard load (2 g). The colon was opened longitudinally, and the lesion was assigned a macroscopic score of 0–10 as described by [23]. The extent of the lesion (cm) and the weight/length ratio (mg/cm) were also measured. Afterwards, the colon was divided longitudinally into fragments for biochemical assays. In the preventive protocol, rats received 25, 50, 100, 200 or 400 mg/kg of P. paniculata extract or 2 mg/kg of prednisolone for 14 days before colitis induction. The drugs were administered by oral route (10 mL/kg). Two additional groups, the non-colitic group and the colitic group, were included for reference and received only the

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vehicle (10 mL/kg saline) orally. The animal body weights and the total food intakes for each group were recorded daily. Animals from all groups (n = 7–8) were killed 48 h after colitis induction. In the curative protocol, the extract was evaluated according to the experimental protocol of curative ulcerative colitis as described by [24]. In this protocol, colonic inflammation was induced with 10 mg of TNBS in 50% ethanol, as described previously. Treatments started 2 h after the administration of TNBS and continued daily for 7 days. The animals were killed on the eighth day. The animals were divided randomly into eight groups and treated daily with the same drugs and doses used in the preventive protocol. The non-colitic and TNBS control groups were similar to those in the preventive protocol.

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subjected to Kruskal–Wallis non-parametric variance analysis followed by Dunns' post test. The proportions were compared using Fisher's exact test. All of the statistical analyses were performed using GraphPad InStat® version 3.02. Differences were considered significant when p ≤ 0.05. 3. Results 3.1. Plant material and extract preparation The collection of P. paniculata provided 1.146 kg of fresh material, which resulted in 609 g (53.14%) after drying. Five hundred grams of plant was used to prepare the hydromethanolic extract, which resulted in 166.38 g (33.28%) of lyophilised extract.

2.7. Histological evaluation and ultrastructure analysis 3.2. Phytochemical analysis A representative colon fragment located 1 cm above the lesion was collected for histological slide preparation and stained with haematoxylin and eosin (HE) for analysis of the microscopic damage. An adapted microscopic damage score was assigned on a 0–27 scale as described by [25]. Images were acquired using Zeiss Imager Axio Vision 4.8.2.0 software. Samples of the colon were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde solutions in 0.1 M phosphate buffer (pH 7.3) for transmission electron microscopy (TEM) analysis. Afterwards, the material was post-fixed in a 1% osmium tetroxide solution in 0.1 M phosphate buffer (pH 7.3) at room temperature for 2 h, dehydrated through a graded series of acetone, and embedded in Araldite® resin. Ultra-thin sections (70 ηm) were double-stained with uranyl acetate and lead citrate. All samples were analysed and images were acquired using a Tecnai Spirit Transmission Electron Microscope from Fei Company, 80 kV. For scanning electron microscopy (SEM) analysis, small fragments of the colon were fixed in a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.3) and post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.3) at room temperature for 2 h. Samples were dehydrated through the serial application of increasingly concentrated ethanol, critical-point-dried with liquid CO2, and sputter-coated with gold (10 ηm). All samples were analysed and images were acquired using a QUANTA 200 Scanning Electron Microscope from Fei Company, 80 kV.

After preparation, a qualitative phytochemical screening of the extract was performed. The results showed that the extract consisted mainly of triterpenes and saponins, though the presence of phenolic compounds, flavonoids and alkaloids was also evidenced in agreement with the previous data [28–32]. Other classes of substances, including tannins, anthraquinones, coumarins and cyanogenic glycosides, were not detected. Thin layer chromatography of P. paniculata was performed in comparison to Pfaffia glomerata. Among the developed stains, the Rf = 0.78 is a feature noted by [33] and [34] that indicates the presence of ecdysterone in crude P. glomerata extracts (data not shown). The chromatographic analysis of the P. paniculata hydromethanolic extract obtained by HPLC–PAD demonstrated the presence of little peaks arising from weak chromophore groups. The UV spectra of the eluted peaks in the region tr = 0–45 min show a single band of approximately 215–220 ηm (Fig. 1C). This result is consistent with the presence of triterpene as well as nortriterpenic and steroidal saponins described in the literature for the roots of these species [28,30,35]. The P. paniculata extract (Fig. 1A) was different from the P. glomerata extract (Fig. 1B), which is consistent with the differences in chemical composition between these species. The measurement of total phenols contained in the crude P. paniculata extract showed 3.02 mg of gallic acid equivalents (GAE)/g extract.

2.8. Biochemical analysis in colon samples

3.3. Antioxidant and scavenger activities

MPO activity was measured according to the technique described by [26] and the results are expressed as MPO units (U) per g of wet tissue. The total GSH content was quantified using the recycling assay described by [27] and the results are expressed as ηmol per g of wet tissue. The cytokine and C-reactive protein levels of all of the treated groups that showed improvements in the macroscopic damage score, extent of the lesion, total glutathione content or myeloperoxidase activity were measured according to the following protocol: colon samples were weighed and diluted to a concentration of 1:5 w/v in 10 mmol/L phosphate buffered saline (pH 7.4) and homogenised with an automatic Heidolph homogeniser for the determination of cytokines levels. The tubes were placed in a shaker submerged in a water bath (37 °C) for 20 min and then centrifuged at 9000 g for 5 min at 4 °C. The supernatants were used to quantify IL-1β, IL-6, TNF-α, INF-γ, IL-10, and CRP levels using a specific ELISA Kit (R&D Systems Inc., USA). The results were expressed as ρg/mL (cytokines) or mg/mL (CRP).

In the protocol to verify the free radical scavenging activity (DPPH-assay), the extract showed weak scavenging activity (EC50 = 5.03 mg/mL) compared to the control gallic acid (EC50 = 1.89 μg/mL). In the lipid peroxidation assay, the extract also showed weak antioxidant activity (IC50 = 4.05 mg/mL) compared to the control quercetin (IC50 = 4.52 μg/mL).

