1. INTRODUCTION
Oxidative stress and inflammation are the main causes of many chronic diseases including rheumatoid arthritis (RA). Oxidative stress plays an important role in the initiation and progression of joint diseases (Knight, 2000). Reactive oxygen species (ROS) cause oxidative damage which accumulates during the life cycle, the radical-related damage affects DNA, proteins and lipids which have been proposed to play a key role in the development of arthritis.
RA is an autoimmune disease that causes chronic inflammation of the joints and surrounding tissue with infiltration of macrophages and activated T cells. The pathogenesis of this disease is predominantly linked with the formation of free radicals at the site of inflammation. The disease progression of RA is associated with chronic soft tissue inflammation, which is often followed by bone and cartilage destruction of inflamed joints (Anderson et al., 2012). Oxidative injury and inflammation is reflected in increased levels of serum TNF-α that has been shown to play a critical role in the onset and progression of RA. The overproduction of TNF-α could stimulate cartilage matrix degradation by inhibiting the production of proteoglycans and type II collagen and up-regulating the production of matrix-degrading enzymes (Goldring, 2000). An excessive accumulation of ROS may cause cellular oxidative damage to nucleic acids, proteins and chromosomal aberrations in the cells of several systems and that could occur in RA as reported previously (Al-Okbi et al., 2011).
The inverse association between fruit and vegetable intake and the risk of chronic diseases related to morbidity and mortality is often attributed to bioactive ingredients that may possess anti-inflammatory and antioxidant activity such as vitamins C and E, carotenoids and polyphenols (Prakash and Gupta, 2009). Plant foods are rich sources of such bioactive ingredients. The pumpkin seed is among the plant foods that contain high levels of bioactive ingredients, including β-carotene, unsaturated fatty acids, phenolic compounds, phytosterols and tocopherols especially in the oil compartment. These functional food ingredients have been reported to possess both anti-inflammatory and antioxidant activities in hyper-cholesterolemic rats (Al-Okbi et al., 2014).
The aim of the present research was to assess the functional food ingredients, β-carotene and phenolic contents, in PSOs of the Egyptian and European varieties. The main objective was to evaluate the PSOs’ anti-inflammatory activity and the related mechanism in a rat model of chronic inflammation that simulates RA in humans.
2. MATERIALS AND METHODS
2.1. Plant materials
Egyptian pumpkin seeds (Cucurbita moschata, L. Family Curcubitaceae) were purchased from the local market, Cairo, Egypt. A European PSO (Cucurbita pepo, L. Family Cucurbitaceae var. styria) was obtained from Graz, Austria.
2.2. Animals
Male white albino rats of 100-110 g body weight were used in the present study. The animals were kept individually in stainless steel cages; water and food were given ad-libitum. The animal experiment was carried out adopting the Ethics Committee of the National Research Centre, Cairo, Egypt, and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).
2.3. Major chemicals
Colchicine and Folin-Ciocalteu reagent were purchased from Sigma (USA) and Sigma-Aldrich, Germany, respectively. Freund’s complete adjuvant (FCA) was supplemented from DIFCO LABORATORIES, Detroit, Michigan USA. All chemicals and solvents used were of high quality analytical grade.
2.4. Preparation of PSO
PSO was prepared from Egyptian pumpkin seeds by extraction using petroleum ether (40–60ºC) (Al-Okbi et al., 2014).
2.5. Determination of total phenolic content (TPC) of the oils
TPC was extracted from PSOs and determined colorimetrically using the Folin-Ciocalteu reagent (Singleton and Rossi, 1965). Absorbance was measured at 765 nm using a UVPC spectrophotometer. Gallic acid was used as a standard and results were calculated as mg gallic acid equivalent (GAE) per gm of oil. The reaction was conducted in triplicate and results were averaged.
