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Anti-inflammatory activity and phenolic composition of prickly pear (Opuntia ficus-indica) flowers
Industrial Crops & Products 112 (2018) 313–319
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Anti-inflammatory activity and phenolic composition of prickly pear (Opuntia ficus-indica) flowers
T
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Imene Ammara, , Maryem Ben Salemb, Bahira Harrabib, Massara Mzidc, Sana Bardaab, Zouheir Sahnounb, Hamadi Attiaa, Monia Ennouria,d a
Valuation, Analysis and Food Safety Laboratory, National School of Engineers of Sfax, Sfax University, 3038, Sfax, Tunisia Laboratory of Pharmacology, Faculty of Medicine of Sfax, Sfax University, Road Majida Boulila, 3028, Sfax, Tunisia c Laboratory of Histology Embryology, Faculty of Medicine of Sfax, Sfax University, Road Majida Boulila, 3028, Sfax, Tunisia d Higher Institute of Applied Sciences & Technology of Mahdia, Monastir University, Sidi Messaoud, 5111, Mahdia, Tunisia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Opuntia ficus-indica Flowers Phenolic acids Flavonols Anti-inflammatory
The in vivo anti-inflammatory activity of methanolic extract of Prickly pear Opuntia ficus-indica flowers was studied using carrageenan induced rat paw edema model. In order to characterize the extract, the qualitative and quantitative analysis of phenolic compounds of the methanol extract of Opuntia ficus-indica flowers (OFIFE) were performed using LC–MS/MS technique. The main components of OFIFE were phenolic acids and flavonoids. It was observed that the OFIFE inhibited significantly the inflammation as confirmed by the hematological and the histological analysis. The anti-inflammatory effect of OFIFE was associated with the reduction of malonaldialdehyde level (MDA as an index of lipid peroxidation) and an increase in activities of catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH). Overall, the results showed that OFIFE might serve as a natural source for the treatment of inflammation.
1. Introduction The plant genus Opuntia ficus-indica has proved to be an important source of therapeutic agents, because of the diversity of pharmacological properties and chemical structures. Opuntia ficus-indica has been used in the traditional medicine of diverse countries for a long time (Park et al., 2001). The O. ficus-indica flowers exert a wide range of pharmacological activities including antioxidant, anti-microbial (Ammar et al., 2012; Ammar et al., 2015), antiulcerogenic and wound healing activities (Alimi et al., 2011; Ammar et al., 2015) as well as anti-inflammatory effect (Ahmed et al., 2005; Benayad et al., 2014). Phytochemical studies have demonstrated the presence of several classes of chemical constituents in the O. ficus-indica flowers. These included isorhamnetin glycosides as the major phenolic compounds, followed by quercetin and kaempferol glycosides, which are identified from methanolic and aqueous extracts of O. ficus indica flowers grown in Italy and Tunisia (Ammar et al., 2015; De Leo et al., 2010; Yeddes et al., 2014). Opuntia ficus-indica has been traditionally used to reduce inflammation and previous studies demonstrated the anti-inflammatory potential of this plant. Indeed, earlier finding showed that the alcohol
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extracts of different organs of Opuntia dillenii (Ker-Gawl) Haw, as well as their subfractions (Ahmed et al., 2005) and the fruit aqueous extract (Loro et al., 1999) attenuated, efficiently, the in vivo carrageenan-induced paw edema. More recently, the anti-inflammatory effects of Opuntia cladodes methanol extract was also studied by Siddiqui et al. (2016). With regard to literature, few studies have been focused on flowers compared to other parts (cladodes, fruits and seeds). This study is the first report on the anti-inflammatory effect of the methanolic extract of Opuntia ficus-indica flowers using the carrageenan-induced paw edema test. The study also explores the qualitative and quantitative phenolic composition of the methanolic extract. 2. Materials and methods 2.1. Chemicals All solvents used for extraction and analysis in this study were of analytical grade. Lamda CARR and INDO were purchased from sigma chemical company (St. Louis, MO, USA). The phenolic standards used for quantification purposes, were supplied by Sigma-Aldrich (Chemie Gmbh, Steinhein, Germany). HPLC grade solvent (methanol and acetic
Corresponding author. E-mail address: [email protected] (I. Ammar).
https://doi.org/10.1016/j.indcrop.2017.12.028 Received 21 July 2017; Received in revised form 28 October 2017; Accepted 11 December 2017 Available online 15 December 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2.6. Carrageenan-induced rat paw edema
acid, formic acid, and acetonitrile) were purchased from Fisher Scientific (U.K.). Double distilled water was used in the LC mobile phase
In this assay, edema was induced by subcutaneous injection of 0.1 ml of 1% freshly prepared solution of carrageenan in saline solution (Ravi et al., 2009). The rats were divided into four groups of six rats which were treated by intraperitoneal injection, one hour before administration of carrageenan. The group (I) was the control group and was treated with 10 ml/kg of isotonic saline solution (9‰) (negative control group). The group (II) was inflamed by carrageenan injection and did undergo any treatment (CARR). The group (III) was used as reference group, that were treated with indomethacin (reference drug, 10 mg/kg) (CARR + INDO) and the group (IV) was treated with methanolic extract of O. ficus-indica flowers (CARR + OFIFE) at the dose of 400 mg/kg. The volumes of the paw edema were measured before and after carrageenan injection using a digital caliper at t = 0, 1, 2, 3, 4 and 5 h. The initial volumes (V0) obtained for each group before any treatment and the subsequent values recorded for each group (Vt) gave the actual edema value, using the following ratio: Percentage vt − v0 edema = v0 × 100. The percentages of inhibition were obtained for each group and at each record, using the following ratio:
2.2. Plant material Opuntia ficus-indica (L.) Miller flowers were collected during the post-flowering stage of the plant in June 2014 from wild populations growing in Sfax, Tunisia (latitude 34 46′29″N, longitude 10 39′73″E; elevation: 41 m), a region which is characterized by a semi arid climate and a mean rainfall of 200 mm/year. Plant authenticity was confirmed by the Alimentary Analysis Laboratory of the National Engineering School of Sfax, Tunisia. Flowers were collected in one batch within the same sampling area and were stored at −20 °C until subsequent analysis. 2.3. Preparation of the plant extracts The flower samples (50 g) were minced and extracted by maceration using 500 ml of methanol. The maceration was carried out on a shaker, at room temperature, 150 rpm and in the dark for 24 h. The extract was filtered through filter paper and centrifuged at 4000 × g for 10 min to remove any floating matters. The remaining residue was then re-extracted under the same conditions. The combined extracts were evaporated at 35 °C (rotary evaporator Büchi R-210, Flawil, Switzerland) to remove methanol. The percentage yield was 14.8% (w/w).
Percentage inhibition =
(vt−v0)control − (vt−v0)treated × 100 (vt−v0)control
2.7. Blood sample collection 2.4. Analyses of phenolic compounds by LC–MS-ESI−MS Five hours after carrageenan induction, rats were anesthetized by ketamine (50 mg/kg body weight), intramuscularly injected, along with midazolam (5 mg/kg body weight). The blood samples were quickly collected through cardiac puncture using 2 ml hypodermic syringes into heparinized bottles and immediately centrifuged at 3000 rpm for 15 min to obtain plasma. They were kept at −20 °C until biochemical analysis.
The flowers extract was re-dissolved in methanol at 5 mg/ml and filtered through a 0.45 μm membrane filter (Millipore, Bedford, MA) for injection. The sample analysis was conducted by an HPLC system (Thermo Fisher Scientific, San Jose, CA) coupled to a quadrupole mass spectrometer equipped with electrospray source (ESI) (Waters, Manchester, UK) and Diode Array Detector (DAD). The separation of phenolic compounds was performed on a Phenomenex Luna C18 (2) column (length 150 mm, internal diameter 3 mm, film thickness 3 μm) operating at 25 °C and a flow rate of 0.4 ml/min and 5 μl of sample was injected. The mobile phase used consisted of 0.1% (v/v) acetonitrile–water (solvent A) and 0.1% (v/v) acetic acid–water (solvent B). The elution gradient program with a ratio of A to B was as follows: from 0 to 12 min (15:85, v/v), from 12 to 14 min (40:60, v/v), from 14 to 18 min (60:40, v/v), from 18 to 20 min (80:20, v/v), from 20 to 26 min (90:10, v/v) and from 26 to 40 min (100:0, v/v). Detection was performed on-line in the UV–vis spectra in the range of 220–380 nm. Mass spectrometry data were acquired in the negative ionisation mode. The phenolic compounds were identified and quantified by interpreting their elution order in the HPLC chromatograms, their UV–vis spectra, their mass spectra (m/z, fragmentation patters in MS/MS) and chromatographically compared with corresponding standards. The standards including protocatechuic acid, ferulic acid, quercetin 3-Orutinoside and apegenin were purchased from Merck (Darmstadt, Germany). The calibration curves concentrations are between 0.125 and 50 mg/ml.