2.9. Statistics The results are expressed as the mean ± S.E.M. or median (interquartile range). Data were either analysed using one-way ANOVA followed by Dunnett's Multiple Comparison test or were

3.4. TNBS model of intestinal inflammation 3.4.1. Clinical and macroscopic evaluation In the preventive protocol, daily assessment over the 14 previous treatments with the P. paniculata extract showed that the animals had a constant feed intake and increasing weight gain until day 13, after which point the animals were fasted for induction the colonic inflammation at day 14 (data not shown). The macroscopic evaluations showed that the treatment with different doses of P. paniculata or with the standard drug prednisolone were not able to decrease the macroscopic damage score, extent of lesion, and weight/length ratio (Table 1 and Supplementary material). During the daily monitoring of the animals in the curative protocol, there was an increase in the food intake after the induction of inflammation (data not shown). The macroscopic evaluation demonstrated that

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Fig. 1. Chromatographic profile of the P. paniculata extract (A) and P. glomerata (B) by HPLC–UV–PAD. UV spectrum representative of the eluted peaks (C) and ecdysterone (D). Column: Phenomenex Synergi-hydro C18 (150 × 4.6 mm d. i., 4 μm); Eluent A: H2O + 0.1% TFA; Eluent B: ACN + 0.1% TFA; Gradient elution 10–75% B in A for 35 min, followed by 75–90% B for 50 min; Flow 1 mL/min, injection volume 20 μL, λ = 214 ηm.

treatments with prednisolone and P. paniculata extract at doses of 100 or 200 mg/kg were able to diminish the extent of lesion, while only the 200 mg/kg dose P. paniculata was able to reduce the macroscopic damage score (Table 1 and Supplementary material).

3.4.2. Histological evaluation and ultrastructure analysis from the curative protocol The microscopic evaluation demonstrated that treatments with the P. paniculata extract at doses of 50 and 100 mg/kg were able to

Table 1 Macroscopic evaluation of the Pfaffia paniculata crude extract at doses of 25, 50, 100, 200 or 400 mg/kg in the preventive and curative experimental protocols of TNBS-induced intestinal inflammation in rats. Model

Groups

Macroscopic damage score (0–10 (IQR))a

Extent of lesion (cm)b

Weight/length ratio (mg/cm)b

Preventive

TNBS control (8) Non-colitic (8) Prednisolone (8)

8 (7.75–8.5) 0* 9.5 (9–10) 9.5 (9–10) 9 (8–10) 8 (7–9) 8 (7.5–10) 9 (8.75–10) 7 (6.75–8) 0** 6 (5–6.5) 6 (5.5–7) 6 (5.5–7.5) 5 (5–5.5) 4.5 (1.75–5)** 7 (6–7.5)

3.6 ± 0.4 0** 4.2 ± 0.3 3.9 ± 0.3 3.9 ± 0.5 3.3 ± 0.4 3.7 ± 0.5 4.4 ± 0.3 3.13 ± 0.3 0** 1.90 ± 0.3* 2.10 ± 0.3 2.23 ± 0.5 1.81 ± 0.2* 1.25 ± 0.4** 2.74 ± 0.3

161.7 ± 8.0 89.5 ± 1.9** 164.5 ± 6.0 151.8 ± 6.6 178.6 ± 10.2 179.3 ± 13.5 182.9 ± 13.3 181.6 ± 22.9 209.5 ± 34.4 89.6 ± 1.5* 160.5 ± 12.2 166.1 ± 5.0 184.3 ± 22.9 165.9 ± 23.7 157.9 ± 9.3 173.1 ± 14.1

Pfaffia paniculata mg/kg (n)

Curative

TNBS control (8) Non-colitic (8) Prednisolone (7)

Pfaffia paniculata mg/kg (n)

8 > > > > < > > > > :

8 > > > > < > > > > :

25 (8) 50 (8) 100 (7) 200 (8) 400 (8)

25 (7) 50 (7) 100 (7) 200 (6) 400 (7)

*p ≤ 0.05, **p ≤ 0.01, relative to the TNBS control. a Score data are expressed as the median (interquartile range) and were analysed by the Kruskal–Wallis test followed by Dunn's post hoc test. b The extent of lesions and colonic weight/length ratio data are expressed as the means ± S.E.M. and were analysed using Analysis of variance (ANOVA) followed by Dunnett's post hoc test.

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significantly reduce the microscopic damage score compared to the TNBS control group. Though the remaining treatments with the plant extract and the standard drug prednisolone did not diminish the total microscopic damage score, decreases in polymorphonuclear cell infiltration, fewer ulceration sites, reduced dilated crypts, and goblet cell depletion were observed in all groups that received P. paniculata extracts. In fact, the groups treated with P. paniculata demonstrated an increase of the number of goblet cells. Prednisolone was only effective in reducing leucocyte infiltration (Fig. 2). The transmission ultrastructure analysis corroborated the histopathological findings. Doses of 50 and 100 mg/kg of P. paniculata were able to increase the regeneration of the mucosa as detected by the recovered microvillus border and normal structural–functional aspects of the epithelium. Treatments with 100 mg/kg of P. paniculata also resulted in perfectly attached colonocytes with normal nuclei and organelles (Fig. 3). The scanning electron microscopy confirmed both the histopathological and transmission ultrastructure analyses. At doses of 50, 100 and 200 mg/kg, P. paniculata recovered the surface hexagonal shape of colonocytes, while treatment with 50 and 100 mg/kg showed normal surface cytoarchitecture of the tubular glands. It is also important to highlight the presence of mucus-adhered bacteria in the groups treated with 100 and especially 200 mg/kg of extract (Fig. 3). Most importantly, the scanning electron microscopy analysis demonstrated copious mucus secretion in all groups treated with the plant material, which