2.6. Assessment of β-carotene in Egyptian and European PSO
β-Carotene was determined using the HPLC method (Hart and Scott, 1995). The exact weights of 2.5 grams from the Egyptian and European PSOs were weighed in two separate glass screw cap tubes. The oils were homogenized with 20 ml ethanol (HPLC) by vortex mixing (5 min), then 10 ml of hexane (HPLC) were added to each tube, mixed for 2 min, centrifuged (5 min, 5000g) and the clear upper layer was carefully transferred to other glass screw cap tubes. The samples were re-extracted twice with hexane. Then 7 ml of the hexane extracts were dried to completion under a nitrogen stream and re-suspended in 1 ml of 50% ethanol for analysis by the HPLC System. HPLC conditions: Waters Melinnium 3.2 software using a system equipped with a binary pump system (Waters 515), an auto-injector (Waters 717 plus), a PDA detector (Waters 996), and a column heater (Spectra Physics SP8792). The compound was separated on a 4.6 x 250 mm, 5 µm, YMC Carotenoid column (C-30 reverse-phase) purchased from Waters (Milford, MA), which was maintained at 35 °C. The following gradient system was used: methanol/water/triethylamine (90:10:0.1v/v/v) (A), and methanol/MTBE/triethylamine (6:90:0.1v/v/v) (B); gradient (min/%A) 0/99, 8/99, 45/0, 50/0, and 53/99. The column was brought back to initial conditions, and allowed to equilibrate for 10 minutes before injection. All solvents were filtered and degassed before use. The concentration of β-carotene in the samples was obtained by comparing its peak area with the peak area of standard β-carotene in relation to concentration.
2.7. Preparation of dosage form
An emulsion of each oil was prepared using gum acacia to easily manage the rats’ oral dose. The same concentration of gum acacia was prepared in water (the vehicle) to be given to control groups of rats.
2.8. Preparation of diet
A balanced diet was prepared containing 11.9 casein (10 protein), 10 sunflower oil, 45.73 corn starch, 22.87 sucrose, 5 cellulose, 1 vitamin mixture and 3.5 salt mixture as g./100g.
2.9. In-vivo study of the anti-inflammatory activity of PSO in rats
The potential anti-inflammatory effect of PSOs was evaluated in an adjuvant arthritis model (chronic inflammation model) of rats. Adjuvant arthritis (AA) was induced by subcutaneous injection of 0.3 ml FCA/rat into the sub-planter region of the right hind paw (Singh et al., 1992).
Thirty-six male albino rats were divided into 6 groups, 6 rats each; all rats were fed a normal balanced diet throughout the experiment. The rats from the first group (control normal) received no medication or injection and were given a daily oral dose of the vehicle for 21 days. Rats of the second group (adjuvant arthritis control) were given a daily oral dose of the vehicle for 21 days; the rats were treated with FCA 8 days from starting vehicle dosing. The rats of group 3 were given a daily oral dose of Egyptian PSO (40 mg/Kg rat body weight) for 21 days along with treatment by FCA 8 days later from starting the oral dose. Rats of group 4 were given a daily oral dose of Egyptian PSO (500 mg/Kg rat body weight) for 21 days while FCA was injected 8 days from starting the oral dose. The rats of groups 5 and 6 were treated like groups 3 and 4, respectively but with applying the European PSO. The food intake and body weight of rats were recorded once a week. At the end of the experiment, total food intake, body weight gain and food efficiency ratio were calculated. Paw thickness was measured immediately before the induction of arthritis (zero time) and at the end of the experiment using Vernier caliper to assess the degree of inflammation. The increase in the thickness of the injected paw (inflammation thickness) of the rats of all groups was calculated. Also, at the end of the experiment, rats were anesthetized and blood samples were withdrawn from the eye vein orbital after an overnight fast (16 h). Blood samples were divided into two parts. One was mixed with trisodium citrate (109 mmol/L) for the determination of the erythrocyte sedimentation rate (Westergren, 1921), the second part was mixed with heparin as anticoagulant, followed by centrifugation at 3000 rpm for 15 min for separation of plasma and determination of TNF-α (Stepaniak et al., 1995), MDA (Satoh, 1978) and total antioxidant capacity (Koracevic et al., 2001). ESR and TNF-α are both considered as inflammatory biomarkers. Plasma MDA and total antioxidant capacity were assessed for evaluating the oxidative stress and/or antioxidant status.
All rats were kept alive for an additional 24 hours and treated by intraperitoneal injection of colchicine at a concentration of 3 mg/kg body weight. After 2 hours of injection, rats were anesthetized with ether and sacrificed; the liver was separated, washed with saline and received upon filter paper to remove any excess blood then wrapped with foil and kept at -20ºC until being analyzed for DNA fragmentation assay. The reproductive tract was exposed and the epididymus were excised. Both epididymus of each rat were received in a tube containing saline. The femurs bones of both legs of each rat were also received on saline in another tube. Both epididymus and the bone marrow of the femur were taken immediately for cytogenetic and chromosomal aberration tests.
For an extra cytogenetic study, two more groups were run parallel to the previous groups; each of 6 rats; a group was given daily oral dose of Egyptian PSO (500mg/Kg rat body weight) and the other group was given the same dose from the European variety for 21 days and fed on balanced diet.
