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In vivo anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes
Industrial Crops and Products 76 (2015) 803–808
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In vivo anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes Marilena Antunes-Ricardo, Janet A. Gutiérrez-Uribe ∗ , Felipe López-Pacheco, Mario M. Alvarez, Sergio O. Serna-Saldívar Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, N.L., Mexico
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Article history: Received 6 January 2015 Received in revised form 7 May 2015 Accepted 27 May 2015 Keywords: Cytokine Flavonoid glycosides Inflammation Isorhamnetin Opuntia ficus-indica (L.) Mill
a b s t r a c t Opuntia ficus-indica (L.) Mill [OFI] has been traditionally used to reduce inflammation. This plant possesses a varied composition of phytochemicals such as flavonoids. The aim of this investigation was to study the anti-inflammatory effects of an OFI extract and two isolated isorhamnetin glycosides using an air-pouch inflammation model. Isorhamnetin glycosides were isolated by semi-preparative chromatography and identified with LC/MSD TOF. Male Wistar rats were divided into five groups (n = 6) receiving isorhamnetin3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), isorhamnetin-3-O-glucosyl-rhamnoside (IGR), OFI extract, indomethacin or vehicle intraperitoneally. IGR showed the best potential to decrease cell infiltration, mainly neutrophils (51.8 ± 10.9%). IGRR and OFI extract inhibited the nitric oxide production (IGRR: 81.4%; OFI extract: 77.2%) and cyclooxygenase-2 activity (IGRR: 76.3%; OFI extract: 77.7%). OFI extract showed the highest inhibition of tumor necrosis factor (TNF)-␣ (85.2%) and interleukin (IL)-6 (53.0%). Results showed that OFI extract and isorhamnetin glycosides possess anti-inflammatory potential. IGRR showed better effect than IGR suggesting that the glycosylation profile affects the bioactivity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Opuntia ficus-indica (L.) Mill is a xerophyte plant belonging to the Cactaceae family. Its cladodes have been traditionally used as a folk medicine due to their hypoglycemic, antioxidant, apoptotic, antibacterial, antifungal and anti-inflammatory activities (Ammar et al., 2012; Antunes-Ricardo et al., 2014; Avila-Nava et al., 2014; Ayadi et al., 2009; López-Romero et al., 2014; Tesoriere et al., 2004). These biological effects have been attributed mainly to the flavonoids content, being the most abundant several types of isorhamnetin glycosides (Santos-Zea et al., 2011). Flavonoids modulate the inflammatory response affecting the nitric oxide (NO) and cyclooxygenase-2 (COX-2) pathways and also by a significant down-regulation of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL6) production (Mahat et al., 2010; Maihöfner et al., 2003; Shalini et al., 2012; Xie et al., 2012).
Abbreviations: OFI, Opuntia ficus-indica (L.) Mill; IsoEq, isorhamnetin equivalents. ∗ Corresponding author. E-mail address: [email protected] (J.A. Gutiérrez-Uribe). http://dx.doi.org/10.1016/j.indcrop.2015.05.089 0926-6690/© 2015 Elsevier B.V. All rights reserved.
Isorhamnetin possesses anti-inflammatory effects through inhibiting the NF-B activation and the COX-2 gene expression (Hämäläinen et al., 2007, 2011; Yang et al., 2013). Isorhamnetin-3O-galactoside, besides inhibiting the NF-B activation, suppressed the production of TNF-␣ (Kim et al., 2013a). The number and position of the sugar moieties affect the anti-inflammatory activity of flavonoids (De Melo et al., 2009; Liu et al., 2012). The purpose of this study was to evaluate the anti-inflammatory effects of an O. ficus-indica (L.) Mill extract and two isolated isorhamnetin glycosides on the inflammatory mediators NO, COX-2, IL-6 and TNF-␣ using an in vivo inflammation model.
2. Materials and methods 2.1. Plant The O. ficus-indica (L.) Mill var. Jalpa plant used was harvested in the region of Montemorelos, Nuevo León, México. The taxonomic identification of the cactus species was done at the School of Agronomy of Universidad Autónoma de Nuevo León (UANL), México. The pads of O. ficus-indica (L.) Mill with stage of maturity of 7 months were sanitized with a chlorine solution, cut, dried and ground to obtain a particle size lower than 180 m. The resulting flour was
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packed in sealed bags, protected from light and transported to the laboratory. 2.2. Reagents Organic solvents, n-hexane, n-butanol, and ethyl acetate used for FCPC separations were of analytical grade (DEQ, García, N.L., MX). For HPLC-PDA and LC/MSD TOF analysis, chromatography grade methanol (VWR International LLC, Radnor, PA) and chromatography grade water (VWR International LLC, West Chester, PA) were used. Formic acid solution HPLC grade, isorhamnetin standard ≥95.0% (HPLC), indomethacin [≥99%] and carrageenan (mixture of lambda and kappa-carrageenans) were bought from Sigma Chemical Company (St. Louis, MO). 2.3. Preparation of O. ficus-indica (L.) Mill extract The extraction was performed by alkaline hydrolysis with 4 N NaOH in a 1:10 (mass to solvent ratio) by stirring at 150 rpm in an incubator shaker (Innova 4000; New Brunswick Scientific Inc., Edison, NJ) at 40 ◦ C during 30 min. The subsequent procedure was performed according to the method described by Antunes-Ricardo et al. (2014). Once finalized the alkaline extraction, the sample was filtered and pH was adjusted to 2 with 6 N HCl. Following, lipids were removed with n-hexane (1:1, v/v) and the resulting aqueous fraction was extracted three times with ethyl acetate (1:1, v/v) to remove non-glycosylated flavonoids. Supernatant was filtered through a Whatman filter paper No. 3 and centrifuged at 1550 × g for 10 min and 4 ◦ C in an IEC Centra MP4R centrifuge (International Equipment Co., Needham Heights, MA). An isorhamnetin glycosides enriched fraction was obtained from the crude extract by solid phase extraction using a 1000 mg/6 mL GracePureTM SPE C18Aq cartridge (Grace-Davison, Deerfield, IL), previously conditioned with methanol and distilled water. The isorhamnetin glycosides enriched fraction recovered in 100% methanol was evaporated to dryness under vacuum (50 ◦ C, 50 min) (Genevac Rocket, Ltd., Ipswich, UK). Then, this OFI extract was stored at −80 ◦ C (Labconco, Kansas City, MO). 