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might be confirmed by the increased number of goblet cells observed in transmission electron and optical microscopy (Figs. 2 and 3). 3.5. Biochemical analysis In the preventative protocol, treatment with P. paniculata extract or prednisolone did not decrease the activity of MPO (Fig. 4 — left panel A), corroborating the macroscopic results. In the curative protocol, treatment with P. paniculata extract at a dose of 200 mg/kg reduced the activity of the MPO. A similar effect was also observed following treatment with prednisolone (Fig. 4 — left panel - B). In the preventive protocol, none of the doses of the extract or prednisolone were able to maintain normal levels of glutathione compared to the non-colitic group (Fig. 4 — right panel - A). However, prednisolone and P. paniculata extract at doses of 50 or 200 mg/kg prevented the depletion of glutathione levels in the curative protocol (Fig. 4 — right panel - B). In addition, colon samples were subjected to some inflammatory markers, considering that P. paniculata extract showed curative antiinflammatory activity at a dose of 200 mg/kg. A significant reduction in IL-1β levels was observed in the groups treated with doses of 50, 100 or 200 mg/kg of P. paniculata extract compared to the control group. There was also a significant reduction in the IFN-γ levels in the groups treated with prednisolone and all doses of P. paniculata extract. Furthermore, there was a significant difference

Fig. 2. Photomicrography of a rat colon submitted to the curative protocol of TNBS-induced intestinal inflammation. The full-thickness histological sections of colon stained with haematoxylin and eosin are shown. In the bottom right corner, the microscopic score was assigned according to the criteria previously described [25]. Mucosa (m); tubular gland (black arrow); submucosa (sm); oedema (oe); disruption of the epithelium (dep); infiltration (in). Data are expressed as the median ± IQ (interquartile range) and were analysed by Kruskal–Wallis followed by Dunn's post hoc test. *p ≤ 0.05, ***p ≤ 0.001 relative to the TNBS control.

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Fig. 3. Electron micrography of a rat colon submitted to the curative protocol of TNBS-induced intestinal inflammation. Scanning and transmission electron microscopy analyses. Crypts (white arrow); Goblet cells (black arrow); Mucin (m); Nucleus (n); Microvillus border (mv).

between the non-colitic and control groups in the TNF-α and IL-6 levels, but only the plant extract at a dose of 200 mg/kg reduced the levels of these cytokines while the standard drug prednisolone only altered the levels of TNF-α (Fig. 5). For the levels of the anti-inflammatory cytokine IL-10, the only significant difference observed was between the control group and the non-colitic group. Neither prednisolone nor the plant extract were effective at altering IL-10 levels. Finally, the levels of C-reactive protein (CRP) were significantly reduced in all treated groups (Fig. 5). 4. Discussion Recent studies have shown that inflammatory bowel disease (IBD) has no cure, and the aims of available treatments are to increase remission periods and improve quality of life [36]. Defects in the immune system, oxidative stress, and the microbial content of the gastrointestinal tract play a role in its pathogenesis [37]. IBD is considered a disease of modern society, and its increased incidence since the mid-twentieth century is associated with a modern, urbanised lifestyle [38,39]. Current treatment strategies for IBD use biological agents, such as monoclonal antibodies against tumour necrosis factor alpha (TNF-α), to target the excessive activity of the adaptive immune system. Although treatment with these anti-TNF-α agents is successful in many

patients, only a third or less will achieve full remission [40]. Based on this evidence and the fact that IBD is a life-long disease, new therapies must be developed [41]. In addition to modern therapeutic procedures, several natural products have been studied in many models of intestinal inflammation. Phenolic compounds in particular, such as flavonoids [42–45], coumarin derivatives [24,46–48] and some medicinal plant extracts [49–51], have been shown to have significant effects. In fact, medicinal plants represent an invaluable source of substances with antioxidant and immunomodulatory properties [52,53]. P. paniculata, or Brazilian ginseng, is the most commonly employed Pfaffia species in commercial preparations [54]. The roots of P. paniculata are used by traditional Brazilian communities as a tonic for its aphrodisiacal and memory-improving properties [10]. According to a study by [55], there are more than fifty preparations containing P. paniculata on the US market. However, despite the large use of the species, there are few studies showing the pharmacological effects [11] of P. paniculata and no information is available regarding its use in IBD. Brazilian ginseng is a part of a special group of herbs called “adaptogenic plants”, a term established by N. Lazarev in 1947 in the Soviet Union. Adaptogenic plants are defined as natural metabolic regulators that increase the body's ability to adapt to and avoid damage caused

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Fig. 4. Effects of P. paniculata extract on the colonic myeloperoxidase (MPO) activity and total glutathione (GSH) content after the preventive (A) or curative (B) protocols were performed on rats with TNBS-induced intestinal inflammation. Data are expressed as the mean ± SEM and were analysed by Analysis of variance (ANOVA) followed by Dunnett's post hoc test and are representative of two independent experiments. *p ≤ 0.05, **p ≤ 0.01 relative to the TNBS control.