2.8. Testing the blood count Complete blood counts KX 21 was used to determine the following hematological parameters: leukocytes, platelets and lymphocytes on an automatic biochemistry analyzer (Vitalab Flexor-E, USA) at the biochemical laboratory of Hedi Cheker Hospital of Sfax. 2.9. Determination of oxidant stress parameters Total superoxide dismutase activity (SOD) was estimated according to Beauchamp and Fridovich (1971). The developed blue color in the reaction was measured at 560 nm. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of Nitroblue tetrazolium (NBT) by 50%, and the activity was expressed as units per milligram of protein. Total catalase activity (CAT) was assayed by the decomposition of hydrogen peroxide according to the method of Aebi (1984). In brief, the reduction of 10 mM H2O2 in 20 mM of phosphate buffer (pH 7) was monitored by measuring the absorbance at 240 nm. The activity was calculated in terms of μmol H2O2 consumed/min/μg of protein. Glutathione level (GSH) in tissue was determined by the method of Ellman (1959) modified by Jollow et al. (1974) based on the development of a yellow color when 5,5-dithiobis-2 nitro benzoic acid (DTNB) was added to compounds containing sulfhydryl groups. The absorbance was measured at 412 nm after 10 min. Glutathione level was expressed as nanomoles/mg of protein. Malondialdehyde (MDA) level in skin was determined according to the method of Draper and Hadley (1990). An amount of 0.5 ml of organ extract supernatant was mixed with 1 ml of trichloroacetic acid solution (20%) and centrifuged at 2500g for 10 min. A 1-ml solution containing
2.5. Animals Male Wistar rats, in the age group of 2–3 months and weighting 186 ± 7 g, were purchased from the local Central Pharmacy, Tunisia and used in the experimental assays of the present study. The rats were housed in hygienic and well ventilated compartments and maintained under standard laboratory conditions (12 h light/dark cycles at 22 ± 2 °C) as approved by the Experimentation and Ethics Committee of the College of Medicine, University of Lagos (CM/COM/08). 314
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Compounds (1, 2, 5–9, 13) were identified as hydroxycinnamic acids derivatives based on retention time order, maximum in UV and mass spectra. Three of them being quinic acid (compound 1) and quinic acid derivatives (Compounds 5, 13), whose identities were assigned based on their MS spectra and fragmentation patterns. Compound 1 (Rt = 3.25 min) showed a [M−H]− ion at m/z 191 and its MS2 fragmentation gave a fragment ion at m/z 127 characteristic of quinic acid. Compounds 5 and 13 released characteristic MS2 fragment ions at m/z 191 (deprotonated quinic acid), 179 (deprotonated caffeic acid), which together with their pseudo molecular [M−H]− ions at m/z 355 and 515, respectively allowed their identification as quinic acid derivatives containing one or two caffeic acid moieties, respectively. Compound 5 was positively identified as 4-O-caffeoylquinic acid according to the fragmentation pattern described by Clifford et al. (2003, 2005). Compound 13 was identified as di-O-caffeoyquinic acid. Indeed, MS2 base peak was at m/z 353, produced by the loss of one of the caffeoyl moieties [M−H−caffeoyl]−, and subsequent fragmentation of this ion yielded the same fragments as 5-caffeoylquinic acid at m/z 191, 179 and 135. Concerning compound 13, it was assigned as 4,5-O-dicaffeoylquinic acid, since its fragmentation was identical to those previously reported by Clifford et al. (2005) and Guimarães et al. (2013). Compound 2 and 6 exhibited [M−H]− ion at m/z 269 and 179. Thus they were tentatively characterized as gallic acid and caffeic acid, respectively. Coumaric acid was identified based on the molecular ion at m/z 163 [M−H]− and the typical fragment of coumaric acid detected at m/z 119. This compound was identified as p-coumaric acid since the fragmentation pattern were consistent with previous report (Belguith-Hadriche et al., 2013; Ristivojevi et al., 2016). Compound 3 was identified as protocatechuic acid by comparing its UV spectrum and retention time with those of an external standard. Compound 4 displayed maximum UV absorption at 320 and exhibited [M−H]− ions at m/z 353. According to the above information, this compound was identified to chlorogenic acid. Compound 8 with MS fragment at m/z 193 ([ferulic acid −H]−) and MS2 fragment at m/z 134 was identified as ferulic acid which was identical with the external standard. In addition to the phenolic acid derivatives described above, there were also identified flavonoid compounds. The flavonoid profile of the OFIFE consisted of quercetin glycosides and its derivatives. Indeed,
0.67% thiobarbituric acid (TBA) and 0.5 ml of supernatant were incubated for 15 min at 80 °C and cooled. Absorbance of TBA–MDA complex was determined at 530 nm using a spectrophotometer (Jenway UV-6305; Essex, England). Lipid peroxidation was expressed as nanomoles of MDA/mg protein. 2.10. Histological examination For histopathological examination, biopsies of paws (n = 3) from control, CARR, CARR + INDO and CARR + OFIFE rats were removed and immediately fixed in 10% neutral buffered formalin solution, embedded in paraffin, sectioned into 5 μm-thick sections using a rotary microtome (Leitz, Germany) and stained with hematoxylin–eosine for evaluation under light microscopy. 2.11. Statistics All the data were expressed as mean ± standard deviation (SD) of six animals in each group. Carrageenan-induced inflammation test data were assessed by one-way analysis of variance (ANOVA), and the significant differences were examined by Duncan’s multiple range post hoc-test using Statistical Package for the Social Sciences (SPSS) software (version 16, SPSS, Inc, Chicago, IL, USA). Differences between groups were considered significant at a level of p < 0.05 and p < 0.01. The student’s t-test using EXCEL (Microsoft Corporation, USA) was used to establish the significance of difference from control group or carrageenan group at the significance level of p < 0.001. 3. Results 3.1. Phenolic compounds analysis of the OFIFE Phenolic compounds were identified according to their retention time, the wavelengths of maximum absorbance (UV λmax at 250–280, 320–360 nm), the deprotonated molecules ([M−H]−) in negative ionisation mode and the characteristic product ions in comparison with those of authentic standards and literature data. Table 1 summarizes the phenolic compounds identified in the OFIFE. The detected phenolic compounds were typical of phenolics acids and flavonoids classes.
Table 1 LC–ESI–MS characteristics of the identified phenolic compounds in the methanol extract of the O. ficus-indica flowers. Peak number
Phenolic acids 1 2 3 4 5 6 7 8 9 Flavonoids 10 11 12 13 14 15 16 17 18 Phenolic acids Flavonoids Total phenolic compounds
Rt (min)
λmax (nm)
Molecular ion [M−H]− (m/ z)
MS/MS fragments (m/ z)
Tentative identification
Quantification (mg/100 g)
3.25 6.73 10.45 11.97 13.28 12.98 16.25 17.55 20.49
320 270 278 320 328 324 300 328 254–328
191 269 153 353 355 179 163 193 359
174, 134, 129, 127 169, 125 109 191, 179. 191, 179, 173. 135 163, 119 134 161,197
Quinic acid Gallic acid Protocatechuic acid Chlorogenic acid 4-O-caffeoylquinic acid Caffeic acid p-coumaric acid Trans ferulic acid Rosmarinic acid
131.85 ± 5.8 1.42 ± 0.002 4.28 ± 0.0085 1.62 ± 0.001 3.50 ± 0.0002 1.61 ± 0.001 11.83 ± 0.005 16.49 ± 1.2 1.96 ± 0.002
19.04 20.98 21.08
354 256–354 255–354
593 609 463
285 611, 465, 303 300
6.46 ± 0.1 390.27 ± 10.2 325.31 ± 50.2
22.25 22.6 24.71 26.83 30.49 36.56
– – 354 350–372 250–332 260–350
515 447 623 477 269 417
353, 191, 179, 173, 135 300 315 315 227, 225, 149, 119 285
Kaempferol-3-O-rutinoside Rutin (quercetin-3-O- rutinoside) Hyperoside (quercetin-3-Ogalactoside) 4.5-di-O-caffeoyl quinic acid Quercetin-3-O-rhamonoside Isorhamnetin-3-O-rutinoside Isorhamnetin 3-O-glucoside Apegenin Kaempferol 3-O-arabinoside
315
3.05 ± 0.0001 134.12 ± 0.5 1.05 ± 0.001 26.83 ± 0.001 0.15 ± 0.00 2.15 ± 0.000 177.62 886.34 1063.96
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The high total leukocytes counting confirmed the inflammatory response after CARR injection. The administration of OFIFE before the injection of CARR caused a significant decrease (p < 0.01) in the number of total leukocytes (49.4%) and lymphocytes (27.4%) when compared to the CARR group. Indomethacin treated group showed 52.27 and 49.17% of leukocytes and lymphocytes reduction, respectively. Thus, the OFFIE could reduce actively the leukocytes and lymphocytes. The number of platelets remained without significant changes.
compounds 11, 12, and 14 were identified as quercetin glycosides. The [M−H]− ion at m/z 609 of compound 11 (Table 1) with significant fragments at m/z 611, m/z 465 and m/z 303 was identified as quercetin 3-O-rutinoside which was identical with the authentic standard. While the [M−H]− ion m/z 463 (UV λmax at 255 nm and 354 nm) of compound 12 was identified as quercetin 3-O-galactoside. Compound 14 was identified as quercetin-3-O-rhamonoside. Another group of detected flavonols was isorhamnetin derivatives according to their UV–vis and mass spectra (MS2 product ion at m/z 315); compounds 15 and 16 release ([M−H]− at m/z 623 and m/z 477, respectively) and one MS2 fragment ion at m/z 315, were tentatively identified as isorhamnetin-3O-rutinoside and isorhamnetin-3-O-glucoside, respectively as previously reported in the studies of De Leo et al. (2010) and Chahdoura et al. (2014). Compound 17 (Rt = 30.49 min) showed a [M−H]− ion at m/z 269. Comparison of the main MS2 fragment ions such as those at m/z 227,149, UV spectrum (250 and 332 nm) and the retention time to those obtained for a standard allowed their identification as apigenin. The total amount of phenolic acids and flavonoids of OFIFE were respectively 177.61 mg and 886.34 mg/100 g extract. Flavonoids, especially quercetin derivatives, were the major phenolic compounds present in these samples. The predominant phenolic acid of the extract was quinic acid (12.4% of total identified phenolics), while quercetin-3-O-rutinoside (36.68% of total identified phenolics) was the main identified flavonoid. It was followed by quercetin-3-O-galactoside (30.57% of total identified phenolics), quercetin-3-O-rhamonoside (12.6% of total identified phenolics) and isorhamnetin 3-O-glucoside with 2.5% of total identified phenolics.