2.4. Purification and identification of isorhamnetin glycosides Purification and quantification of isorhamnetin glycosides was performed using an HPLC equipped with a photodiode array detector (PDA) (Agilent 1100 Series Santa Clara, CA) according to the method described by Antunes-Ricardo et al. (2014). Chromatograms of OFI extract were obtained at 365 nm. Purification was performed by semi-preparative chromatography using a semipreparative Zorbax SB-C18 (9.4 × 250 mm, 5 m) column operating at a temperature of 17 ◦ C with a flow rate of 2.0 mL/min. The injection volume was 50 L. The mobile phase used was (A) water with 0.1% formic acid (Sigma, St. Louis, MO) and (B) methanol 80%. Separation was achieved starting with 35% of B, increasing to 60% until 2 min, increasing to 90% until 19 min, and then decreasing to 0% of B for the next 3 min. Isorhamnetin glycosides were collected using peak-based mode with maximum peak duration of 0.3 min. Purity of the isolated compounds was greater than 90%. Identification of isorhamnetin glycosides was carried out by LC/MSD TOF (Model G1969A Agilent 1100 Santa Clara, CA) according to the method described by Santos-Zea et al. (2011). 2.5. Animals This study was approved by the Institutional Committee on Care and Use of Experimental Animals at the Tecnológico de Monterrey (Protocol number 2014-RE-001/June 11, 2014). Male Wistar rats (250–300 g) were procured from Bioinvert (Mexico DF, Mexico).
Animals were randomly divided into groups of six animals each and kept in a room with controlled temperature (25 ◦ C) and relative humidity (50 ± 5%) for 12 h light/dark cycles with free access to lab chow pelleted diet and water.
2.6. Carrageenan-induced air-pouch inflammation Animals were grouped randomly into five groups (n = 6), then the air pouches were produced by subcutaneous injection of 20 mL of sterile air into their intra-scapular area and maintained by reinjection of 10 mL of air three days later (Duarte et al., 2012). On the sixth day, O. ficus-indica (L.) Mill extract (5 mg/kg), isorhamnetin-3O-glucosyl-rhamnosyl-rhamnoside (IGRR, 5 mg/kg), isorhamnetin3-O-glucosyl-rhamnoside (IGR, 5 mg/kg), indomethacin (5 mg/kg) or vehicle (0.9% NaCl) were administered intraperitoneally (i.p.). Indomethacin was used as positive control of anti-inflammatory drug. Inflammation was induced after 60 min by the injection of 2 mL of 1% sterile carrageenan directly into the pouch. After 24 h, the animals were euthanized with sodium pentobarbital (60 mg/kg, i.p.) followed by cervical dislocation. A small incision was made in the pouch, and the exudate was carefully removed according to the procedure suggested by Duarte et al. (2012). The total volume of the exudate was measured and the cells were separated by centrifugation (1000 × g for 10 min, at 4 ◦ C). Total and differential counts were performed by microscopic observation. Blood was collected in K2 EDTA Lavender HemogardTM tubes (BD, Franklin Lakes, NJ). Blood samples were centrifuged (1000 × g × 10 min, 4 ◦ C) to obtain plasma. The plasma was stored in small aliquots at −20 ◦ C and used for cytokine assays.
2.7. Cytokines, COX-2 and NO determination The activities of cytokines (IL-6 and TNF-␣) were determined by enzyme-linked immunosorbent assay with commercially available kits (Invitrogen Corp., Camarillo, CA), according to the manufacturer’s instructions. TNF-␣ and IL-6 levels were measured in plasma and air-pouch exudates. The inhibition of COX-2 was determined by the COX fluorescent inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI). NO production was determined with the Griess Reagent System (Promega, Madison, WI) and was expressed as percentage of inhibition of nitric oxide production.
2.8. Statistical analysis Experiments were performed at least by triplicate and results were analyzed with JMP 11.0 software (SAS Institute Inc., Cary, NC) using one-way ANOVA followed by Tukey’s HSD tests. For each data set, p < 0.05 was considered statistically significant.
3. Results and discussion 3.1. Identification and quantification of isorhamnetin glycosides in O. ficus-indica (L.) Mill extract The main flavonoids quantified in the OFI extract corresponded to isorhamnetin diglycosides and triglycosides such as isorhamnetin-3-O-glucosyl-rhamnoside (IGR) and isorhamnetin3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), respectively (Fig. 1). The most abundant triglycoside was IGRR with 77.3 ± 3.8 mg IsoEq/g OFI extract. IGR was the most abundant diglycoside with 30.4 ± 2.6 mg IsoEq/g OFI extract. The fragmentation pattern analysis indicated that the only difference between these two compounds was an additional rhamnose residue in IGRR (Fig. 2).
M. Antunes-Ricardo et al. / Industrial Crops and Products 76 (2015) 803–808
OCH3 OH O
HO
O-R OH
O
-R IGRR: Glu-Rha-Rha IGR: Glu-Rha
Fig. 1. Structure of isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR) and its corresponding diglycoside isorhamnetin-3-O-glucosyl-rhamnoside (IGR) isolated from O. ficus-indica extract. I: isorhamnetin; GLU: glucose; RHA: rhamnose.