2.10. Assessment of cytogenetic parameters
2.10.1. Chromosomal aberrations test
Bone marrow metaphases were prepared (Yosida and Amano, 1965). Bone-marrow cells from both femurs were collected in a 6-8 ml hypotonic solution of KCl (0.075 M). The cell suspension was incubated for about 20 min. at 37 °C then centrifuged at 1000 rpm for 10 min. After discarding the supernatant, cells were fixed by re-suspending in freshly-prepared cold fixative methanol/ acetic acid (3:1), and centrifuged again for 10 min. at 1000 rpm. The fixation step was repeated at least three times. Finally, the cells were re-suspended in the appropriate volume of fixative and were spread by dropping the concentrated cell suspensions onto slides that had just been removed from the freezer. Five to seven slides were prepared for each rat. The slides were stained with 7% Giemsa in phosphate buffer (pH 6.8). One hundred well-spread Metaphases were analyzed per animal. Different chromosome aberrations including gaps, breaks, fragments and deletions were recorded.
2.10.2. Sperm shape abnormalities
The protocol recommended previously (Wyrobek, 1978) was used. Both epididymus from each rat were minced together with small scissors in physiological saline, pipetted up and down and then filtered into small test tubes to exclude large tissue fragments to which the volume was made up to 2 ml. Smears were prepared on clean dry slides. The slides were stained in 1% Eosin Y (aqueous). Six rats were taken for each treatment and at least three slides were prepared for each rat to study sperm abnormalities for microscopical examination. For each dose, at least 5000 sperms were assessed for morphological abnormalities of the sperm shape.
2.10.3. DNA fragmentation assay
The method of DNA fragmentation was carried out (Perandones et al., 1993). Reagents used were hypotonic lysis buffer pH 8.0, 10% Trichloroaceticacid (TCA), 5% TCA and color reagent. Hypotonic lysis buffer was made from 0.2%Triton X-100, 10mM Tris and 1mM EDTA while the color reagent consisted of 0.088M Diphenylamine (DPA), 98% v/v Glacial acetic acid, 1.5% v/v Sulphuric acid and 0.5% v/v 1.6% Acetaldehyde solution. The liver tissues (50 mg) were mechanically dissociated in 400 µL hypotonic lysis buffer to obtain cell lysate. The cell lysate was centrifuged at 13.800 xg for 15 minutes. The supernatant containing small DNA fragments was separated immediately, the supernatant and the pellet containing large pieces of DNA, were used for the diphenylamine (DPA) assay.
2.10.3.1. Diphenylamine assay (DPA)
The colorimetric determination of DNA content was assessed (Perandones et al., 1993). Both the supernatant and the pellet were used for DPA assay after acid extraction of DNA. The pellet containing large fragments of DNA and cell debris was re-suspended in a 400 µl hypotonic lysis buffer. TCA (10%) was added (400 μl) to both the supernatant and the re-suspended pellet. The tubes were centrifuged at 2000 rpm for 10 min. The precipitate was re-suspended in 400 µl of 5% TCA. The tubes were incubated at 80 ºC for 30 min. The supernatant containing the extracted DNA was left to cool at room temperature. Two volumes of the color reagent were added to one volume of extracted DNA. The samples were stored at 4 ºC for 48h till the blue color was developed. The blue color was measured colorimetrically using a spectrophotometer at 578 nm. The percentage of DNA fragmentation in each sample was expressed by the formula: % DNA fragmentation = OD of supernatant x 100⁄OD of supernatant + OD of pellet, where OD is the optical density.
2.11. Statistical analysis
Results of total phenolic content were expressed as mean ± SE with application of t-Students test. The results of the animal experiments were presented as the mean ± SE and were analyzed statistically using one-way analysis of variance ANOVA followed by LSD test. In all cases p < 0.05 was used as the criterion for statistical significance. For the statistical analysis of chromosomal aberrations in bone marrow cells, the Chi-square test (X
2 contingency table) was applied. One-way analysis of variance (ANOVA test) was applied to the data of sperm shape abnormalities and DNA fragmentation in liver cells.
4. DISCUSSION
In the present research, the anti-inflammatory activity of two varieties of PSO, an Egyptian and a European, was studied in a chronic inflammatory model of adjuvant arthritis in rats. These types of oils were selected as they are expected to be rich in bioactive ingredients which could have antioxidant, free radical scavenging capacity and anti-inflammatory effects.