3.4. Effect of OFIFE on oxidative stress parameters The activities of CAT, SOD and GSH as well as the MDA level, in paw edema, were evaluated (Table 4). Results revealed that the CARR group of rats has the lowest activities of CAT, SOD and GSH, while they showed the highest MDA level, proving the presence of an oxidative stress state in rats. In fact, the induction of inflammation with CARR decrease the activities of SOD, CAT, and GSH in edema by 47.7, 74.0 and 64.41%, respectively, in comparison to the control group (p < 0.001). However, the OFIFE treatment increase the activities of SOD to 51.19%, CAT to 226.3% and GSH to 328.75%, respectively, compared to that observed in the CARR group. Indomethacin treatment also exhibited increase effects in the activities of SOD (46.7%), CAT (171.11%) and GSH (359.49%) in comparison to the CARR group. These data imply that treatment of induced edema with OFIFE improved, significantly these activities compared to CARR group. The inflammation induced a highly significant increase in the level of MDA, at the skin edema of normal control group (34.18%). The treated groups by OFIFE and INDO showed significant reduction in the tissue levels of MDA (33.43 and 37.16%, respectively) when compared to the CARR group. Pretreatment with OFIFE prevented the increase in level of MDA, while it restored significantly (P < 0.01) the CAT, SOD, and GSH activities in induced edema compared with the CARR group (Table 4).
3.2. Effect of OFIFE on the carrageenan (CARR) induced paw edema The anti-inflammatory activity of OFIFE was investigated in vivo by applying a carrageenan-induced paw edema model in rats. As the results showed in Fig. 1, the CARR injection causes edema development which increase progressively with time over the 5 h of treatment. After 1 h of the induction, the edema development began and the edema percentage increase significantly to reach 23.4%, 22.4% and 19.7% in the CARR group, OFIFE and INDO treated group, respectively. The edema development of OFIFE and INDO treated group decreased significantly in comparison with the CARR group. Thus, the percentages of edema inhibition calculated for each group for OFFIE and INDO are reported in Table 2. The highest significant inhibitory activity (52.31% of inhibition) is shown in the OFIFE group over a period of 4 h after induction of inflammation in comparison with INDO group (54.43%).
3.5. Histological analysis The macroscopic results of the paw edema were confirmed through the histological examination. The histological analysis of the edema skin (Fig. 2), revealed that the control group showed normal histological section (Fig. 2a), however, the most severe inflammatory changes is showed in the CARR group as reflected by vasodilatation, neutrophils accumulation and connective tissue disorganization. Arrows in Fig. 2(b) indicate prominent areas of edema with massive signs of inflammation (inflammatory cells, particularly neutrophils) and edema disruption of tissue structure. The treatment with OFIFE proved to be able to reduce the sign of acute inflammation with a decrease in the inflammatory cells number and improvements in edema tissue (Fig. 2d). Indomethacin treated
3.3. Effect of OFIFE on hematological parameters of blood of rats The anti-inflammatory effect of the OFIFE was evaluated by studying changes in leukocytes, lymphocytes and platelets (Table 3).
Fig. 1. Percentage inhibition of carrageenan (CARR) induced-paw edema in rats treated with O. ficus indica flowers methanol extract (OFIFE) and indomethacin (INDO). Each value represents as mean ± SD (n = 6). **p < 0.001 as compared with the CARR group.
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Table 2 Percentage inhibition of O. ficus indica flowers extract and indomethacin on carrageenan-induced paw edema in rat. Treatment
CARR + OFIFE CARR + INDO
% inhibition of inflammation 1h
2h
3h
4h
4.22 ± 0.7 a 16.58 ± 1.5 b
38.22 ± 2.1 a 40.04 ± 1.63 a
45.19 ± 1.4 a 50.25 ± 2.36 b
42.31 ± 0.75 44.43 ± 2.14
5h a a
35.29 ± 0.45 a 32.1 ± 1.79 b
Values are means ± SD. Statistical analysis was done by a one-way ANOVA with Duncan’s multiple comparisons post-test (p < 0.05).
dillenii revealed 48.9% inhibition, 4 h after carrageenan induction of rat paw edema (200 mg/kg) (Ahmed et al., 2005). In order to confirm the results obtained from the edema size inhibition, the test blood cells and plateltes count of the induced paw edema were performed. The treatment with OFIFE reduced significantly the inflammatory cells number (leukocytes and lymphocytes) (p < 0.01) in comparison with the CARR group. These hematological parameters confirmed that the OFIFE has an anti-inflammatory effect on edema tissue following CARR injection, with a significant reduction (p < 0.01) of total leukocytes (49.4%). This inhibitory effect is higher than that reported for the cladodes extract of Opuntia ficus-indica (5 mg/kg) with a 29.4% of leukocytes reduction (Antunes-Ricardo et al., 2015). Another important component responsible of inflammation is oxidative stress, which derived when reactive species (ROS) are overproduced, exceeding the capacity of the endogenous antioxidant system (Nathan, 2006). The SOD, CAT, and GSH provide the first line of cellular defense against toxic free radicals. The primary role of GSH is to detoxify the damaging radicals either by directly scavenging them or by acting as a co substrate in the glutathione peroxidase (GPx)-catalyzed reduction of hydrogen peroxide and lipid peroxides. CAT is known to detoxify the H2O2 generated into water and oxygen. While SOD activity protects cells and the extracellular matrix from the harmful effects of superoxide anion (O2 %-) and its derivatives such as hydroxyl radical (% OH) (Afonso et al., 2007). In the present study, it was shown that the induction of inflammation significantly decrease (p < 0.001) the activities of SOD, CAT, GSH and increase the production of MDA in comparison with the normal control group (Table 4). In contrast pre-treatment of rats with OFIFE restored the normal activities of SOD, CAT and GSH and decrease the MDA level. This implied that OFIFE reduced the inflammatory oxidation by promoting the activities of antioxidant system in edema paw. There are various studies suggesting that polyphenolic compounds and mainly flavonoids play an important role, as protective factors against free radicals and reactive oxygen species compounds, that which results natural inhibitors of inflammation (Conti et al., 2013; Moon et al., 2006). According to previous reports, O. ficus indica flowers have been considered to be an excellent source of polyphenolic compounds and natural antioxidants (Alimi et al., 2011; Ammar et al., 2015). Consequently, the anti-inflammatory effect showed in this study can be attributed to the synergistic effect of phenolic compounds present in the OFIFE to act as free radical scavenging generated by the inflammation. Furthermore, the phenolic compounds determined by
group showed a marked effect in reducing the inflammation features (Fig. 2c). The histopathological analysis of the OFIFE was close to that of INDO group. 4. Discussion The obtained results in the phenolic analysis are in agreement with previous studies reporting the presence of phenolic acids and flavonoids in methanol extract of O. ficus-indica Thornless form flowers from Tunisia (Yeddes et al., 2014), as well as in methanol/water extract of O.microdasys flowers (Chahdoura et al., 2014). The quinic acid is the major phenolic acid identified in this study which accounted for 12.4% of total identified phenolics. This compound is also present in the Moroccan O. ficus-indica flowers at a lower amount (3.65% of total identified phenolics) (Benayad et al., 2014). The OFIFE is dominated by the flavonol glycoside and the flavonol 3-O-glycosides (quercetin, kaempferol and isorhamnetin) were the most characteristic flavonoids. These compounds have been previously identified in closely related to Opuntia flowers (De Leo et al., 2010). The identified compounds in OFIFE have been reported to exhibit a wide range of therapeutic properties including anti-bacterial, anti-carcinogenic, and anti-inflammatory effects (Ou and Kwok, 2004). The evaluation of the anti-inflammatory activity of OFIFE in this study was carried out using the CARR induced edema test. It has been reported that this inflammation model provokes a local inflammatory reaction and involves several mediators that is a suitable to evaluate the anti-inflammatory effects of natural products (Woldesellassie et al., 2011). This edema model involves the synthesis and the release of inflammatory mediators at the site of injury like histamine and serotonin for the initial phase of inflammation (during the first 2 h after CARR injection) and prostaglandins for the second phase of inflammation (3–4 h after CARR injection) (Orhan et al., 2007). The reduction of edema size is a good indicator to determine the protective action of anti-inflammatory agents. According to Fig. 1, the OFIFE (400 mg/kg) inhibited significantly the development of edema at 3 h after CARR induction (p < 0.001). Besides, there is no significant difference (p < 0.001) existed between the OFIFE and INDO groups, at 2–5 h after CARR treatment, which suggests that OFIFE has potential antiinflammatory effect. The OFIFE showed also, significant inhibition of rat paw edema (p < 0.05) (Table 2). This result is in parallel with earlier finding in which alcohol extract of fresh flowers of Opuntia
Table 3 Effect of treatments (carrageenan, indomethacin and OFIFE) on hematological parameters of the rat’s blood. Parameter
Leukocytes (*103/mm3) Lymphocytes (%) Platelets (*103/mm3)
Treatments Control
CARR + INDO
CARR + OFIFE
CARR
8.05 ± 0.05 32.1 ± 0.9 500 ± 25.3
8.2 ± 0.03b (% = 52.27) 21.5 ± 0.05a,b (% = 49.17) 610 ± 12.8
8.7 ± 0.02b (% = 49.4) 30.7 ± 0.3b (% = 27.4) 622 ± 8.9
17.18 ± 2.1a 42.3 ± 1.5a 685 ± 20.3
Each value represents as mean ± SD (n = 6). a, b, c Different letters indicate significant differences between treatments. CARR. carrageenan; OFIFE. methanol extract of Opuntia-ficus indica and INDO. indomethacin. a p < 0.001 significantly different from the control group. b p < 0.01 significantly different from the carrageenan group (one way ANOVA).