by environmental factors by exerting nonspecific antioxidant, immunomodulatory, hypoglycemic, hypocholesterolemic and other effects [56–58]. Different compounds, such as pfaffic acid, nortriterpenoid saponins derived from the pfaffic acid and denominated pfaffosides A, B, C, D, E, and F, which are chemically related to ginsenosides, have been isolated from P. paniculata, along with phytosterols, such as the β-sitosterol, stigmasterol and their glucosides [28–32,55]. The phytochemical analysis performed in our study showed that the P. paniculata extract contained a series of compounds belonging to these chemical classes. These substances have been found to be responsible for a number of biological activities, such as analgesic and anti-inflammatory properties [31,59–62], as well as antitumour effects [28,63–66]. Importantly, none of the phytochemical analyses showed the presence of β-ecdysterone in the studied extract (Fig. 1). β-Ecdysterone is a marker that allows for the differentiation between P. glomerata and P. paniculata [14,34], and its characterisation is necessary because both Pfaffia species are commercialized as the same product due to the difficulties in their botanical identification [34]. To evaluate the antioxidant activity of the extract and its ability to combat free radicals, an assay to verify the presence of polyphenols, which are substances with potent antioxidant activities [67,68], was performed. Polyphenols are also anti-inflammatory compounds that stimulate stress-related cellular signalling pathways and modulate the intestinal microbiota [68]. Notably, the antioxidant activity of the phenols is not directly correlated to the total amount of phenolic compounds [67]. Thus, the antioxidant activity was also verified in rat brain membranes and through the DPPH assay. Though the results

showed low antioxidant activity in vitro, a finding that does not support the antioxidant activity of the extract, the possible anti-inflammatory activity has not yet been excluded. To verify the immunomodulatory and anti-inflammatory activities of the extract, the experimental protocol of TNBS-induced intestinal inflammation was performed. TNBS administration caused intense injury to the colon that can be histologically characterised by infiltration of the mucosa and submucosa of the colon by polymorphonuclear cells, macrophages and mast cells [69,70]. This infiltration causes the alteration of two markers: myeloperoxidase (MPO) is an enzyme present in the granules of neutrophils [71,72] that, when increased in the inflammatory process, leads to a production of free radicals, which in turn promote a decrease in the levels of glutathione (GSH) [73,74]. Treatment with P. paniculata extract or the standard drug prednisolone in the preventive model did not decrease the MPO activity. These results confirm the observed macroscopic data. By contrast, in the curative model, a 200 mg/kg dose of either the extract or the prednisolone significantly reduced the MPO activity. This reduction in MPO activity may be related to a decrease in inflammatory cell infiltration as a consequence of the anti-inflammatory effects of the compounds contained in the P. paniculata extract. These compounds are chemically similar to the ginsenosides, which are substances with demonstrated antiinflammatory actions according to studies by [61,62,75–77]. Glutathione is a low molecular weight tripeptide that is biologically active in its reduced form [78]. The reaction between free radicals and reduced glutathione result in the neutralization of free radicals and increased levels of the oxidized form of glutathione in the cytoplasm. Because oxidized glutathione can more easily diffuse out of the cell,

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Fig. 5. Effects of P. paniculata extract on colonic levels of inflammatory markers (TNF-α, IFN-γ, IL-1β, IL-6, IL-10, and CRP) after the curative protocol was performed on rats with TNBSinduced intestinal inflammation. Data are expressed as the means ± SEM and were analysed by Analysis of variance (ANOVA) followed by Dunnett's post hoc test. *p ≤ 0.05, **p ≤ 0.01 relative to the TNBS control.

pathological conditions, such as IBD, which produces large amounts of free radicals, can deplete intracellular glutathione [79,80]. This fact is reflected in the preventive model, where neither the extract nor prednisolone was able to prevent the depletion of glutathione levels. By contrast, in the curative model, 50 and 200 mg/kg doses of either prednisolone or the extract were able to prevent the depletion of glutathione. The mechanisms of pathogenesis, development, or relapses of IBD are not fully understood [1–3]. One of the problems physicians face in diagnosing and treating IBD is the identification of early recurrence; for instance, microscopic abnormalities have been found in colons that appear macroscopically to be disease-free [81]. In the micro and ultrastructural analyses of the curative protocol, it was clear that epithelium regenerated first even though the inflammation was still active in the mucosa and the other layers of colon. This observation might explain

why a colonoscopy or any other macroscopic evaluation must not be the only parameter of analysis for diagnosing recurrences of IBD. Optical and electron microscopic evidences reinforce and explain the above biochemical and macroscopic results because P. paniculata treated groups at doses of 50, 100 or 200 mg/kg showed a reduction in the amount of polimorphonuclear cell infiltration, in the ulceration sites and in crypts' damage, although lymphocyte infiltration was not decreased by any dose of P. paniculata. In addition to the parameters evaluated, inflammatory markers of the colon samples that were quantified for according to the P. paniculata extract showed good curative anti-inflammatory activity at a dose of 200 mg/kg, as previously described. Cytokines are immune system molecules necessary for the maintenance of gut homeostasis. The imbalance between pro-inflammatory cytokines, such as TNF-α, IL-1β and IFN-γ, and anti-inflammatory