3.2. Effect of isorhamnetin glycosides and OFI extract on cells count of the air pouches exudates The intraperitoneal administration of IGR 60 min before the injection of carrageenan caused a significant decrease (p < 0.05)
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in the number of total leukocytes (54% decrease) (Fig. 3). IGRR showed a leukocytes reduction of 17.9%, meanwhile indomethacin and OFI extract showed 28.9% and 29.4% decrease, respectively. Contrary to showed by Morikawa et al. (2003), who reported that quercetin-3-O-rhamnoside (quercetin monoglycoside) and quercetin-3-O-rutinoside (quercetin diglycoside) shown no significant differences in leukocyte reduction in air-pouches exudates, in this study differences were observed between isorhamnetin triglycoside IGRR and the isorhamnetin diglycoside IGR. On the other hand, De Melo et al. (2009) demonstrated that kaempferol-3-Orhamnosyl-glucopyranoside-7-O-␣-rhamnopyranoside inhibited 50.4% the leukocyte infiltration whereas kaempferol-3,7-O-dirhamnoside inhibited 42.9%. In this investigation, isorhamnetin diglycoside IGR reduced more actively the leukocytes migration compared to the isorhamnetin triglycoside IGRR. As expected and in agreement with Jain and Parmar (2011), cell infiltration after 24 h of carrageenan administration was characterized by the presence of neutrophils. Similarly to the observed with total leukocytes, the number of neutrophils in the air pouch exudates was significantly reduced when treated with
Fig. 2. Fragmentation pattern of isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR) (A) and its corresponding diglycoside isorhamnetin-3-O-glucosyl-rhamnoside (IGR) (B) detected by LC/MSD TOF analysis (ESI+). I: isorhamnetin; GLU: glucose; RHA: rhamnose.
M. Antunes-Ricardo et al. / Industrial Crops and Products 76 (2015) 803–808
Total leukocytes count (cells/mm3)
806
100000 80000 a 60000
b b
b c
40000 20000 0 Control
Neutrophils Monocytes Lymphocytes Eosinophils Basophils
Control 74.3 ± 2.4*a 2.7 ± 0.1b 23.2 ± 1.7a 0.4 ± 0.1a 0.5 ± 0.1ab
Indomethacin
Indomethacin 73.7 ± 9.2a 3.6 ± 1.1b 13.5 ± 2.1a 0.4 ± 0.0a 0.8 ± 0.4ab
IGRR
Treatment IGRR 74.2 ± 3.1a 5.0 ± 0.5ab 16.8 ± 1.7a 0.2 ± 0.1b 1.4 ± 0.3a
IGR
OFI extract
IGR 51.8 ± 10.9b 5.4 ± 1.6ab 20.0 ± 8.3a 0.2 ± 0.1b 1.3 ± 0.4a
OFI extract 58.1 ± 6.1ab 6.2 ± 0.6a 23.3 ± 4.2a 0.2 ± 0.1b 0.3 ± 0.1b
*Data expressed as percentage Fig. 3. Comparison in the number of total leukocytes (neutrophils, monocytes, lymphocytes, eosinophils and basophils) into air-pouch exudates of the groups; control, indomethacin, isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), isorhamnetin-3-O-glucosyl-rhamnoside (IGR) and OFI extract. Vertical bars represent the average (n = 6) with the corresponding standard deviation. a,b,c Different letters indicate significant differences between treatments.
IGR (51.8 ± 10.9%) when compared to the control group (Fig. 3). This inhibitory effect was comparable to that reported for the naringenin-7-O-neohesperidoside at 30 mg/kg in a similar study (53.8%) and higher than the reduction exerted by cyanidin-3-Oglucoside at 40 mg/kg in a peritonitis model (39%) (Hassimotto et al., 2013; Jain and Parmar, 2011). The number of monocytes (macrophages precursors) and lymphocytes remained without significant changes.
3.3. Effect of OFI extract and isorhamnetin glycosides on the inhibition of NO and COX-2 activity induced by carrageenan Administration of IGRR and the OFI extract significantly lowered NO production induced by carrageenan administration with 81.4% and 77.2% of inhibition, respectively (Table 1). Isorhamnetin glycosides and the OFI extract had a better effect on NO inhibition than indomethacin, which was expected considering that this drug is a non-steroidal anti-inflammatory drug (NSAID) that mediate the inhibition of cyclooxygenases 1 and 2 (COX-1 and COX-2). Previous studies showed that the anti-inflammatory mechanism of O. ficus-indica (L.) Mill and isorhamnetin glycosides involved the suppression of the iNOS expression, which is directly related to NO production (Kim et al., 2013b; Lee et al., 2006). Moreover, IGR reduced to 69.6% the NO production, a greater inhibition than that produced by naringenin-7-O-neohesperidoside (15.8 mg/kg/5 days) which decrease to 50% the NO levels (Amaro et al., 2009). The COX-2 inhibition percentages obtained with the OFI extract and the isorhamnetin glycosides were comparable with results obtained by Liu et al. (2012), although the concentration of OFI extract and isorhamnetin glycosides tested was lower in 50 and 5 times, respectively. The aqueous extract from Trigonella foenum-graecum tested at 250 g/mL inhibited in 63.0% the COX-2 activity while the apigenin 6-C-di-ˇ-glucopyranoside (25 g/mL) exhibited an inhibition of 70.0% (Liu et al., 2012). In contrast, in our study the effect of OFI extract on the COX-2 activity inhibition (77.7%) was equal than IGRR (76.3%), but greater than the IGR effect (59.6%) (Table 1). Isorhamnetin with an additional