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Table 4 Effects of methanol extract of Opuntia-ficus indica (OFIFE) and indomethacin (INDO) on the catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) activities in paw edema. Groups
SOD (U/mg protein)
CAT (μmol H2O2/min/μg de Prot)
GSH (nmol/mg protein)
MDA (nmol/mg protein)
Normal control CARR group (Untreated) CARR + INDO CARR + OFIFE
15.98 13.01 23.45 24.16
50.58 13.15 35.65 42.90
43 ± 1.2 15.3 ± 1.1a(64.41%) 70.3 ± 3.1b (359.49%) 65.6 ± 4.9b (328.75%)
23.4 ± 0.85 31.4 ± 1.52a (34.18%) 20.9 ± 1.0b (33.43%) 19.73 ± 1.8b (37.16%)
± ± ± ±
0.36 0.9a (47.7%) 1.2b(46.7%) 1.78b (51.19%)
± ± ± ±
2.3 0.6a (74.0%) 1.3b (171.11%) 2.38b (226.23%)
Each value represents as mean ± SD (n = 6). Carr. carrageenan. a p < 0.001 as compared with the control group. b p < 0.01 as compared with the carrageenan group (oneway ANOVA).
2012; Trendafilova et al., 2011). Lipid peroxidation is a process in which free radicals damage cell structures and tissues. MDA is the major final product of lipid peroxidation that accumulates at the inflammatory site (Paul et al., 2012). The decrease of MDA level in OFIFE treated group also suggested that this extract might neutralize the ROS mediated lipid peroxidation which leads to a marked reduction of lipids damage in cell membranes of tissues. In consistency with our results, the extract of the flowers of O. ficus-indica have shown to attenuate the lipid peroxidation products of inflammation processes that are related to nitric oxide (NO) production (Benayad et al., 2014). Considering the obtained results, it seems reasonable that the benefic action of the OFIFE against inflammation is more likely
LC–MS/MS analysis of the OFIFE showed the presence of quercetin, as the predominant compound, isorhamnetin and kaempferol besides the presence of quinic acid as the major compound of phenolic acids. It was previously reported that quercetin proved a potential radical-scavenging activity, based on its ability to donate electrons from their hydroxide group (Inal and Kahraman, 2000) and it was also demonstrated that quercetin has the ability of minimizing the oxidative damage by modulating expression of antioxidant activities (Zerin et al., 2013). The promising inflammatory activity of kaempferol and isorhamnetin glycoside has been also reported (Shaker et al., 2010). Besides, it was shown that 1,5 dicaffeoyl quinic acid, dicaffeoyl derivative and quercetin-3-O-galactoside have strong free radical scavenging capacities, which could effectively reduce the oxidative stress (Beekmann et al.,
Fig. 2. Representative photomicrographs of histological sections of edema paws of the control showed normal tissue (a), the carrageenan reated group (b), carrageenan + OFIF extract (c) and carrageenan + indomethacin treated group (d). Tissues were stained with hematoxylin–eosin; (a), (b) visualized at 200× magnifications and (c), (d) visualized at 400× magnifications. massive signs of inflammation.
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attributed to the occurrence of flavonoids compounds. The phenolic compounds and mainly flavonoids apart from displaying a significant antioxidant effect, also contribute to ameliorate the inflammatory processes. Hence, previous studies have shown that anti-inflammatory can be a result of the high polyphenol content of plants especially phenolics and flavonoids (Lee et al., 2006; Orhan et al., 2007). For example, Opuntia dillenii flowers extract showed high levels of flavonoids which determined the reported anti-inflammatory activity (Ahmed et al., 2005).
2012. A state-of-the-art overview of the effect of metabolic conjugation on the biological activity of flavonoids. Food Funct. 3, 1008–1018. Belguith-Hadriche, O., Bouaziz, M., Jamoussi, K., Simmonds, M.S.J., El Feki, A., MakniAyedi, F., 2013. Comparative study on hypocholesterolemic and antioxidant activities of various extracts of fenugreek seeds. Food Chem. 138 (2–3), 1448–1453. Benayad, Z., Martinez-villaluenga, C., Frias, J., Gomez-cordoves, C., Essafi, N.E., 2014. Phenolic composition, antioxidant and anti-inflammatory activities of extracts from Moroccan Opuntia ficus-indica flowers obtained by different extraction methods. Ind. Crops Prod. 62, 412–420. Chahdoura, H., Barreira, J.C.M., Barros, L., Santos-Buelga, C., Ferreira, I.C.F.R., Achour, L., 2014. Phytochemical characterization and antioxidant activity of Opuntia microdasys (Lehm.) pfeiff flowers in different stages of maturity. J. Funct. Foods 9, 27–37. Clifford, M.N., Johnston, K.L., Knight, S., Kuhnert, N.A., 2003. A hierarchical scheme for LC–MSn identification of chlorogenic acids. J. Agric. Food Chem. 51, 2900–2911. Clifford, M.N., Knight, S., Kuhnert, N.A., 2005. Discriminating between the six isomers of dicaffeoylquinic acid by LC–MSn. J. Agric. Food Chem. 53, 3821–3832. Conti, P., Varvara, G., Murmura, G., Tete, S., Sabatino, G., Saggini, A., et al., 2013. Comparison of beneficial actions of non-steroidal anti-inflammatory drugs to flavonoids. J. Biol. Regul. Homeost. 27, 1–7. De Leo, M., Abreu De, M.B., Pawlowska, A.M., Cioni, P.L., Braca, A., 2010. Profiling the chemical content of Opuntia ficus-indica flowers by HPLC−PDA-ESI–MS and GC/ EIMS analyses. Phytochem. Lett. 3, 48–52. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 86, 421–431. Ellman, G.L., 1959. Tissue sulphydryl groups. Arch. Biochem. 82, 70–77. Guimarães, R., Barros, L., Dueñas, M., Calhelha, R.C., Carvalho, A.M., Santos-Buelga, C., Queiroz, M.J.R., Ferreira, I.C.F.R., 2013. Infusion and decoction of wild German chamomile: bioactivity and characterization of organic acids and phenolic compounds. Food Chem. 136, 947–954. Inal, M.E., Kahraman, A., 2000. The protective effect of flavonol quercetin against ultraviolet a induced oxidative stress in rats. Toxicology 154, 21–29. Jollow, D.J., Mitchell, J.R., Zampaglione, N., Gillete, J.R., 1974. Bromobenzene induced liver necrosis: protective role of glutathione and evidence for 3,4 bromobenzeneoxide as the hepatotoxic intermediate. Pharmacology 11, 151–169. Lee, M.H., Kim, J.Y., Yoon, J.H., Lim, H.J., Kim, T.H., Jin, C., Kwak, W.J., Han, C.K., Ryu, J.H., 2006. Inhibition of nitric oxide synthase expression inactivated microglia and peroxynitrite scavenging activityby Opuntia ficus indica var. saboten. Phytother. Res. 20, 742–747. Loro, J.F., del Rio, I., Pérez-Santana, L., 1999. Preliminary studies of analgesic and antiinflammatory properties of Opuntia dillenii aqueous extract. J. Ethnopharmacol. 67, 213–218. Moon, Y.J., Wang, X., Morris, M.E., 2006. Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicol. In Vitro. 20, 187–210. Nathan, C., 2006. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6, 173–182. Orhan, D.D., Hartevioglu, A., Küpeli, E., Yesilada, E., 2007. In vivo anti-inflammatory and antinociceptive activity of the crude extract and fractions from Rosa canina L. fruits. J. Ethnopharmacol. 112, 394–400. Ou, S., Kwok, K.C., 2004. Ferulic acid: pharmaceutical functions, preparation and applications in foods. J. Sci. Food Agric. 84, 1261–1269. Park, E.-H., Kahng, J.-H., Lee, S.H., Shin, K.-H., 2001. An anti-inflammatory principle from cactus. Fitoterapia 72, 288–290. Paul, S., Shin, H.S., Kang, S.C., 2012. Inhibition of inflammations and macrophage activation by ginsenoside-reisolated from Korean ginseng (Panax ginseng C.A. Meyer). Food Chem. Toxicol. 50, 1354–1361. Ravi, V., Saleem, T.S.M., Patel, S.S., Raamamurthy, J., Gauthama, K., 2009. Anti-inflammatory effect of methanolic extract of Solanum nigrum Linn. Berrie. Int. J. Appl. Res. Nat. Prod. 2, 33–36. Ristivojevi, P., Janakiev, T., Beri, T., Trifkovi, J., 2016. Phenolic profiles and antimicrobial activity of various plant resins as potential botanical sources of Serbian propolis. Ind. Crops Prod. 94, 856–871. Shaker, E., Mahmoud, H., Mnaa, S., 2010. Anti-inflammatory and anti-ulcer activity of the extract from Alhagi maurorum (camelthorn). Food Chem. Toxicol. 48, 2785–2790. Siddiqui, F., Naqvi, S., Abidi, L., Faizi, S., Lubna, Avesi, L., et al., 2016. Opuntia dillenii cladode: opuntiol and opuntioside attenuated cytokines and eicosanoids mediated inflammation. J. Ethnopharmacol. 182, 221–234. Trendafilova, A., Todorova, M., Nikolova, M., Gavrilova, A., Vitkova, A., 2011. Flavonoid constituents and free radical scavenging activity of Alchemilla mollis. Nat. Prod. Commun. 6, 1851–1854. Woldesellassie, M., Eyasu, M., Kelbessa, U., 2011. In vivo antiinflammatory activities of leaf extracts of Ocimum lamiifolium in mice model. J. Ethnopharmacol. 134, 32–36. Yeddes, N., Chérif, J.K., Guyot, S., Baron, A., Trabelsi-Ayadi, M., 2014. Phenolic profile of tunisian Opuntia ficus indica thornless form flowers via chromatographic and spectral analysis by reversed phase-high performance liquid chromatography-UV-photodiode array and electrospray ionization-mass spectrometer. Int. J. Food Prop. 17, 741–751. Zerin, T., Kim, Y.-S., Hong, S.-Y., Song, H.-Y., 2013. Quercetin reduces oxidative damage induced by paraquat via modulating expression of antioxidant genes in A549 cells. J. Appl. Toxicol. 33, 1460–1467.