In the present study, the induction of AA in rats produced high oxidative stress reflected in elevated plasma MDA and reduced total antioxidant capacity. Also inflammatory biomarkers represented by ESR and plasma TNF-α were significantly elevated in AA rats with the induction of foot paw inflammation. In support of the present work, arthritis is described as being a condition that involves systemic oxidative stress (Strosova et al., 1995). In previous studies, it was shown that plasma oxidative stress and inflammatory biomarkers increased in both AA rats and in patients with RA (Al-Okbi et al., 2000a; 2000b; 2011; Stepaniak et al., 1995).
TNF-α is a potent pro-inflammatory cytokine that plays an important role in inflammation. It binds to its cellular receptor TNF receptor 1, which triggers signaling cascades that activate NF-κB and activator protein 1 transcription factors. Pro-inflammatory cytokines like IL-1β and TNF-α could thus help to propagate the extension of a local or systemic inflammatory process by activating NF-κB, forming a positive feedback mechanism which exaggerates the inflammatory process (Sonis, 2002).
Overproduction of lipid peroxide oxidative stress marker, MDA, was noticed in AA rats (Zhang et al., 2014). AA was monitored by hind paw volume and high level of MDA. During inflammatory joint diseases, like adjuvant-induced arthritis, phagocytes accumulate in the joints and produce superoxide and hydroxyl radicals as well as hydrogen peroxide. Such reactive oxygen species might contribute to the elevated ESR, plasma MDA, and TNF-α observed in the arthritic model of inflammation. An elevation of the oxidative damage marker such as blood MDA with reduction in total antioxidants in the present study demonstrated a disturbance in the oxidant to antioxidant balance in favor of lipid peroxidation, which could lead to the tissue damage seen in the disease. These results suggest the importance of therapeutic co-administration of antioxidants with conventional drugs in such condition.
Paw swelling, body weight loss and overproduction of serum TNF-α and MDA were reported as the main pathological signs in FCA-induced arthritis in rats (Zhang et al., 2014). Paw volume and ESR were evaluated as arthritic markers (Banji et al., 2014). During arthritis, TNF-α has been implicated in the pathological mechanisms of synovial tissue proliferation, joint destruction and apoptosis. Hemshekhar et al. (2013) demonstrated a significant reduction in arthritis-induced paw swelling and body weight regain, establishing a close relationship between inflammation and loss of body weight. TNF-α played a role in the increased prevalence of low body mass which has been reported in arthritic individuals (Kaufmann et al., 2003). The decrease in body weight gain and food efficiency ratio in the control AA rats in the present study is similar to that in rheumatoid arthritis patients where the loss of lean tissues could be due to tissue destruction. Elevated TNF-α in arthritis could contribute in cartilage and bone degeneration through the activation of extracellular matrix degrading enzymes. The increase in TNF-α in the present study could thus explain the loss in weight gain. The enhanced degradation of muscle protein and muscle wasting in experimental arthritis might have a hand in body weight reduction. The decrease in total food intake in adjuvant arthritis rats in the current study is similar to the observed anorexia in RA patients and could participate in reducing body weight gain.
The administration of both PSOs to arthritic rats in the present study produced significant improvements in inflammatory and oxidative stress biomarkers together with a reduction in inflammation thickness. These effects are certainly attributed to the presence of antioxidant and anti-inflammatory constituents such as tocopherols, carotenoids, phytosterols, and phenolic compounds in the POs. The variation in degree of improvements may be related to the different contents and concentration of bioactive constituents and their synergistic effects. The present study showed the presence of phenolic compounds and beta- carotene in both varieties of PSOs. Also, the two varieties of PSOs were reported previously to contain phytosterols, unsaturated fatty acids, and tocopherols (Al-Okbi et al., 2014). These ingredients could collectively render the oils their antioxidant and anti-inflammatory activity.
Egyptian and European PSOs significantly inhibited the elevated plasma levels of TNF-α and MDA, thereby reducing the severity of inflammation as noticed from the reduced paw inflammation thickness. Eventually, elevated ROS promotes liver damage by elevating the levels of lipid peroxides. Lipid peroxides, represented in the present study by MDA, could behave as a secondary toxic trigger causing further damage by modulating membrane fluidity, permeability and transport in arthritic patients. Thus the level of MDA may be a sensitive marker of oxidative damage during arthritis and treatment with PSO which reflects the potent antioxidant and free radical scavenging activity of PSO.
PSOs have a significant inhibitory effect on foot inflammation and this ability to reduce edema may be related to its inhibitory action on TNF-α and ESR which reflect their anti-inflammatory activity. Also both Egyptian and European PSO doses significantly elevate the antioxidant capacity and thus they may play an important role in reducing the oxidative stress associated with the arthritic condition, and therefore has the potential to be used as an anti-arthritic agent.