sugar substitution significantly increased the anti-inflammatory mechanism exhibited by IGR. Data published by Handoussa et al. (2013) showed that the aqueous extract of Corchorus olitorius (100 g/mL) and its major compound, quercetin-3-O-ˇ-D-galactoside (25 g/mL), exerted a similar effect inhibiting the COX-2 activity (62.4% and 60.7%, respectively). In this research, the anti-inflammatory effect of IGRR was the same as observed for the OFI extract, although the amount of IGRR contained in the OFI extract was about thirteen times lower than the dose of IGRR tested. Data suggest that the antiinflammatory effect observed with OFI extract could be influenced by the content of IGRR. 3.4. Effect of isorhamnetin glycosides and OFI extract on cytokines TNF-˛ and IL-6 induced by carrageenan OFI extract showed the highest inhibition (85.2%) on the secretion of TNF-␣ in the exudate (Table 1), but in plasma, TNF-␣ concentration was below the detection limit of the ELISA kit used. This inhibition was higher than the exerted by Eryngium bourgatii extract (100 g/mL) tested in vitro (Cádiz-Gurrea et al., 2013). Regarding to IL-6 content in exudates, OFI extract and IGRR were the best treatments with 53% and 42% of inhibition, respectively (Table 1). In plasma, IGRR and indomethacin inhibited the IL-6 production by 76% and 78%, respectively (Table 1). Results agree with Morikawa et al. (2003) who proposed that quercetin glycosides modulated the TNF-␣ production along with other anti-inflammatory mechanisms. Likewise, Fang et al. (2005) demonstrated that kaempferol glycosides inhibited the TNF-␣ production, observing differences between their effects derived from the type of sugar moieties. The most potent kaempferol glycoside was the kaempferol-3-O--D-apiofuranosyl-(1 → 2)␣-l-arabinofuranosyl-7-O-␣-l-rhamnopyranoside that showed a significant difference TNF-␣ production when tested at 10 M. Kim et al. (2013b) reported that myricetin-3-O-(2 -O-galloyl)-D-galactopyranoside reduced 50% the TNF-␣ production at 8.65 ± 1.62 M, while myricetin-3-O-(2 -O-galloyl)-␣-lrhamnopyranoside and myricetin-3-O-rhamnoside needed
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Table 1 Inhibitory effect, using indomethacin as control, of isorhamnetin glycosides (IGRR, IGR) and OFI extract on the production of nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-␣, and interleukin (IL)-6 in the exudates and plasma obtained after 24 h of induced inflammation in a rat air-pouch model. Exudate Nitric oxide (M) Control Indomethacin IGRR IGR OFI extract
31.5 18.6 5.8 9.6 7.2
± ± ± ± ±
9.3 0.5 (40.8)* c 1.0 (81.4)a 1.0 (69.6)b 0.9 (77.2)a
Plasma COX-2 inhibition (%)
TNF-␣ (pg/mL)
– (64.9)b (76.3)a (59.6)b (77.7)a
177.1 36.7 34.4 33.8 26.4
± ± ± ± ±
9.2 1.4 (79.3)b 0.0 (80.6)b 2.7 (80.9)b 0.5 (85.2)a
IL-6 (ng/mL) 21.3 19.8 12.3 20.6 10.0
± ± ± ± ±
0.2 0.4 (6.8)c 0.4 (42.0)b 0.4 (3.0)c 0.3 (53.0)a
IL-6 (pg/mL) 168.4 37.4 40.1 90.9 73.7
± ± ± ± ±
1.2 5.1 (77.8)a 2.6 (76.2)a 4.6 (46.0)c 1.4 (56.2)b
IGRR: isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside, IGR: isorhamnetin-3-O-glucosyl-rhamnoside, OFI extract: O. ficus-indica extract. * Values in parenthesis correspond to the inhibition percentage with respect to control. a,b,c Different letters in each column indicate significant differences.
12.90 ± 0.84 and 25.20 ± 0.54 M, respectively. In contrast, the behavior of these compounds on IL-6 production was different since myricetin-3-O-(2 -O-galloyl)-␣-l-rhamnopyranoside showed a better effect. In our study, differences in IL-6 inhibition between IGR and IGRR were also observed, showing better effect the triglycoside IGRR because it strongly inhibited the signaling pathways involved in cytokine production. 4. Conclusions Opuntia ficus-indica (L.) Mill extract and their isorhamnetin glycosides, IGRR and IGR, exhibited significant anti-inflammatory potential through different mechanisms of action such as the suppression of cellular infiltration, inhibiting both the production of nitric oxide and activity of COX-2, and decreased secretion of cytokines. Also, we demonstrated that the anti-inflammatory effect was greater with the OFI extract treatment probably because the synergistic effects of their phytochemicals. However, we can only speculate that the effect observed with the OFI extract is mainly due to synergistic effect between isorhamnetin glycosides, particularly isorhamnetin triglycosides. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements Authors acknowledge the financial support from the Consejo Nacional de Ciencia y Tecnología (CONACYT-CB Research Project 1168708 and Scholarship Program) and the Tecnológico de Monterrey (Nutriomics Research Group). Also we acknowledge the donation of Opuntia ficus-indica (L.) Mill flour by Alimentos Funcionales S. de R.L. M.I. References Amaro, M.I., Rocha, J., Vila-Real, H., Eduardo-Figueira, M., Mota-Filipe, H., Sepodes, B., Ribeiro, M.H., 2009. Anti-inflammatory activity of naringin and the biosynthesised naringenin by naringinase immobilized in microstructured materials in a model of DSS-induced colitis in mice. Food Res. Int. 42, 1010–1017, http:// dx.doi.org/10.1016/j.foodres.2009.04.016 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. Crop. Prod. 37, 34–40, http://dx.doi.org/10.1016/j. indcrop.2011.11.027 Antunes-Ricardo, M., Moreno-García, B.E., Gutiérrez-Uribe, J.A., Aráiz-Hernández, D., Alvarez, M.M., Serna-Saldivar, S.O., 2014. Induction of apoptosis in colon cancer cells treated with isorhamnetin glycosides from Opuntia ficus-indica pads. Plant Foods Hum. Nutr. 69, 331–336, http://dx.doi.org/10.1007/s11130-014-0438-5 Ayadi, M.A., Abdelmaksoud, W., Ennouri, M., Attia, H., 2009. Cladodes from Opuntia ficus indica as a source of dietary fiber: effect on dough characteristics and cake making. Ind. Crop. Prod. 30, 40–47, http://dx.doi.org/10.1016/j.indcrop.2009.01. 003
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In vivo anti-inflammatory effects of isorhamnetin glycosides isolated from Opuntia ficus-indica (L.) Mill cladodes Marilena Antunes-Ricardo, Janet A. Gutiérrez-Uribe ∗ , Felipe López-Pacheco, Mario M. Alvarez, Sergio O. Serna-Saldívar Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, C.P. 64849 Monterrey, N.L., Mexico