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cytokines, such as IL-10 and IL-4, is considered to be the central mechanism by which inflammation of the bowel is modulated in response to exposure to various gut pathogens [82]. Activated macrophages are thought to be the major producers of inflammatory cytokines in the gut, and an imbalance of cytokines contributes to the pathogenesis of IBD [83]. Our results showed that the highest dose of the extract (200 mg/kg) was able to reduce the levels of the evaluated pro-inflammatory cytokines relative to the TNBS control group. [84] showed that ginseng saponins reduced the production of pro-inflammatory cytokines such as TNF-α and IL-1β, suggesting that the saponins (pfaffosides) in the extract may be responsible for these effects. Furthermore, in a mouse model of intestinal inflammation, phytosterols, such as β-sitosterol, which has a number of beneficial effects in inflammatory models, decreased the activity of colonic MPO by 28% and the expression of IL-1β, TNF-α, and IL-6 [85]. As expected, there was a significant difference between the TNBS control and the non-colitic groups for all of the evaluated cytokines, except INF-γ. Recent studies have shown that in intestinal inflammation, INF-γ levels are higher early in the inflammatory process and decline over time [86–88]. It is possible that the lack of a significant difference between the non-colitic and TNBS control animals was due to this decline. The anti-inflammatory cytokine IL-10 appeared to increase in colitic animals and showed a tendency to decrease in the animals treated with the higher doses of the extract or the standard drug. We expect that the levels of IL-10 would be higher in animals subjected to the inflammation process relative to animals that received treatment. Our results reveal that the inflammatory process was solved. Low levels of IL-10 have been shown to increase the severity of Crohn's disease in patients compared to high levels of IL-10 [89]. However, according to [90], IL-10 concentration levels increase during the phase of disease resolution and decline thereafter regardless of the treatment modality. The results of the cytokine evaluation suggest that the lymphocyte infiltration observed in the microscopic analyses actually promoted the anti-inflammatory response. Thus, the macroscopic, microscopic and immunologic parameters must all be verified to accurately diagnose and predict a recurrence. Finally, we measured the levels of another inflammatory marker, C-reactive protein (CRP), which is considered to be the leading protein with an upregulated expression during the acute phase of IBD in humans. Though the functions of CRP are still not completely understood, it is known to act in the opsonization of infectious agents and damaged cells [91]. The production of CRP is stimulated mainly by hepatocytes and the pro-inflammatory cytokines IL-6, IL-1β and TNF-α produced at the site of inflammation [91]. In our study, all of the treatment groups showed a reduction in CRP levels compared to the TNBS group. These data are consistent with the reduced levels of the cytokines IL-6, IL-1β, and TNF-α previously described. In sum, the results encourage us to continue evaluating the antiinflammatory activities of the P. paniculata extract. We will also evaluate an enriched partition of terpenoid saponins, compounds thought to exert anti-inflammatory effects [59,92]. In conclusion, the crude extract from P. paniculata was not able to prevent TNBS-induced intestinal inflammation but was able to reduce the colonic inflammation, suggesting a curative potential. This may represent a new and valuable treatment for IBD in humans. Conflict of interest The authors declare that they have no conflicts of interest. Authors' contributions This paper was part of the post-doctoral research of Celso A.R.A. Costa, who drafted the manuscript, was responsible for animal care

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and management, supervised and accompanied all of the experiments, and performed the appropriate statistical treatments. Alexandre Tanimoto contributed to the draft, helped with all of the experiments, and supervised the histological analyses. Ana E.V. Quaglio contributed to the draft, helped with all of the experiments and supervised the biochemical and molecular analyses. Luiz D.A. Junior helped with all of the experiments, contributed to the ideas and was responsible for the animal care and management. Juliana A. Severi performed the HPLC analysis of the P. paniculata extract. Luiz C. Di Stasi is the senior author and contributed to the draft, supervised the entire work and gave final approval of the manuscript. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2015.07.002.

Acknowledgements The authors acknowledge the technical support provided by Aline W. Fantinati, Alexandre S. Chagas, Adriana Del Ben, Tainan F. S. Curimbaba, Juliana R. Ribeiro, and Flávia Bonamin. Ilio Montanari Júnior from the Chemical, Biological and Agricultural Research Centre at CPQBA, University of Campinas, UNICAMP provided us with the plant. Celso A.R.A. Costa was the recipient of a fellowship from FAPESP (2011/50847-4).

References [1] J. Mawdsley, D. Rampton, Psychological stress in IBD: new insights into pathogenic and therapeutic implications, Gut 54 (2005) 1481–1491. [2] D. Rampton, Does stress influence inflammatory bowel disease? The clinical data, Dig. Dis. 27 (Suppl. 1) (2009) 76–79. [3] A. Ananthakrishnan, Environmental risk factors for inflammatory bowel disease, Gastroenterol. Hepatol. 9 (2013) 367–374. [4] J. Yamamoto-Furusho, Innovative therapeutics for inflammatory bowel disease, World J. Gastroenterol. 13 (2007) 1893–1896. [5] S. Danese, New therapies for inflammatory bowel disease: from the bench to the bedside, Gut 61 (2012) 918–932. [6] A. Stallmach, S. Hagel, T. Bruns, Adverse effects of biologics used for treating IBD, Best Pract. Res. Clin. Gastroenterol. 24 (2010) 167–182. [7] S. Vakili, M. Taher, N. Daryani, Update on the management of ulcerative colitis, Acta Med. Iran. 50 (2012) 363–372. [8] T. Hansel, H. Kropshofer, T. Singer, J. Mitchell, A. George, The safety and side effects of monoclonal antibodies, Nat. Rev. Drug Discov. 9 (2010) 325–338. [9] L. Liu, Y. Li, The unexpected side effects and safety of therapeutic monoclonal antibodies, Drugs of Today (Barcelona, Spain: 1998)502014. 33–50. [10] F. de Oliveira, Pfaffia paniculata (Martius) Kuntze: o ginseng-brasileiro, Rev. Bras. Farm. 1 (1986) 8692. [11] F. Mendes, E. Carlini, Brazilian plants as possible adaptogens: an ethnopharmacological survey of books edited in Brazil, J. Ethnopharmacol. 109 (2007) 493500. [12] G. Vilela, L. Ming, M. Marques, S. da Silva, Junior I. Montanari, Agronomical and phytochemical aspects of fafia, Hortic. Bras. 31 (2013) 133138. [13] A. Dasgupta, M. Reyes, Effect of Brazilian, Indian, Siberian, Asian, and North American ginseng on serum digoxin measurement by immunoassays and binding of digoxin-like immunoreactive components of ginseng with fab fragment of antidigoxin antibody (Digibind), Am. J. Clin. Pathol. 124 (2005) 229236. [14] S. Rates, G. Gosmann, Gênero Pfaffia: aspectos químicos, farmacológicos e implicações para o seu emprego terapêutico, Brazil. J. Pharm. 12 (2002) 85–93. [15] A. Panossian, G. Wikman, Effects of adaptogens on the central nervous system and the molecular mechanisms associated with their stress—protective activity, Pharmaceuticals 3 (2010) 188224. [16] F. Gonzalez, L. Di Stasi, Anti-ulcerogenic and analgesic activities of the leaves of Wilbrandia ebracteata in mice, Phytomedicine 9 (2002) 125–134. [17] H. Wagner, S. Bladt, E. Zgainsky, Plant Drug Analysis: A Thin Layer Chromatography Atlas, Springer Verlag, Berlin, 1984. [18] V. Singleton, R. Orthofer, R. Lamuela-Raventós, Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent, in: P. Lester (Ed.), Methods in Enzymology, Academic Press 1999, pp. 152–178. [19] J. Galvez, J. de la Cruz, A. Zarzuelo, F. Sanchez de la Cuesta, Flavonoid inhibition of enzymic and nonenzymic lipid peroxidation in rat liver differs from its influence on the glutathione-related enzymes, Pharmacology 51 (1995) 127–133. [20] M. Blois, Antioxidant determinations by the use of a stable free radical, Nature 181 (1958) 1199–1200. [21] W. Brand-Williams, M. Cuvelier, C. Berset, Use of a free radical method to evaluate antioxidant activity, LWT Food Sci. Technol. 28 (1995) 25–30. [22] G. Morris, P. Beck, M. Herridge, W. Depew, M. Szewczuk, J. Wallace, Hapten-induced model of chronic inflammation and ulceration in the rat colon, Gastroenterology 96 (1989) 795–803.