It was noticed that all treatments produced a significant increase in body weight gain and food efficiency ratio which is reflected in the improvement of AA condition.
The presence of α, δ and γ-tocopherol in PSO (Al-Okbi et al., 2014) could have a significant impact on improving the changes in AA rats. The phenolic content and the considerable concentration from both tocopherol and β-carotene in PSO may explain the antioxidant and anti-inflammatory effects. The presence of a high phenolic content was reported to play an anti-arthritic role through lowering ESR and restoring body weight (Gupta et al., 2014).
Antioxidants play an important role in relieving arthritis in rats (Zhang et al., 2014). Chronic administration of PSO in the present study caused a remarkable elevation of serum antioxidant levels and a marked inhibition of paw edema that could be attributed to its high content of the aforementioned antioxidants.
PSO administration improved biochemical parameters studied in the present research during chronic arthritis which could be ascribed to the high level of unsaturated fatty acids in PSO and other antioxidants. Previously, extra-virgin olive oil was reported to reduce inflammation and cartilage-matrix degradation in a mice model of arthritis which was assigned to the adequate fatty acid profile and the presence of high phenolic content in the oil (Rosillo et al., 2016). Similarly, evening primrose oil could reduce the pro-inflammatory mediators in a model of fibromyalgia syndrome in mice (Montserrat-de la Paz et al., 2013) and in RA patients (Al-Okbi et al., 2000a) which might most probably be due to the presence of polyunsaturated fatty acids particularly gamma- linolenic acid. In another study, functional foods containing either fish oil or primrose oil showed beneficial effects in a rat model of arthritis due to their high levels of polyunsaturated fatty acids (Al-Okbi et al., 2012).
The present study indicated that AA has a highly significant genotoxic effect on rat bone marrow cells as well as sperm and liver DNA. Our results are in agreement with a previous study which reported the induction of structural and numerical aberrations in the synovial fibroblasts in RA patients and other inflammatory joint diseases (Wyllie et al., 1980). The current work also indicated DNA fragmentation induced by AA in rat liver cells similar to the work of Wyllie et al. (1980).
Oxidative metabolism is considerably enhanced in the liver of AA rats. The livers of adjuvant-induced arthritis rats show a pronounced high oxidative stress which is the consequence of both a stimulated pro-oxidant system and reduction in antioxidant defense represented by the lowering in catalase and glutathione peroxidase activities (Kelmer-Bracht et al., 2003). This liver high oxidative stress could be the cause of the observed DNA damage in the AA rats in the present study. The mRNA levels of mdr1a and mdr1b encoding P-glycoprotein (P-gp) decreased significantly in the livers of the AA rats (Kawase et al., 2014). In arthritic rats, it was observed previously that there was a significant elevation in DNA damage and genotoxicity (Al-Okbi et al., 2011). Mutation rates might be increased due to the elevated level of DNA damage in adjuvant arthritis. The present study showed the protective role of the tested PSOs by inhibiting the genotoxic effects induced by adjuvant arthritis and the protection of cells against oxidative DNA damage. The bioactivity of PO could be attributed to the presence of the aforementioned bioactive constituents that could impart antioxidant and anti-inflammatory activity.
It was reported that carotenoids reduced the induction of micronuclei in polychromatic erythrocytes in mice bone-marrow cells (Rauscher et al., 1998). The protective effect of nut oil was demonstrated against free radical capacity due to the presence of tocopherols. Tocopherol itself was shown previously to possess protective effects against the induced genotoxicity in mice bone-marrow cells (Arranz et al., 2008). So tocopherols in combination with the different functional ingredients of PSO in the present study could have synergistic anti-genotoxicity. In the current research, the mutagenic effect and DNA fragmentation in AA was minimized by the oral administration of POs which significantly inhibited the sperm shape abnormalities, DNA fragmentation and chromosomal aberrations of somatic and germ cells. Total phenolics have been reported to have anti-mutagenic effects (Batista et al., 2016), so the phenolic content of PSO could have a similar effect.
The essential fatty acid, linoleic acid accounts for nearly one-third of the total fatty acid in pumpkin seeds while the contribution of α-linolenic acid was considerably low (Glew et al., 2006; Tsaknis et al., 1997). These fatty acids could participate in the bioactivity of PSO.
Mini Paulista and Nova Caravela pumpkin varieties showed high amounts of total phenolic compounds in the lipid fractions and in the seeds. It was also found that γ-tocopherol is the isomer that stood out in the lipid fractions and in the seeds, mainly in Menina Brasileira (Veronezi and Jorge, 2012).