a r t i c l e
i n f o
Article history: Received 6 January 2015 Received in revised form 7 May 2015 Accepted 27 May 2015 Keywords: Cytokine Flavonoid glycosides Inflammation Isorhamnetin Opuntia ficus-indica (L.) Mill
a b s t r a c t Opuntia ficus-indica (L.) Mill [OFI] has been traditionally used to reduce inflammation. This plant possesses a varied composition of phytochemicals such as flavonoids. The aim of this investigation was to study the anti-inflammatory effects of an OFI extract and two isolated isorhamnetin glycosides using an air-pouch inflammation model. Isorhamnetin glycosides were isolated by semi-preparative chromatography and identified with LC/MSD TOF. Male Wistar rats were divided into five groups (n = 6) receiving isorhamnetin3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), isorhamnetin-3-O-glucosyl-rhamnoside (IGR), OFI extract, indomethacin or vehicle intraperitoneally. IGR showed the best potential to decrease cell infiltration, mainly neutrophils (51.8 ± 10.9%). IGRR and OFI extract inhibited the nitric oxide production (IGRR: 81.4%; OFI extract: 77.2%) and cyclooxygenase-2 activity (IGRR: 76.3%; OFI extract: 77.7%). OFI extract showed the highest inhibition of tumor necrosis factor (TNF)-␣ (85.2%) and interleukin (IL)-6 (53.0%). Results showed that OFI extract and isorhamnetin glycosides possess anti-inflammatory potential. IGRR showed better effect than IGR suggesting that the glycosylation profile affects the bioactivity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Opuntia ficus-indica (L.) Mill is a xerophyte plant belonging to the Cactaceae family. Its cladodes have been traditionally used as a folk medicine due to their hypoglycemic, antioxidant, apoptotic, antibacterial, antifungal and anti-inflammatory activities (Ammar et al., 2012; Antunes-Ricardo et al., 2014; Avila-Nava et al., 2014; Ayadi et al., 2009; López-Romero et al., 2014; Tesoriere et al., 2004). These biological effects have been attributed mainly to the flavonoids content, being the most abundant several types of isorhamnetin glycosides (Santos-Zea et al., 2011). Flavonoids modulate the inflammatory response affecting the nitric oxide (NO) and cyclooxygenase-2 (COX-2) pathways and also by a significant down-regulation of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-␣) and interleukin-6 (IL6) production (Mahat et al., 2010; Maihöfner et al., 2003; Shalini et al., 2012; Xie et al., 2012).
Abbreviations: OFI, Opuntia ficus-indica (L.) Mill; IsoEq, isorhamnetin equivalents. ∗ Corresponding author. E-mail address: [email protected] (J.A. Gutiérrez-Uribe). http://dx.doi.org/10.1016/j.indcrop.2015.05.089 0926-6690/© 2015 Elsevier B.V. All rights reserved.
Isorhamnetin possesses anti-inflammatory effects through inhibiting the NF-B activation and the COX-2 gene expression (Hämäläinen et al., 2007, 2011; Yang et al., 2013). Isorhamnetin-3O-galactoside, besides inhibiting the NF-B activation, suppressed the production of TNF-␣ (Kim et al., 2013a). The number and position of the sugar moieties affect the anti-inflammatory activity of flavonoids (De Melo et al., 2009; Liu et al., 2012). The purpose of this study was to evaluate the anti-inflammatory effects of an O. ficus-indica (L.) Mill extract and two isolated isorhamnetin glycosides on the inflammatory mediators NO, COX-2, IL-6 and TNF-␣ using an in vivo inflammation model.
2. Materials and methods 2.1. Plant The O. ficus-indica (L.) Mill var. Jalpa plant used was harvested in the region of Montemorelos, Nuevo León, México. The taxonomic identification of the cactus species was done at the School of Agronomy of Universidad Autónoma de Nuevo León (UANL), México. The pads of O. ficus-indica (L.) Mill with stage of maturity of 7 months were sanitized with a chlorine solution, cut, dried and ground to obtain a particle size lower than 180 m. The resulting flour was
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packed in sealed bags, protected from light and transported to the laboratory. 2.2. Reagents Organic solvents, n-hexane, n-butanol, and ethyl acetate used for FCPC separations were of analytical grade (DEQ, García, N.L., MX). For HPLC-PDA and LC/MSD TOF analysis, chromatography grade methanol (VWR International LLC, Radnor, PA) and chromatography grade water (VWR International LLC, West Chester, PA) were used. Formic acid solution HPLC grade, isorhamnetin standard ≥95.0% (HPLC), indomethacin [≥99%] and carrageenan (mixture of lambda and kappa-carrageenans) were bought from Sigma Chemical Company (St. Louis, MO). 2.3. Preparation of O. ficus-indica (L.) Mill extract The extraction was performed by alkaline hydrolysis with 4 N NaOH in a 1:10 (mass to solvent ratio) by stirring at 150 rpm in an incubator shaker (Innova 4000; New Brunswick Scientific Inc., Edison, NJ) at 40 ◦ C during 30 min. The subsequent procedure was performed according to the method described by Antunes-Ricardo et al. (2014). Once finalized the alkaline extraction, the sample was filtered and pH was adjusted to 2 with 6 N HCl. Following, lipids were removed with n-hexane (1:1, v/v) and the resulting aqueous fraction was extracted three times with ethyl acetate (1:1, v/v) to remove non-glycosylated flavonoids. Supernatant was filtered through a Whatman filter paper No. 3 and centrifuged at 1550 × g for 10 min and 4 ◦ C in an IEC Centra MP4R centrifuge (International Equipment Co., Needham Heights, MA). An isorhamnetin glycosides enriched fraction was obtained from the crude extract by solid phase extraction using a 1000 mg/6 mL GracePureTM SPE C18Aq cartridge (Grace-Davison, Deerfield, IL), previously conditioned with methanol and distilled water. The isorhamnetin glycosides enriched fraction recovered in 100% methanol was evaporated to dryness under vacuum (50 ◦ C, 50 min) (Genevac Rocket, Ltd., Ipswich, UK). Then, this OFI extract was stored at −80 ◦ C (Labconco, Kansas City, MO). 