5. Conclusion The methanolic extract of Opuntia ficus-indica was phytochemically studied for their total phenolics and flavonoids contents, as well as the anti-inflammatory activity was evaluated on carrageenan in vivo model. The chemical characterization of the OFIFE with LC-ESI–MS/MS allowed us to conclude to the wide range of phenolic compounds. The most common flavonoide was identified as being quercetin. The anti-inflammatory activity is evidenced by the noted decrease in rat paw edema, the reduction of the inflammatory cells number (leukocytes and lymphocytes) and the amelioration of inflammatory histological tissue structure of rat paw edema. Besides the biochemical assays shows that the OFIFE treatment restored the CAT, SOD and GSH activities and MDA level. Taken together, these results are good indicators that the Opuntia ficus-indica flowers could be useful for the prevention and treatment of inflammation. This study explains the advantages of O. ficus-indica flowers better and safer anti-inflammatory profile which is attributed to phenolic and mainly flavonoids compounds. Further studies are needed to purify the main constituent responsible for the anti-inflammatory activity in order to reveal the detailed mechanism of action and the responsible compounds. Conflict of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2017.12.028. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 126. Afonso, V., Champy, R., Mitrovic, D., Collin, P., Lomri, A., 2007. Reactive oxygen species and superoxide dismutases: role in joint diseases, Joint. Bone Spine. 74, 324–329. Ahmed, M.S., El Tanbouly, N.D., Islam, W.T., Sleem, A.A., El Senousy, A.S., 2005. Antiinflammatory flavonoids from Opuntia dillenii (Ker-Gawl) haw: flowers growing in Egypt. Phytothe. Res. 19, 807–809. Alimi, H., Hfaiedh, N., Bouoni, Z., Sakly, M., Ben Rhouma, K., 2011. Evaluation of antioxidant and antiulcerogenic activities of Opuntia ficus indica f. inermis flowers extract in rats. Environ. Toxicol. Pharmacol. 32, 406–416. Ammar, I., Ennouri, M., Khemakhem, B., Yangui, T., Attia, H., 2012. Variation in chemical composition and biological activities of two species of Opuntia flowers at four stages of flowering. Ind. Crops Prod. 37, 34–40. Ammar, I., Ennouri, M., Attia, H., 2015. Phenolic content and antioxidant activity of cactus (Opuntia ficus-indica L.) flowers are modified according to the extraction method. Ind. Crops Prod. 64, 97–104. Antunes-Ricardo, M., Gutiérrez-Uribe, J.A., López-Pacheco, F., Alvarez, M.M., SernaSaldívar, S.O., 2015. In vivo anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes. Ind. Crops Prod. 808. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gel. Anal. Biochem. 44, 276–287. Beekmann, K., Actis-Goretta, L., Van Bladeren, P., Dionisi, F., Destaillats, F., Rietjens, I.,
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Anti-inflammatory activity and phenolic composition of prickly pear (Opuntia ficus-indica) flowers
T
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Imene Ammara, , Maryem Ben Salemb, Bahira Harrabib, Massara Mzidc, Sana Bardaab, Zouheir Sahnounb, Hamadi Attiaa, Monia Ennouria,d a
Valuation, Analysis and Food Safety Laboratory, National School of Engineers of Sfax, Sfax University, 3038, Sfax, Tunisia Laboratory of Pharmacology, Faculty of Medicine of Sfax, Sfax University, Road Majida Boulila, 3028, Sfax, Tunisia c Laboratory of Histology Embryology, Faculty of Medicine of Sfax, Sfax University, Road Majida Boulila, 3028, Sfax, Tunisia d Higher Institute of Applied Sciences & Technology of Mahdia, Monastir University, Sidi Messaoud, 5111, Mahdia, Tunisia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Opuntia ficus-indica Flowers Phenolic acids Flavonols Anti-inflammatory
The in vivo anti-inflammatory activity of methanolic extract of Prickly pear Opuntia ficus-indica flowers was studied using carrageenan induced rat paw edema model. In order to characterize the extract, the qualitative and quantitative analysis of phenolic compounds of the methanol extract of Opuntia ficus-indica flowers (OFIFE) were performed using LC–MS/MS technique. The main components of OFIFE were phenolic acids and flavonoids. It was observed that the OFIFE inhibited significantly the inflammation as confirmed by the hematological and the histological analysis. The anti-inflammatory effect of OFIFE was associated with the reduction of malonaldialdehyde level (MDA as an index of lipid peroxidation) and an increase in activities of catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH). Overall, the results showed that OFIFE might serve as a natural source for the treatment of inflammation.
1. Introduction The plant genus Opuntia ficus-indica has proved to be an important source of therapeutic agents, because of the diversity of pharmacological properties and chemical structures. Opuntia ficus-indica has been used in the traditional medicine of diverse countries for a long time (Park et al., 2001). The O. ficus-indica flowers exert a wide range of pharmacological activities including antioxidant, anti-microbial (Ammar et al., 2012; Ammar et al., 2015), antiulcerogenic and wound healing activities (Alimi et al., 2011; Ammar et al., 2015) as well as anti-inflammatory effect (Ahmed et al., 2005; Benayad et al., 2014). Phytochemical studies have demonstrated the presence of several classes of chemical constituents in the O. ficus-indica flowers. These included isorhamnetin glycosides as the major phenolic compounds, followed by quercetin and kaempferol glycosides, which are identified from methanolic and aqueous extracts of O. ficus indica flowers grown in Italy and Tunisia (Ammar et al., 2015; De Leo et al., 2010; Yeddes et al., 2014). Opuntia ficus-indica has been traditionally used to reduce inflammation and previous studies demonstrated the anti-inflammatory potential of this plant. Indeed, earlier finding showed that the alcohol
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extracts of different organs of Opuntia dillenii (Ker-Gawl) Haw, as well as their subfractions (Ahmed et al., 2005) and the fruit aqueous extract (Loro et al., 1999) attenuated, efficiently, the in vivo carrageenan-induced paw edema. More recently, the anti-inflammatory effects of Opuntia cladodes methanol extract was also studied by Siddiqui et al. (2016). With regard to literature, few studies have been focused on flowers compared to other parts (cladodes, fruits and seeds). This study is the first report on the anti-inflammatory effect of the methanolic extract of Opuntia ficus-indica flowers using the carrageenan-induced paw edema test. The study also explores the qualitative and quantitative phenolic composition of the methanolic extract. 2. Materials and methods 2.1. Chemicals All solvents used for extraction and analysis in this study were of analytical grade. Lamda CARR and INDO were purchased from sigma chemical company (St. Louis, MO, USA). The phenolic standards used for quantification purposes, were supplied by Sigma-Aldrich (Chemie Gmbh, Steinhein, Germany). HPLC grade solvent (methanol and acetic
Corresponding author. E-mail address: [email protected] (I. Ammar).
https://doi.org/10.1016/j.indcrop.2017.12.028 Received 21 July 2017; Received in revised form 28 October 2017; Accepted 11 December 2017 Available online 15 December 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.
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2.6. Carrageenan-induced rat paw edema
acid, formic acid, and acetonitrile) were purchased from Fisher Scientific (U.K.). Double distilled water was used in the LC mobile phase
In this assay, edema was induced by subcutaneous injection of 0.1 ml of 1% freshly prepared solution of carrageenan in saline solution (Ravi et al., 2009). The rats were divided into four groups of six rats which were treated by intraperitoneal injection, one hour before administration of carrageenan. The group (I) was the control group and was treated with 10 ml/kg of isotonic saline solution (9‰) (negative control group). The group (II) was inflamed by carrageenan injection and did undergo any treatment (CARR). The group (III) was used as reference group, that were treated with indomethacin (reference drug, 10 mg/kg) (CARR + INDO) and the group (IV) was treated with methanolic extract of O. ficus-indica flowers (CARR + OFIFE) at the dose of 400 mg/kg. The volumes of the paw edema were measured before and after carrageenan injection using a digital caliper at t = 0, 1, 2, 3, 4 and 5 h. The initial volumes (V0) obtained for each group before any treatment and the subsequent values recorded for each group (Vt) gave the actual edema value, using the following ratio: Percentage vt − v0 edema = v0 × 100. The percentages of inhibition were obtained for each group and at each record, using the following ratio:
2.2. Plant material Opuntia ficus-indica (L.) Miller flowers were collected during the post-flowering stage of the plant in June 2014 from wild populations growing in Sfax, Tunisia (latitude 34 46′29″N, longitude 10 39′73″E; elevation: 41 m), a region which is characterized by a semi arid climate and a mean rainfall of 200 mm/year. Plant authenticity was confirmed by the Alimentary Analysis Laboratory of the National Engineering School of Sfax, Tunisia. Flowers were collected in one batch within the same sampling area and were stored at −20 °C until subsequent analysis. 2.3. Preparation of the plant extracts The flower samples (50 g) were minced and extracted by maceration using 500 ml of methanol. The maceration was carried out on a shaker, at room temperature, 150 rpm and in the dark for 24 h. The extract was filtered through filter paper and centrifuged at 4000 × g for 10 min to remove any floating matters. The remaining residue was then re-extracted under the same conditions. The combined extracts were evaporated at 35 °C (rotary evaporator Büchi R-210, Flawil, Switzerland) to remove methanol. The percentage yield was 14.8% (w/w).
Percentage inhibition =
(vt−v0)control − (vt−v0)treated × 100 (vt−v0)control
2.7. Blood sample collection 2.4. Analyses of phenolic compounds by LC–MS-ESI−MS Five hours after carrageenan induction, rats were anesthetized by ketamine (50 mg/kg body weight), intramuscularly injected, along with midazolam (5 mg/kg body weight). The blood samples were quickly collected through cardiac puncture using 2 ml hypodermic syringes into heparinized bottles and immediately centrifuged at 3000 rpm for 15 min to obtain plasma. They were kept at −20 °C until biochemical analysis.