468

C.A.R.A. Costa et al. / International Immunopharmacology 28 (2015) 459–469

[23] C. Bell, D. Gall, J. Wallace, Disruption of colonic electrolyte transport in experimental colitis, Am. J. Physiol. 268 (1995) G622–G630. [24] L. Di Stasi, D. Camuesco, A. Nieto, W. Vilegas, A. Zarzuelo, J. Galvez, Intestinal antiinflammatory activity of paepalantine, an isocoumarin isolated from the capitula of Paepalanthus bromelioides, in the trinitrobenzenesulphonic acid model of rat colitis, Planta Med. 70 (2004) 315–320. [25] A. Stucchi, S. Shofer, S. Leeman, O. Materne, E. Beer, J. McClung, et al., NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats, Am. J. Physiol. Gastrointest. Liver Physiol. 279 (2000) G1298–G1306. [26] J. Krawisz, P. Sharon, W. Stenson, Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models, Gastroenterology 87 (1984) 1344–1350. [27] M. Anderson, Determination of glutathione and glutathione disulfide in biological samples, Methods Enzymol. 113 (1985) 548–555. [28] S. Nakai, N. Takagi, H. Miichi, S. Hayashi, N. Nishimoto, T. Takemoto, et al., Pfaffosides, nortriterpenoid saponins, from Pfaffia paniculata, Phytochemistry 23 (1984) 1703–1705. [29] N. Nishimoto, S. Nakai, N. Takagi, S. Hayashi, T. Takemoto, S. Odashima, et al., Pfaffosides and nortriterpenoid saponins from Pfaffia paniculata, Phytochemistry 23 (1984) 139–142. [30] T. Takemoto, N. Nishimoto, S. Nakai, N. Takagi, S. Hayashi, S. Odashima, et al., Pfaffic acid, a novel nortriterpene from Pfaffia paniculata Kuntze, Tetrahedron Lett. 24 (1983) 1057–1060. [31] G. Mazzanti, L. Braghiroli, Analgesic antiinflammatory action of Pfaffia paniculata (Martius) kuntze, Phytother. Res. 8 (1994) 413–416. [32] M. Oshima, Y. Gu, Pfaffia paniculata-induced changes in plasma estradiol-17beta, progesterone and testosterone levels in mice, J. Reprod. Dev. 49 (2003) 175–180. [33] R. Flores, V. Cezarotto, D. Brondani, S. Giacomelli, F. Nicoloso, Análise de B-ecdisona em plantas in vivo e in vitro de Pfaffia glomerata (Spreng.) Pedersen, através da Cromatografia em Camada Delgada, Rev. Bras. Plant. Med. 11 (2009) 368–371. [34] C. Vigo, E. Narita, M. Milaneze-gutierre, L. Marques, Caracterização farmacognóstica comparativa de Pfaffia glomerata (Spreng.) Pedersen e Hebanthe paniculata Martius– Amaranthaceae Kuntze, 2004. [35] Y. Shiobara, S. Inoue, K. Kato, Y. Nishiguchi, Y. Oishi, N. Nishimoto, et al., A nortriterpenoid, triterpenoids and ecdysteroids from Pfaffia glomerata, Phytochemistry 32 (1993) 1527–1530. [36] C. Fiocchi, Towards a ‘cure’ for IBD, Dig. Dis. (Basel, Switzerland) 30 (2012) 428–433. [37] E. Mayer, The neurobiology of stress and gastrointestinal disease, Gut 47 (2000) 861–869. [38] S. Danese, C. Fiocchi, Ulcerative colitis, N. Engl. J. Med. 365 (2011) 1713–1725. [39] B. Crohn, L. Ginzburg, G. Oppenheimer, Regional ileitis: a pathologic and clinical entity. 1932, Mt Sinai J. Med. 67 (2000) 263–268. [40] L. Peyrin-Biroulet, M. Lemann, Review article: remission rates achievable by current therapies for inflammatory bowel disease, Aliment. Pharmacol. Ther. 33 (2011) 870–879. [41] S. Danese, J. Colombel, W. Reinisch, P. Rutgeerts, Review article: infliximab for Crohn's disease treatment — shifting therapeutic strategies after 10 years of clinical experience, Aliment. Pharmacol. Ther. 33 (2011) 857–869. [42] F. Sanchez de Medina, J. Galvez, J. Romero, A. Zarzuelo, Effect of quercitrin on acute and chronic experimental colitis in the rat, J. Pharmacol. Exp. Ther. 278 (1996) 771–779. [43] M. Crespo, J. Galvez, T. Cruz, M. Ocete, A. Zarzuelo, Anti-inflammatory activity of diosmin and hesperidin in rat colitis induced by TNBS, Planta Med. 65 (1999) 651–653. [44] F. Sanchez de Medina, B. Vera, J. Galvez, A. Zarzuelo, Effect of quercitrin on the early stages of hapten induced colonic inflammation in the rat, Life Sci. 70 (2002) 3097–3108. [45] R. Gonzalez, F. Sanchez de Medina, J. Galvez, M. Rodriguez-Cabezas, J. Duarte, A. Zarzuelo, Dietary vitamin E supplementation protects the rat large intestine from experimental inflammation, Int. J. Vitam. Nutr. Res. 71 (2001) 243–250. [46] A. Luchini, D. de Oliveira, C. Pellizzon, L. Di Stasi, J. Gomes, Relationship between mast cells and the colitis with relapse induced by trinitrobenzesulphonic acid in Wistar rats, Mediat. Inflamm. 2009 (2009) 432493. [47] A. Witaicenis, A. Luchini, C. Hiruma-Lima, S. Felisbino, N. Garrido-Mesa, P. Utrilla, et al., Suppression of TNBS-induced colitis in rats by 4-methylesculetin, a natural coumarin: comparison with prednisolone and sulphasalazine, Chem. Biol. Interact. (2012) 195. [48] A. Witaicenis, L. Seito, L. Di Stasi, Intestinal anti-inflammatory activity of esculetin and 4-methylesculetin in the trinitrobenzenesulphonic acid model of rat colitis, Chem. Biol. Interact. 186 (2010) 211–218. [49] S. Cestari, J. Bastos, L. Di StasI, Intestinal anti-inflammatory activity of Baccharis dracunculifolia in the trinitrobenzenesulphonic acid model of rat colitis. Evidencebased complementary and alternative medicine, eCAM 2011 (2011) 524349. [50] F. Algieri, P. Zorrilla, A. Rodriguez-Nogales, N. Garrido-Mesa, O. Banuelos, M. Gonzalez-Tejero, et al., Intestinal anti-inflammatory activity of hydroalcoholic extracts of Phlomis purpurea L. and Phlomis lychnitis L. in the trinitrobenzenesulphonic acid model of rat colitis, J. Ethnopharmacol. 146 (2013) 750–759. [51] P. Orsi, L. Seito, L. Di Stasi, Hymenaea stigonocarpa Mart. ex Hayne: a tropical medicinal plant with intestinal anti-inflammatory activity in TNBS model of intestinal inflammation in rats, J. Ethnopharmacol. 151 (2014) 380385. [52] F. Koehn, G. Carter, The evolving role of natural products in drug discovery, Nat. Rev. Drug Discov. 4 (2005) 206–220. [53] S. Dev, Impact of natural products in modern drug development, Indian J. Exp. Biol. 48 (2010) 191–198. [54] J. Vasconcelos, Estudo taxonômico sobre Amaranthaceae no RS, Universidade Federal do Rio Grande do Sul, Brasil, 1982.