2.4. Purification and identification of isorhamnetin glycosides Purification and quantification of isorhamnetin glycosides was performed using an HPLC equipped with a photodiode array detector (PDA) (Agilent 1100 Series Santa Clara, CA) according to the method described by Antunes-Ricardo et al. (2014). Chromatograms of OFI extract were obtained at 365 nm. Purification was performed by semi-preparative chromatography using a semipreparative Zorbax SB-C18 (9.4 × 250 mm, 5 m) column operating at a temperature of 17 ◦ C with a flow rate of 2.0 mL/min. The injection volume was 50 L. The mobile phase used was (A) water with 0.1% formic acid (Sigma, St. Louis, MO) and (B) methanol 80%. Separation was achieved starting with 35% of B, increasing to 60% until 2 min, increasing to 90% until 19 min, and then decreasing to 0% of B for the next 3 min. Isorhamnetin glycosides were collected using peak-based mode with maximum peak duration of 0.3 min. Purity of the isolated compounds was greater than 90%. Identification of isorhamnetin glycosides was carried out by LC/MSD TOF (Model G1969A Agilent 1100 Santa Clara, CA) according to the method described by Santos-Zea et al. (2011). 2.5. Animals This study was approved by the Institutional Committee on Care and Use of Experimental Animals at the Tecnológico de Monterrey (Protocol number 2014-RE-001/June 11, 2014). Male Wistar rats (250–300 g) were procured from Bioinvert (Mexico DF, Mexico).
Animals were randomly divided into groups of six animals each and kept in a room with controlled temperature (25 ◦ C) and relative humidity (50 ± 5%) for 12 h light/dark cycles with free access to lab chow pelleted diet and water.
2.6. Carrageenan-induced air-pouch inflammation Animals were grouped randomly into five groups (n = 6), then the air pouches were produced by subcutaneous injection of 20 mL of sterile air into their intra-scapular area and maintained by reinjection of 10 mL of air three days later (Duarte et al., 2012). On the sixth day, O. ficus-indica (L.) Mill extract (5 mg/kg), isorhamnetin-3O-glucosyl-rhamnosyl-rhamnoside (IGRR, 5 mg/kg), isorhamnetin3-O-glucosyl-rhamnoside (IGR, 5 mg/kg), indomethacin (5 mg/kg) or vehicle (0.9% NaCl) were administered intraperitoneally (i.p.). Indomethacin was used as positive control of anti-inflammatory drug. Inflammation was induced after 60 min by the injection of 2 mL of 1% sterile carrageenan directly into the pouch. After 24 h, the animals were euthanized with sodium pentobarbital (60 mg/kg, i.p.) followed by cervical dislocation. A small incision was made in the pouch, and the exudate was carefully removed according to the procedure suggested by Duarte et al. (2012). The total volume of the exudate was measured and the cells were separated by centrifugation (1000 × g for 10 min, at 4 ◦ C). Total and differential counts were performed by microscopic observation. Blood was collected in K2 EDTA Lavender HemogardTM tubes (BD, Franklin Lakes, NJ). Blood samples were centrifuged (1000 × g × 10 min, 4 ◦ C) to obtain plasma. The plasma was stored in small aliquots at −20 ◦ C and used for cytokine assays.
2.7. Cytokines, COX-2 and NO determination The activities of cytokines (IL-6 and TNF-␣) were determined by enzyme-linked immunosorbent assay with commercially available kits (Invitrogen Corp., Camarillo, CA), according to the manufacturer’s instructions. TNF-␣ and IL-6 levels were measured in plasma and air-pouch exudates. The inhibition of COX-2 was determined by the COX fluorescent inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI). NO production was determined with the Griess Reagent System (Promega, Madison, WI) and was expressed as percentage of inhibition of nitric oxide production.
2.8. Statistical analysis Experiments were performed at least by triplicate and results were analyzed with JMP 11.0 software (SAS Institute Inc., Cary, NC) using one-way ANOVA followed by Tukey’s HSD tests. For each data set, p < 0.05 was considered statistically significant.
3. Results and discussion 3.1. Identification and quantification of isorhamnetin glycosides in O. ficus-indica (L.) Mill extract The main flavonoids quantified in the OFI extract corresponded to isorhamnetin diglycosides and triglycosides such as isorhamnetin-3-O-glucosyl-rhamnoside (IGR) and isorhamnetin3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), respectively (Fig. 1). The most abundant triglycoside was IGRR with 77.3 ± 3.8 mg IsoEq/g OFI extract. IGR was the most abundant diglycoside with 30.4 ± 2.6 mg IsoEq/g OFI extract. The fragmentation pattern analysis indicated that the only difference between these two compounds was an additional rhamnose residue in IGRR (Fig. 2).
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OCH3 OH O
HO
O-R OH
O
-R IGRR: Glu-Rha-Rha IGR: Glu-Rha
Fig. 1. Structure of isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR) and its corresponding diglycoside isorhamnetin-3-O-glucosyl-rhamnoside (IGR) isolated from O. ficus-indica extract. I: isorhamnetin; GLU: glucose; RHA: rhamnose.