The flowers extract was re-dissolved in methanol at 5 mg/ml and filtered through a 0.45 μm membrane filter (Millipore, Bedford, MA) for injection. The sample analysis was conducted by an HPLC system (Thermo Fisher Scientific, San Jose, CA) coupled to a quadrupole mass spectrometer equipped with electrospray source (ESI) (Waters, Manchester, UK) and Diode Array Detector (DAD). The separation of phenolic compounds was performed on a Phenomenex Luna C18 (2) column (length 150 mm, internal diameter 3 mm, film thickness 3 μm) operating at 25 °C and a flow rate of 0.4 ml/min and 5 μl of sample was injected. The mobile phase used consisted of 0.1% (v/v) acetonitrile–water (solvent A) and 0.1% (v/v) acetic acid–water (solvent B). The elution gradient program with a ratio of A to B was as follows: from 0 to 12 min (15:85, v/v), from 12 to 14 min (40:60, v/v), from 14 to 18 min (60:40, v/v), from 18 to 20 min (80:20, v/v), from 20 to 26 min (90:10, v/v) and from 26 to 40 min (100:0, v/v). Detection was performed on-line in the UV–vis spectra in the range of 220–380 nm. Mass spectrometry data were acquired in the negative ionisation mode. The phenolic compounds were identified and quantified by interpreting their elution order in the HPLC chromatograms, their UV–vis spectra, their mass spectra (m/z, fragmentation patters in MS/MS) and chromatographically compared with corresponding standards. The standards including protocatechuic acid, ferulic acid, quercetin 3-Orutinoside and apegenin were purchased from Merck (Darmstadt, Germany). The calibration curves concentrations are between 0.125 and 50 mg/ml.
2.8. Testing the blood count Complete blood counts KX 21 was used to determine the following hematological parameters: leukocytes, platelets and lymphocytes on an automatic biochemistry analyzer (Vitalab Flexor-E, USA) at the biochemical laboratory of Hedi Cheker Hospital of Sfax. 2.9. Determination of oxidant stress parameters Total superoxide dismutase activity (SOD) was estimated according to Beauchamp and Fridovich (1971). The developed blue color in the reaction was measured at 560 nm. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of Nitroblue tetrazolium (NBT) by 50%, and the activity was expressed as units per milligram of protein. Total catalase activity (CAT) was assayed by the decomposition of hydrogen peroxide according to the method of Aebi (1984). In brief, the reduction of 10 mM H2O2 in 20 mM of phosphate buffer (pH 7) was monitored by measuring the absorbance at 240 nm. The activity was calculated in terms of μmol H2O2 consumed/min/μg of protein. Glutathione level (GSH) in tissue was determined by the method of Ellman (1959) modified by Jollow et al. (1974) based on the development of a yellow color when 5,5-dithiobis-2 nitro benzoic acid (DTNB) was added to compounds containing sulfhydryl groups. The absorbance was measured at 412 nm after 10 min. Glutathione level was expressed as nanomoles/mg of protein. Malondialdehyde (MDA) level in skin was determined according to the method of Draper and Hadley (1990). An amount of 0.5 ml of organ extract supernatant was mixed with 1 ml of trichloroacetic acid solution (20%) and centrifuged at 2500g for 10 min. A 1-ml solution containing
2.5. Animals Male Wistar rats, in the age group of 2–3 months and weighting 186 ± 7 g, were purchased from the local Central Pharmacy, Tunisia and used in the experimental assays of the present study. The rats were housed in hygienic and well ventilated compartments and maintained under standard laboratory conditions (12 h light/dark cycles at 22 ± 2 °C) as approved by the Experimentation and Ethics Committee of the College of Medicine, University of Lagos (CM/COM/08). 314
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Compounds (1, 2, 5–9, 13) were identified as hydroxycinnamic acids derivatives based on retention time order, maximum in UV and mass spectra. Three of them being quinic acid (compound 1) and quinic acid derivatives (Compounds 5, 13), whose identities were assigned based on their MS spectra and fragmentation patterns. Compound 1 (Rt = 3.25 min) showed a [M−H]− ion at m/z 191 and its MS2 fragmentation gave a fragment ion at m/z 127 characteristic of quinic acid. Compounds 5 and 13 released characteristic MS2 fragment ions at m/z 191 (deprotonated quinic acid), 179 (deprotonated caffeic acid), which together with their pseudo molecular [M−H]− ions at m/z 355 and 515, respectively allowed their identification as quinic acid derivatives containing one or two caffeic acid moieties, respectively. Compound 5 was positively identified as 4-O-caffeoylquinic acid according to the fragmentation pattern described by Clifford et al. (2003, 2005). Compound 13 was identified as di-O-caffeoyquinic acid. Indeed, MS2 base peak was at m/z 353, produced by the loss of one of the caffeoyl moieties [M−H−caffeoyl]−, and subsequent fragmentation of this ion yielded the same fragments as 5-caffeoylquinic acid at m/z 191, 179 and 135. Concerning compound 13, it was assigned as 4,5-O-dicaffeoylquinic acid, since its fragmentation was identical to those previously reported by Clifford et al. (2005) and Guimarães et al. (2013). Compound 2 and 6 exhibited [M−H]− ion at m/z 269 and 179. Thus they were tentatively characterized as gallic acid and caffeic acid, respectively. Coumaric acid was identified based on the molecular ion at m/z 163 [M−H]− and the typical fragment of coumaric acid detected at m/z 119. This compound was identified as p-coumaric acid since the fragmentation pattern were consistent with previous report (Belguith-Hadriche et al., 2013; Ristivojevi et al., 2016). Compound 3 was identified as protocatechuic acid by comparing its UV spectrum and retention time with those of an external standard. Compound 4 displayed maximum UV absorption at 320 and exhibited [M−H]− ions at m/z 353. According to the above information, this compound was identified to chlorogenic acid. Compound 8 with MS fragment at m/z 193 ([ferulic acid −H]−) and MS2 fragment at m/z 134 was identified as ferulic acid which was identical with the external standard. In addition to the phenolic acid derivatives described above, there were also identified flavonoid compounds. The flavonoid profile of the OFIFE consisted of quercetin glycosides and its derivatives. Indeed,
0.67% thiobarbituric acid (TBA) and 0.5 ml of supernatant were incubated for 15 min at 80 °C and cooled. Absorbance of TBA–MDA complex was determined at 530 nm using a spectrophotometer (Jenway UV-6305; Essex, England). Lipid peroxidation was expressed as nanomoles of MDA/mg protein. 2.10. Histological examination For histopathological examination, biopsies of paws (n = 3) from control, CARR, CARR + INDO and CARR + OFIFE rats were removed and immediately fixed in 10% neutral buffered formalin solution, embedded in paraffin, sectioned into 5 μm-thick sections using a rotary microtome (Leitz, Germany) and stained with hematoxylin–eosine for evaluation under light microscopy. 2.11. Statistics All the data were expressed as mean ± standard deviation (SD) of six animals in each group. Carrageenan-induced inflammation test data were assessed by one-way analysis of variance (ANOVA), and the significant differences were examined by Duncan’s multiple range post hoc-test using Statistical Package for the Social Sciences (SPSS) software (version 16, SPSS, Inc, Chicago, IL, USA). Differences between groups were considered significant at a level of p < 0.05 and p < 0.01. The student’s t-test using EXCEL (Microsoft Corporation, USA) was used to establish the significance of difference from control group or carrageenan group at the significance level of p < 0.001. 3. Results 3.1. Phenolic compounds analysis of the OFIFE Phenolic compounds were identified according to their retention time, the wavelengths of maximum absorbance (UV λmax at 250–280, 320–360 nm), the deprotonated molecules ([M−H]−) in negative ionisation mode and the characteristic product ions in comparison with those of authentic standards and literature data. Table 1 summarizes the phenolic compounds identified in the OFIFE. The detected phenolic compounds were typical of phenolics acids and flavonoids classes.
Table 1 LC–ESI–MS characteristics of the identified phenolic compounds in the methanol extract of the O. ficus-indica flowers. Peak number
Phenolic acids 1 2 3 4 5 6 7 8 9 Flavonoids 10 11 12 13 14 15 16 17 18 Phenolic acids Flavonoids Total phenolic compounds
Rt (min)
λmax (nm)
Molecular ion [M−H]− (m/ z)
MS/MS fragments (m/ z)
Tentative identification
Quantification (mg/100 g)
3.25 6.73 10.45 11.97 13.28 12.98 16.25 17.55 20.49
320 270 278 320 328 324 300 328 254–328
191 269 153 353 355 179 163 193 359
174, 134, 129, 127 169, 125 109 191, 179. 191, 179, 173. 135 163, 119 134 161,197
Quinic acid Gallic acid Protocatechuic acid Chlorogenic acid 4-O-caffeoylquinic acid Caffeic acid p-coumaric acid Trans ferulic acid Rosmarinic acid
131.85 ± 5.8 1.42 ± 0.002 4.28 ± 0.0085 1.62 ± 0.001 3.50 ± 0.0002 1.61 ± 0.001 11.83 ± 0.005 16.49 ± 1.2 1.96 ± 0.002
19.04 20.98 21.08
354 256–354 255–354
593 609 463
285 611, 465, 303 300
6.46 ± 0.1 390.27 ± 10.2 325.31 ± 50.2
22.25 22.6 24.71 26.83 30.49 36.56
– – 354 350–372 250–332 260–350
515 447 623 477 269 417
353, 191, 179, 173, 135 300 315 315 227, 225, 149, 119 285
Kaempferol-3-O-rutinoside Rutin (quercetin-3-O- rutinoside) Hyperoside (quercetin-3-Ogalactoside) 4.5-di-O-caffeoyl quinic acid Quercetin-3-O-rhamonoside Isorhamnetin-3-O-rutinoside Isorhamnetin 3-O-glucoside Apegenin Kaempferol 3-O-arabinoside
315
3.05 ± 0.0001 134.12 ± 0.5 1.05 ± 0.001 26.83 ± 0.001 0.15 ± 0.00 2.15 ± 0.000 177.62 886.34 1063.96
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The high total leukocytes counting confirmed the inflammatory response after CARR injection. The administration of OFIFE before the injection of CARR caused a significant decrease (p < 0.01) in the number of total leukocytes (49.4%) and lymphocytes (27.4%) when compared to the CARR group. Indomethacin treated group showed 52.27 and 49.17% of leukocytes and lymphocytes reduction, respectively. Thus, the OFFIE could reduce actively the leukocytes and lymphocytes. The number of platelets remained without significant changes.