[55] J. Li, A. Jadhav, I. Khan, Triterpenoids from Brazilian ginseng, Pfaffia paniculata, Planta Med. 76 (2010) 635–639. [56] A. Panossian, G. Wikman, H. Wagner, Plant adaptogens. III. Earlier and more recent aspects and concepts on their mode of action, Phytomedicine 6 (1999) 287–300. [57] N. Rege, U. Thatte, S. Dahanukar, Adaptogenic properties of six rasayana herbs used in Ayurvedic medicine, Phytother. Res. 13 (1999) 275–291. [58] M. Davydov, A. Krikorian, Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. (Araliaceae) as an adaptogen: a closer look, J. Ethnopharmacol. 72 (2000) 345393. [59] G. Yuan, M. Wahlqvist, G. He, M. Yang, D. Li, Natural products and anti-inflammatory activity, Asia Pac. J. Clin. Nutr. 15 (2006) 143–152. [60] J. Lee, J. Hah,, H. Kim, The effect of red ginseng extract on inflammatory cytokines after chemotherapy in children, J. Ginseng Res. 36 (2012) 383–390. [61] X. Yang, T. Guo, Y. Wang, Y. Huang, X. Liu, X. Wang, et al., Ginsenoside Rd attenuates the inflammatory response via modulating p38 and JNK signaling pathways in rats with TNBS-induced relapsing colitis, Int. Immunopharmacol. 12 (2012) 408–414. [62] X. Yang, T. Guo, Y. Wang, M. Gao, H. Qin, Y. Wu, Therapeutic effect of ginsenoside Rd in rats with TNBS-induced recurrent ulcerative colitis, Arch. Pharm. Res. 35 (2012) 1231–1239. [63] K. Pinello, S. Fonseca Ede, G. Akisue, A. Silva, S. Salgado Oloris, M. Sakai, et al., Effects of Pfaffia paniculata (Brazilian ginseng) extract on macrophage activity, Life Sci. 78 (2006) 12871292. [64] T. Watanabe, M. Watanabe, Y. Watanabe, C. Hotta, Effects of oral administration of Pfaffia paniculata (Brazilian ginseng) on incidence of spontaneous leukemia in AKR/J mice, Cancer Detect. Prev. 24 (2000) 173–178. [65] T. da Silva, B. Cogliati, A. da Silva, H. Fukumasu, G. Akisue, M. Nagamine, et al., Pfaffia paniculata (Brazilian ginseng) roots decrease proliferation and increase apoptosis but do not affect cell communication in murine hepatocarcinogenesis, Exp. Toxicol. Pathol. 62 (2010) 145155. [66] P. Matsuzaki, G. Akisue, S. Salgado Oloris, S. Gorniak, Dagli M. Zaidan, Effect of Pfaffia paniculata (Brazilian ginseng) on the Ehrlich tumor in its ascitic form, Life Sci. 74 (2003) 573–579. [67] D. Jacobo-Velázquez, L. Cisneros-Zevallos, Correlations of antioxidant activity against phenolic content revisited: a new approach in data analysis for food and medicinal plants, J. Food Sci. 74 (2009) 13. [68] G. Chiva-Blanch, F. Visioli, Polyphenols and health: moving beyond antioxidants, J. Berry Res. 2 (2012) 63–71. [69] S. Fiorucci, J. Wallace, A. Mencarelli, E. Distrutti, G. Rizzo, S. Farneti, et al., A betaoxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 15736–15741. [70] J. Hoffmann, K. Peters, S. Henschke, B. Herrmann, K. Pfister, J. Westermann, et al., Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after gammadelta T cell depletion and no effect of alphabeta T cell depletion, Gut 48 (2001) 489–495. [71] M. Nakamuta, M. Ohashi, Y. Tanabe, K. Hiroshige, H. Nawata, High plasma concentration of myeloperoxidase in cirrhosis: a possible marker of hypersplenism, Hepatology 18 (1993) 1377–1383. [72] G. Oberg, G. Lindmark, L. Moberg, P. Venge, The peroxidase activity and cellular content of granule proteins in PMN during pregnancy, Br. J. Haematol. 55 (1983) 701–708. [73] F. Sanchez de Medina, O. Martinez-Augustin, R. Gonzalez, I. Ballester, A. Nieto, J. Galvez, et al., Induction of alkaline phosphatase in the inflamed intestine: a novel pharmacological target for inflammatory bowel disease, Biochem. Pharmacol. 68 (2004) 2317–2326. [74] S. Sanchez-Fidalgo, I. Villegas, A. Martin, M. Sanchez-Hidalgo, C. Alarcon de la Lastra, PARP inhibition reduces acute colonic inflammation in rats, Eur. J. Pharmacol. 563 (2007) 216–223. [75] L. Wang, Y. Zhang, Z. Wang, S. Li, G. Min, L. Wang, et al., Inhibitory effect of ginsenoside-Rd on Carrageenan-induced inflammation in rats, Can. J. Physiol. Pharmacol. 90 (2012) 229–236. [76] L. Christensen, Ginsenosides chemistry, biosynthesis, analysis, and potential health effects, Adv. Food Nutr. Res. 55 (2009) 1–99. [77] J. Park, J. Cho, Anti-inflammatory effects of ginsenosides from Panax ginseng and their structural analogs, Afr. J. Biotechnol. (2009) 3682–3690. [78] M. Circu, T. Aw, Glutathione and modulation of cell apoptosis, Biochim. Biophys. Acta 2012 (1823) 1767–1777. [79] S. Karp, T. Koch, Oxidative stress and antioxidants in inflammatory bowel disease, Dis. Mon. 52 (2006) 199–207. [80] A. White, V. Thannickal, B. Fanburg, Glutathione deficiency in human disease, J. Nutr. Biochem. 5 (1994) 218–226. [81] E. Nagel, M. Bartels, R. Pichlmayr, Scanning electron-microscopic lesions in Crohn's disease: relevance for the interpretation of postoperative recurrence, Gastroenterology 108 (1995) 376–382. [82] S. Hur, S. Kang, H. Jung, S. Kim, H. Jeon, I. Kim, et al., Review of natural products actions on cytokines in inflammatory bowel disease, Nutr. Res. 32 (2012) 801–816. [83] J. Barahona-Garrido, J. Yamamoto-Furusho, New treatment options in the management of IBD — focus on colony stimulating factors, Biologics 2 (2008) 501–504. [84] S. Kim, S. Shim, D. Choi, J. Kim, Y. Kwon, J. Kwon, Modulation of LPS-stimulated astroglial activation by ginseng total saponins, J. Ginseng Res. 35 (2011) 8085. [85] J. Kim, H. Ahn, B. Han, S. Lee, Y. Cho, C. Kim, et al., Korean red ginseng extracts inhibit NLRP3 and AIM2 inflammasome activation, Immunol. Lett. 158 (2014) 143150. [86] S. Fichtner-Feigl, W. Strober, E. Geissler, H. Schlitt, Cytokines mediating the induction of chronic colitis and colitis-associated fibrosis, Mucosal. Immunol. 1 (Suppl. 1) (2008) 7.