3.2. Effect of isorhamnetin glycosides and OFI extract on cells count of the air pouches exudates The intraperitoneal administration of IGR 60 min before the injection of carrageenan caused a significant decrease (p < 0.05)
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in the number of total leukocytes (54% decrease) (Fig. 3). IGRR showed a leukocytes reduction of 17.9%, meanwhile indomethacin and OFI extract showed 28.9% and 29.4% decrease, respectively. Contrary to showed by Morikawa et al. (2003), who reported that quercetin-3-O-rhamnoside (quercetin monoglycoside) and quercetin-3-O-rutinoside (quercetin diglycoside) shown no significant differences in leukocyte reduction in air-pouches exudates, in this study differences were observed between isorhamnetin triglycoside IGRR and the isorhamnetin diglycoside IGR. On the other hand, De Melo et al. (2009) demonstrated that kaempferol-3-Orhamnosyl-glucopyranoside-7-O-␣-rhamnopyranoside inhibited 50.4% the leukocyte infiltration whereas kaempferol-3,7-O-dirhamnoside inhibited 42.9%. In this investigation, isorhamnetin diglycoside IGR reduced more actively the leukocytes migration compared to the isorhamnetin triglycoside IGRR. As expected and in agreement with Jain and Parmar (2011), cell infiltration after 24 h of carrageenan administration was characterized by the presence of neutrophils. Similarly to the observed with total leukocytes, the number of neutrophils in the air pouch exudates was significantly reduced when treated with
Fig. 2. Fragmentation pattern of isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR) (A) and its corresponding diglycoside isorhamnetin-3-O-glucosyl-rhamnoside (IGR) (B) detected by LC/MSD TOF analysis (ESI+). I: isorhamnetin; GLU: glucose; RHA: rhamnose.
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Total leukocytes count (cells/mm3)
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100000 80000 a 60000
b b
b c
40000 20000 0 Control
Neutrophils Monocytes Lymphocytes Eosinophils Basophils
Control 74.3 ± 2.4*a 2.7 ± 0.1b 23.2 ± 1.7a 0.4 ± 0.1a 0.5 ± 0.1ab
Indomethacin
Indomethacin 73.7 ± 9.2a 3.6 ± 1.1b 13.5 ± 2.1a 0.4 ± 0.0a 0.8 ± 0.4ab
IGRR
Treatment IGRR 74.2 ± 3.1a 5.0 ± 0.5ab 16.8 ± 1.7a 0.2 ± 0.1b 1.4 ± 0.3a
IGR
OFI extract
IGR 51.8 ± 10.9b 5.4 ± 1.6ab 20.0 ± 8.3a 0.2 ± 0.1b 1.3 ± 0.4a
OFI extract 58.1 ± 6.1ab 6.2 ± 0.6a 23.3 ± 4.2a 0.2 ± 0.1b 0.3 ± 0.1b
*Data expressed as percentage Fig. 3. Comparison in the number of total leukocytes (neutrophils, monocytes, lymphocytes, eosinophils and basophils) into air-pouch exudates of the groups; control, indomethacin, isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside (IGRR), isorhamnetin-3-O-glucosyl-rhamnoside (IGR) and OFI extract. Vertical bars represent the average (n = 6) with the corresponding standard deviation. a,b,c Different letters indicate significant differences between treatments.
IGR (51.8 ± 10.9%) when compared to the control group (Fig. 3). This inhibitory effect was comparable to that reported for the naringenin-7-O-neohesperidoside at 30 mg/kg in a similar study (53.8%) and higher than the reduction exerted by cyanidin-3-Oglucoside at 40 mg/kg in a peritonitis model (39%) (Hassimotto et al., 2013; Jain and Parmar, 2011). The number of monocytes (macrophages precursors) and lymphocytes remained without significant changes.
3.3. Effect of OFI extract and isorhamnetin glycosides on the inhibition of NO and COX-2 activity induced by carrageenan Administration of IGRR and the OFI extract significantly lowered NO production induced by carrageenan administration with 81.4% and 77.2% of inhibition, respectively (Table 1). Isorhamnetin glycosides and the OFI extract had a better effect on NO inhibition than indomethacin, which was expected considering that this drug is a non-steroidal anti-inflammatory drug (NSAID) that mediate the inhibition of cyclooxygenases 1 and 2 (COX-1 and COX-2). Previous studies showed that the anti-inflammatory mechanism of O. ficus-indica (L.) Mill and isorhamnetin glycosides involved the suppression of the iNOS expression, which is directly related to NO production (Kim et al., 2013b; Lee et al., 2006). Moreover, IGR reduced to 69.6% the NO production, a greater inhibition than that produced by naringenin-7-O-neohesperidoside (15.8 mg/kg/5 days) which decrease to 50% the NO levels (Amaro et al., 2009). The COX-2 inhibition percentages obtained with the OFI extract and the isorhamnetin glycosides were comparable with results obtained by Liu et al. (2012), although the concentration of OFI extract and isorhamnetin glycosides tested was lower in 50 and 5 times, respectively. The aqueous extract from Trigonella foenum-graecum tested at 250 g/mL inhibited in 63.0% the COX-2 activity while the apigenin 6-C-di-ˇ-glucopyranoside (25 g/mL) exhibited an inhibition of 70.0% (Liu et al., 2012). In contrast, in our study the effect of OFI extract on the COX-2 activity inhibition (77.7%) was equal than IGRR (76.3%), but greater than the IGR effect (59.6%) (Table 1). Isorhamnetin with an additional