compounds 11, 12, and 14 were identified as quercetin glycosides. The [M−H]− ion at m/z 609 of compound 11 (Table 1) with significant fragments at m/z 611, m/z 465 and m/z 303 was identified as quercetin 3-O-rutinoside which was identical with the authentic standard. While the [M−H]− ion m/z 463 (UV λmax at 255 nm and 354 nm) of compound 12 was identified as quercetin 3-O-galactoside. Compound 14 was identified as quercetin-3-O-rhamonoside. Another group of detected flavonols was isorhamnetin derivatives according to their UV–vis and mass spectra (MS2 product ion at m/z 315); compounds 15 and 16 release ([M−H]− at m/z 623 and m/z 477, respectively) and one MS2 fragment ion at m/z 315, were tentatively identified as isorhamnetin-3O-rutinoside and isorhamnetin-3-O-glucoside, respectively as previously reported in the studies of De Leo et al. (2010) and Chahdoura et al. (2014). Compound 17 (Rt = 30.49 min) showed a [M−H]− ion at m/z 269. Comparison of the main MS2 fragment ions such as those at m/z 227,149, UV spectrum (250 and 332 nm) and the retention time to those obtained for a standard allowed their identification as apigenin. The total amount of phenolic acids and flavonoids of OFIFE were respectively 177.61 mg and 886.34 mg/100 g extract. Flavonoids, especially quercetin derivatives, were the major phenolic compounds present in these samples. The predominant phenolic acid of the extract was quinic acid (12.4% of total identified phenolics), while quercetin-3-O-rutinoside (36.68% of total identified phenolics) was the main identified flavonoid. It was followed by quercetin-3-O-galactoside (30.57% of total identified phenolics), quercetin-3-O-rhamonoside (12.6% of total identified phenolics) and isorhamnetin 3-O-glucoside with 2.5% of total identified phenolics.
3.4. Effect of OFIFE on oxidative stress parameters The activities of CAT, SOD and GSH as well as the MDA level, in paw edema, were evaluated (Table 4). Results revealed that the CARR group of rats has the lowest activities of CAT, SOD and GSH, while they showed the highest MDA level, proving the presence of an oxidative stress state in rats. In fact, the induction of inflammation with CARR decrease the activities of SOD, CAT, and GSH in edema by 47.7, 74.0 and 64.41%, respectively, in comparison to the control group (p < 0.001). However, the OFIFE treatment increase the activities of SOD to 51.19%, CAT to 226.3% and GSH to 328.75%, respectively, compared to that observed in the CARR group. Indomethacin treatment also exhibited increase effects in the activities of SOD (46.7%), CAT (171.11%) and GSH (359.49%) in comparison to the CARR group. These data imply that treatment of induced edema with OFIFE improved, significantly these activities compared to CARR group. The inflammation induced a highly significant increase in the level of MDA, at the skin edema of normal control group (34.18%). The treated groups by OFIFE and INDO showed significant reduction in the tissue levels of MDA (33.43 and 37.16%, respectively) when compared to the CARR group. Pretreatment with OFIFE prevented the increase in level of MDA, while it restored significantly (P < 0.01) the CAT, SOD, and GSH activities in induced edema compared with the CARR group (Table 4).
3.2. Effect of OFIFE on the carrageenan (CARR) induced paw edema The anti-inflammatory activity of OFIFE was investigated in vivo by applying a carrageenan-induced paw edema model in rats. As the results showed in Fig. 1, the CARR injection causes edema development which increase progressively with time over the 5 h of treatment. After 1 h of the induction, the edema development began and the edema percentage increase significantly to reach 23.4%, 22.4% and 19.7% in the CARR group, OFIFE and INDO treated group, respectively. The edema development of OFIFE and INDO treated group decreased significantly in comparison with the CARR group. Thus, the percentages of edema inhibition calculated for each group for OFFIE and INDO are reported in Table 2. The highest significant inhibitory activity (52.31% of inhibition) is shown in the OFIFE group over a period of 4 h after induction of inflammation in comparison with INDO group (54.43%).
3.5. Histological analysis The macroscopic results of the paw edema were confirmed through the histological examination. The histological analysis of the edema skin (Fig. 2), revealed that the control group showed normal histological section (Fig. 2a), however, the most severe inflammatory changes is showed in the CARR group as reflected by vasodilatation, neutrophils accumulation and connective tissue disorganization. Arrows in Fig. 2(b) indicate prominent areas of edema with massive signs of inflammation (inflammatory cells, particularly neutrophils) and edema disruption of tissue structure. The treatment with OFIFE proved to be able to reduce the sign of acute inflammation with a decrease in the inflammatory cells number and improvements in edema tissue (Fig. 2d). Indomethacin treated
3.3. Effect of OFIFE on hematological parameters of blood of rats The anti-inflammatory effect of the OFIFE was evaluated by studying changes in leukocytes, lymphocytes and platelets (Table 3).
Fig. 1. Percentage inhibition of carrageenan (CARR) induced-paw edema in rats treated with O. ficus indica flowers methanol extract (OFIFE) and indomethacin (INDO). Each value represents as mean ± SD (n = 6). **p < 0.001 as compared with the CARR group.
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Table 2 Percentage inhibition of O. ficus indica flowers extract and indomethacin on carrageenan-induced paw edema in rat. Treatment
CARR + OFIFE CARR + INDO
% inhibition of inflammation 1h
2h
3h
4h
4.22 ± 0.7 a 16.58 ± 1.5 b
38.22 ± 2.1 a 40.04 ± 1.63 a
45.19 ± 1.4 a 50.25 ± 2.36 b
42.31 ± 0.75 44.43 ± 2.14
5h a a
35.29 ± 0.45 a 32.1 ± 1.79 b
Values are means ± SD. Statistical analysis was done by a one-way ANOVA with Duncan’s multiple comparisons post-test (p < 0.05).
dillenii revealed 48.9% inhibition, 4 h after carrageenan induction of rat paw edema (200 mg/kg) (Ahmed et al., 2005). In order to confirm the results obtained from the edema size inhibition, the test blood cells and plateltes count of the induced paw edema were performed. The treatment with OFIFE reduced significantly the inflammatory cells number (leukocytes and lymphocytes) (p < 0.01) in comparison with the CARR group. These hematological parameters confirmed that the OFIFE has an anti-inflammatory effect on edema tissue following CARR injection, with a significant reduction (p < 0.01) of total leukocytes (49.4%). This inhibitory effect is higher than that reported for the cladodes extract of Opuntia ficus-indica (5 mg/kg) with a 29.4% of leukocytes reduction (Antunes-Ricardo et al., 2015). Another important component responsible of inflammation is oxidative stress, which derived when reactive species (ROS) are overproduced, exceeding the capacity of the endogenous antioxidant system (Nathan, 2006). The SOD, CAT, and GSH provide the first line of cellular defense against toxic free radicals. The primary role of GSH is to detoxify the damaging radicals either by directly scavenging them or by acting as a co substrate in the glutathione peroxidase (GPx)-catalyzed reduction of hydrogen peroxide and lipid peroxides. CAT is known to detoxify the H2O2 generated into water and oxygen. While SOD activity protects cells and the extracellular matrix from the harmful effects of superoxide anion (O2 %-) and its derivatives such as hydroxyl radical (% OH) (Afonso et al., 2007). In the present study, it was shown that the induction of inflammation significantly decrease (p < 0.001) the activities of SOD, CAT, GSH and increase the production of MDA in comparison with the normal control group (Table 4). In contrast pre-treatment of rats with OFIFE restored the normal activities of SOD, CAT and GSH and decrease the MDA level. This implied that OFIFE reduced the inflammatory oxidation by promoting the activities of antioxidant system in edema paw. There are various studies suggesting that polyphenolic compounds and mainly flavonoids play an important role, as protective factors against free radicals and reactive oxygen species compounds, that which results natural inhibitors of inflammation (Conti et al., 2013; Moon et al., 2006). According to previous reports, O. ficus indica flowers have been considered to be an excellent source of polyphenolic compounds and natural antioxidants (Alimi et al., 2011; Ammar et al., 2015). Consequently, the anti-inflammatory effect showed in this study can be attributed to the synergistic effect of phenolic compounds present in the OFIFE to act as free radical scavenging generated by the inflammation. Furthermore, the phenolic compounds determined by
group showed a marked effect in reducing the inflammation features (Fig. 2c). The histopathological analysis of the OFIFE was close to that of INDO group. 4. Discussion The obtained results in the phenolic analysis are in agreement with previous studies reporting the presence of phenolic acids and flavonoids in methanol extract of O. ficus-indica Thornless form flowers from Tunisia (Yeddes et al., 2014), as well as in methanol/water extract of O.microdasys flowers (Chahdoura et al., 2014). The quinic acid is the major phenolic acid identified in this study which accounted for 12.4% of total identified phenolics. This compound is also present in the Moroccan O. ficus-indica flowers at a lower amount (3.65% of total identified phenolics) (Benayad et al., 2014). The OFIFE is dominated by the flavonol glycoside and the flavonol 3-O-glycosides (quercetin, kaempferol and isorhamnetin) were the most characteristic flavonoids. These compounds have been previously identified in closely related to Opuntia flowers (De Leo et al., 2010). The identified compounds in OFIFE have been reported to exhibit a wide range of therapeutic properties including anti-bacterial, anti-carcinogenic, and anti-inflammatory effects (Ou and Kwok, 2004). The evaluation of the anti-inflammatory activity of OFIFE in this study was carried out using the CARR induced edema test. It has been reported that this inflammation model provokes a local inflammatory reaction and involves several mediators that is a suitable to evaluate the anti-inflammatory effects of natural products (Woldesellassie et al., 2011). This edema model involves the synthesis and the release of inflammatory mediators at the site of injury like histamine and serotonin for the initial phase of inflammation (during the first 2 h after CARR injection) and prostaglandins for the second phase of inflammation (3–4 h after CARR injection) (Orhan et al., 2007). The reduction of edema size is a good indicator to determine the protective action of anti-inflammatory agents. According to Fig. 1, the OFIFE (400 mg/kg) inhibited significantly the development of edema at 3 h after CARR induction (p < 0.001). Besides, there is no significant difference (p < 0.001) existed between the OFIFE and INDO groups, at 2–5 h after CARR treatment, which suggests that OFIFE has potential antiinflammatory effect. The OFIFE showed also, significant inhibition of rat paw edema (p < 0.05) (Table 2). This result is in parallel with earlier finding in which alcohol extract of fresh flowers of Opuntia
Table 3 Effect of treatments (carrageenan, indomethacin and OFIFE) on hematological parameters of the rat’s blood. Parameter
Leukocytes (*103/mm3) Lymphocytes (%) Platelets (*103/mm3)
Treatments Control
CARR + INDO
CARR + OFIFE
CARR
8.05 ± 0.05 32.1 ± 0.9 500 ± 25.3
8.2 ± 0.03b (% = 52.27) 21.5 ± 0.05a,b (% = 49.17) 610 ± 12.8
8.7 ± 0.02b (% = 49.4) 30.7 ± 0.3b (% = 27.4) 622 ± 8.9
17.18 ± 2.1a 42.3 ± 1.5a 685 ± 20.3
Each value represents as mean ± SD (n = 6). a, b, c Different letters indicate significant differences between treatments. CARR. carrageenan; OFIFE. methanol extract of Opuntia-ficus indica and INDO. indomethacin. a p < 0.001 significantly different from the control group. b p < 0.01 significantly different from the carrageenan group (one way ANOVA).