C.A.R.A. Costa et al. / International Immunopharmacology 28 (2015) 459–469 [87] Y. Jin, Y. Lin, L. Lin, C. Zheng, IL-17/IFN-γ interactions regulate intestinal inflammation in TNBS-induced acute colitis, J. Interf. Cytokine Res. off. J. Int. Soc. Interf Cytokine Res. 32 (2012) 548–556. [88] D. Luger, P. Silver, J. Tang, D. Cua, Z. Chen, Y. Iwakura, et al., Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category, The Journal of Experimental Medicine. 205 (2008) 799–810. [89] G. Marlow, D. van Gent, L. Ferguson, Why interleukin-10 supplementation does not work in Crohn's disease patients, World J. Gastroenterol. 19 (2013) 3931–3941.

469

[90] K. Mitsuyama, N. Tomiyasu, K. Takaki, J. Masuda, H. Yamasaki, K. Kuwaki, et al., Interleukin-10 in the pathophysiology of inflammatory bowel disease: increased serum concentrations during the recovery phase, Mediat. Inflamm. 2006 (2006) 17. [91] S. Vermeire, G. Van Assche, P. Rutgeerts, Laboratory markers in IBD: useful, magic, or unnecessary toys? Gut 55 (2006) 426–431. [92] S. Sparg, M. Light, J. van Staden, Biological activities and distribution of plant saponins, J. Ethnopharmacol. (2004) 94.