sugar substitution significantly increased the anti-inflammatory mechanism exhibited by IGR. Data published by Handoussa et al. (2013) showed that the aqueous extract of Corchorus olitorius (100 g/mL) and its major compound, quercetin-3-O-ˇ-D-galactoside (25 g/mL), exerted a similar effect inhibiting the COX-2 activity (62.4% and 60.7%, respectively). In this research, the anti-inflammatory effect of IGRR was the same as observed for the OFI extract, although the amount of IGRR contained in the OFI extract was about thirteen times lower than the dose of IGRR tested. Data suggest that the antiinflammatory effect observed with OFI extract could be influenced by the content of IGRR. 3.4. Effect of isorhamnetin glycosides and OFI extract on cytokines TNF-˛ and IL-6 induced by carrageenan OFI extract showed the highest inhibition (85.2%) on the secretion of TNF-␣ in the exudate (Table 1), but in plasma, TNF-␣ concentration was below the detection limit of the ELISA kit used. This inhibition was higher than the exerted by Eryngium bourgatii extract (100 g/mL) tested in vitro (Cádiz-Gurrea et al., 2013). Regarding to IL-6 content in exudates, OFI extract and IGRR were the best treatments with 53% and 42% of inhibition, respectively (Table 1). In plasma, IGRR and indomethacin inhibited the IL-6 production by 76% and 78%, respectively (Table 1). Results agree with Morikawa et al. (2003) who proposed that quercetin glycosides modulated the TNF-␣ production along with other anti-inflammatory mechanisms. Likewise, Fang et al. (2005) demonstrated that kaempferol glycosides inhibited the TNF-␣ production, observing differences between their effects derived from the type of sugar moieties. The most potent kaempferol glycoside was the kaempferol-3-O--D-apiofuranosyl-(1 → 2)␣-l-arabinofuranosyl-7-O-␣-l-rhamnopyranoside that showed a significant difference TNF-␣ production when tested at 10 M. Kim et al. (2013b) reported that myricetin-3-O-(2 -O-galloyl)-D-galactopyranoside reduced 50% the TNF-␣ production at 8.65 ± 1.62 M, while myricetin-3-O-(2 -O-galloyl)-␣-lrhamnopyranoside and myricetin-3-O-rhamnoside needed
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Table 1 Inhibitory effect, using indomethacin as control, of isorhamnetin glycosides (IGRR, IGR) and OFI extract on the production of nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-␣, and interleukin (IL)-6 in the exudates and plasma obtained after 24 h of induced inflammation in a rat air-pouch model. Exudate Nitric oxide (M) Control Indomethacin IGRR IGR OFI extract
31.5 18.6 5.8 9.6 7.2
± ± ± ± ±
9.3 0.5 (40.8)* c 1.0 (81.4)a 1.0 (69.6)b 0.9 (77.2)a
Plasma COX-2 inhibition (%)
TNF-␣ (pg/mL)
– (64.9)b (76.3)a (59.6)b (77.7)a
177.1 36.7 34.4 33.8 26.4
± ± ± ± ±
9.2 1.4 (79.3)b 0.0 (80.6)b 2.7 (80.9)b 0.5 (85.2)a
IL-6 (ng/mL) 21.3 19.8 12.3 20.6 10.0
± ± ± ± ±
0.2 0.4 (6.8)c 0.4 (42.0)b 0.4 (3.0)c 0.3 (53.0)a
IL-6 (pg/mL) 168.4 37.4 40.1 90.9 73.7
± ± ± ± ±
1.2 5.1 (77.8)a 2.6 (76.2)a 4.6 (46.0)c 1.4 (56.2)b
IGRR: isorhamnetin-3-O-glucosyl-rhamnosyl-rhamnoside, IGR: isorhamnetin-3-O-glucosyl-rhamnoside, OFI extract: O. ficus-indica extract. * Values in parenthesis correspond to the inhibition percentage with respect to control. a,b,c Different letters in each column indicate significant differences.
12.90 ± 0.84 and 25.20 ± 0.54 M, respectively. In contrast, the behavior of these compounds on IL-6 production was different since myricetin-3-O-(2 -O-galloyl)-␣-l-rhamnopyranoside showed a better effect. In our study, differences in IL-6 inhibition between IGR and IGRR were also observed, showing better effect the triglycoside IGRR because it strongly inhibited the signaling pathways involved in cytokine production. 4. Conclusions Opuntia ficus-indica (L.) Mill extract and their isorhamnetin glycosides, IGRR and IGR, exhibited significant anti-inflammatory potential through different mechanisms of action such as the suppression of cellular infiltration, inhibiting both the production of nitric oxide and activity of COX-2, and decreased secretion of cytokines. Also, we demonstrated that the anti-inflammatory effect was greater with the OFI extract treatment probably because the synergistic effects of their phytochemicals. However, we can only speculate that the effect observed with the OFI extract is mainly due to synergistic effect between isorhamnetin glycosides, particularly isorhamnetin triglycosides. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements Authors acknowledge the financial support from the Consejo Nacional de Ciencia y Tecnología (CONACYT-CB Research Project 1168708 and Scholarship Program) and the Tecnológico de Monterrey (Nutriomics Research Group). Also we acknowledge the donation of Opuntia ficus-indica (L.) Mill flour by Alimentos Funcionales S. de R.L. M.I. References Amaro, M.I., Rocha, J., Vila-Real, H., Eduardo-Figueira, M., Mota-Filipe, H., Sepodes, B., Ribeiro, M.H., 2009. Anti-inflammatory activity of naringin and the biosynthesised naringenin by naringinase immobilized in microstructured materials in a model of DSS-induced colitis in mice. Food Res. Int. 42, 1010–1017, http:// dx.doi.org/10.1016/j.foodres.2009.04.016 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. Crop. Prod. 37, 34–40, http://dx.doi.org/10.1016/j. indcrop.2011.11.027 Antunes-Ricardo, M., Moreno-García, B.E., Gutiérrez-Uribe, J.A., Aráiz-Hernández, D., Alvarez, M.M., Serna-Saldivar, S.O., 2014. Induction of apoptosis in colon cancer cells treated with isorhamnetin glycosides from Opuntia ficus-indica pads. Plant Foods Hum. Nutr. 69, 331–336, http://dx.doi.org/10.1007/s11130-014-0438-5 Ayadi, M.A., Abdelmaksoud, W., Ennouri, M., Attia, H., 2009. Cladodes from Opuntia ficus indica as a source of dietary fiber: effect on dough characteristics and cake making. Ind. Crop. Prod. 30, 40–47, http://dx.doi.org/10.1016/j.indcrop.2009.01. 003
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