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Table 4 Effects of methanol extract of Opuntia-ficus indica (OFIFE) and indomethacin (INDO) on the catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) activities in paw edema. Groups
SOD (U/mg protein)
CAT (μmol H2O2/min/μg de Prot)
GSH (nmol/mg protein)
MDA (nmol/mg protein)
Normal control CARR group (Untreated) CARR + INDO CARR + OFIFE
15.98 13.01 23.45 24.16
50.58 13.15 35.65 42.90
43 ± 1.2 15.3 ± 1.1a(64.41%) 70.3 ± 3.1b (359.49%) 65.6 ± 4.9b (328.75%)
23.4 ± 0.85 31.4 ± 1.52a (34.18%) 20.9 ± 1.0b (33.43%) 19.73 ± 1.8b (37.16%)
± ± ± ±
0.36 0.9a (47.7%) 1.2b(46.7%) 1.78b (51.19%)
± ± ± ±
2.3 0.6a (74.0%) 1.3b (171.11%) 2.38b (226.23%)
Each value represents as mean ± SD (n = 6). Carr. carrageenan. a p < 0.001 as compared with the control group. b p < 0.01 as compared with the carrageenan group (oneway ANOVA).
2012; Trendafilova et al., 2011). Lipid peroxidation is a process in which free radicals damage cell structures and tissues. MDA is the major final product of lipid peroxidation that accumulates at the inflammatory site (Paul et al., 2012). The decrease of MDA level in OFIFE treated group also suggested that this extract might neutralize the ROS mediated lipid peroxidation which leads to a marked reduction of lipids damage in cell membranes of tissues. In consistency with our results, the extract of the flowers of O. ficus-indica have shown to attenuate the lipid peroxidation products of inflammation processes that are related to nitric oxide (NO) production (Benayad et al., 2014). Considering the obtained results, it seems reasonable that the benefic action of the OFIFE against inflammation is more likely
LC–MS/MS analysis of the OFIFE showed the presence of quercetin, as the predominant compound, isorhamnetin and kaempferol besides the presence of quinic acid as the major compound of phenolic acids. It was previously reported that quercetin proved a potential radical-scavenging activity, based on its ability to donate electrons from their hydroxide group (Inal and Kahraman, 2000) and it was also demonstrated that quercetin has the ability of minimizing the oxidative damage by modulating expression of antioxidant activities (Zerin et al., 2013). The promising inflammatory activity of kaempferol and isorhamnetin glycoside has been also reported (Shaker et al., 2010). Besides, it was shown that 1,5 dicaffeoyl quinic acid, dicaffeoyl derivative and quercetin-3-O-galactoside have strong free radical scavenging capacities, which could effectively reduce the oxidative stress (Beekmann et al.,
Fig. 2. Representative photomicrographs of histological sections of edema paws of the control showed normal tissue (a), the carrageenan reated group (b), carrageenan + OFIF extract (c) and carrageenan + indomethacin treated group (d). Tissues were stained with hematoxylin–eosin; (a), (b) visualized at 200× magnifications and (c), (d) visualized at 400× magnifications. massive signs of inflammation.
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attributed to the occurrence of flavonoids compounds. The phenolic compounds and mainly flavonoids apart from displaying a significant antioxidant effect, also contribute to ameliorate the inflammatory processes. Hence, previous studies have shown that anti-inflammatory can be a result of the high polyphenol content of plants especially phenolics and flavonoids (Lee et al., 2006; Orhan et al., 2007). For example, Opuntia dillenii flowers extract showed high levels of flavonoids which determined the reported anti-inflammatory activity (Ahmed et al., 2005).
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Inhibition of inflammations and macrophage activation by ginsenoside-reisolated from Korean ginseng (Panax ginseng C.A. Meyer). Food Chem. Toxicol. 50, 1354–1361. Ravi, V., Saleem, T.S.M., Patel, S.S., Raamamurthy, J., Gauthama, K., 2009. Anti-inflammatory effect of methanolic extract of Solanum nigrum Linn. Berrie. Int. J. Appl. Res. Nat. Prod. 2, 33–36. Ristivojevi, P., Janakiev, T., Beri, T., Trifkovi, J., 2016. Phenolic profiles and antimicrobial activity of various plant resins as potential botanical sources of Serbian propolis. Ind. Crops Prod. 94, 856–871. Shaker, E., Mahmoud, H., Mnaa, S., 2010. Anti-inflammatory and anti-ulcer activity of the extract from Alhagi maurorum (camelthorn). Food Chem. Toxicol. 48, 2785–2790. Siddiqui, F., Naqvi, S., Abidi, L., Faizi, S., Lubna, Avesi, L., et al., 2016. Opuntia dillenii cladode: opuntiol and opuntioside attenuated cytokines and eicosanoids mediated inflammation. J. Ethnopharmacol. 182, 221–234. 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5. Conclusion The methanolic extract of Opuntia ficus-indica was phytochemically studied for their total phenolics and flavonoids contents, as well as the anti-inflammatory activity was evaluated on carrageenan in vivo model. The chemical characterization of the OFIFE with LC-ESI–MS/MS allowed us to conclude to the wide range of phenolic compounds. The most common flavonoide was identified as being quercetin. The anti-inflammatory activity is evidenced by the noted decrease in rat paw edema, the reduction of the inflammatory cells number (leukocytes and lymphocytes) and the amelioration of inflammatory histological tissue structure of rat paw edema. Besides the biochemical assays shows that the OFIFE treatment restored the CAT, SOD and GSH activities and MDA level. Taken together, these results are good indicators that the Opuntia ficus-indica flowers could be useful for the prevention and treatment of inflammation. This study explains the advantages of O. ficus-indica flowers better and safer anti-inflammatory profile which is attributed to phenolic and mainly flavonoids compounds. Further studies are needed to purify the main constituent responsible for the anti-inflammatory activity in order to reveal the detailed mechanism of action and the responsible compounds. Conflict of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2017.12.028. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 126. Afonso, V., Champy, R., Mitrovic, D., Collin, P., Lomri, A., 2007. Reactive oxygen species and superoxide dismutases: role in joint diseases, Joint. Bone Spine. 74, 324–329. Ahmed, M.S., El Tanbouly, N.D., Islam, W.T., Sleem, A.A., El Senousy, A.S., 2005. Antiinflammatory flavonoids from Opuntia dillenii (Ker-Gawl) haw: flowers growing in Egypt. Phytothe. Res. 19, 807–809. Alimi, H., Hfaiedh, N., Bouoni, Z., Sakly, M., Ben Rhouma, K., 2011. Evaluation of antioxidant and antiulcerogenic activities of Opuntia ficus indica f. inermis flowers extract in rats. Environ. Toxicol. Pharmacol. 32, 406–416. Ammar, I., Ennouri, M., Khemakhem, B., Yangui, T., Attia, H., 2012. Variation in chemical composition and biological activities of two species of Opuntia flowers at four stages of flowering. Ind. Crops Prod. 37, 34–40. Ammar, I., Ennouri, M., Attia, H., 2015. Phenolic content and antioxidant activity of cactus (Opuntia ficus-indica L.) flowers are modified according to the extraction method. Ind. Crops Prod. 64, 97–104. Antunes-Ricardo, M., Gutiérrez-Uribe, J.A., López-Pacheco, F., Alvarez, M.M., SernaSaldívar, S.O., 2015. In vivo anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes. Ind. Crops Prod. 808. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gel. Anal. Biochem. 44, 276–287. Beekmann, K., Actis-Goretta, L., Van Bladeren, P., Dionisi, F., Destaillats, F., Rietjens, I.,
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