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Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis
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Research paper
Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis Zeinab Nikniaza , Reza Mahdavib,* , Alireza Ostadrahimib , Aliasghar Ebrahimic , Leila Nikniazd, Amirmansour Vatankhahe a
Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Nutrition Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran c Connective Tissue Diseases Research Centre, Tabriz University of Medical Sciences, Imam Reza Hospital, Iran d Tabriz Health Services Management Research Center, Tabriz University of Medical Sciences, Tabriz, Iran e Drug Applied Research Center, Tabriz University of Medical Science, Tabriz, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 June 2015 Received in revised form 14 July 2015 Accepted 1 September 2015 Available online xxx
Aim: The present study aimed to determine the effects of Elaeagnus angustifolia L. supplement on serum antioxidant status in females with knee osteoarthritis. Methods: In this 8-week randomized clinical trial, 90 females with knee osteoarthritis were randomly assigned to one of three groups—medulla powder (15 g medulla powder), whole fruit powder (15 g whole fruit powder) or placebo. Information on dietary intake was collected using 24-h recall method for 3 days (one weekend day included). The serum levels of total antioxidant capacity (TAC) and malondialdehyde (MDA) were measured using the Randox1 assay and the fluorometry method respectively. Paired sample t-test, ANOVA and ANCOVA were used for statistical analysis. Results: The mean serum TAC level in the medulla, whole fruit and placebo groups increased from 1.12 0.34 to 1.28 0.27, 1.30 0.28 to 1.50 0.28 and 1.18 0.19 to 1.30 0.22 mmol/L respectively. In terms of the TAC level, the significant increase was only observed in the whole fruit group (P = 0.001). The mean serum MDA level in the medulla, whole fruit and placebo groups decreased from 3.80 0.33 to 3.30 0.41, 3.25 0.51 to 2.45 0.43 and 3.38 0.63 to 2.56 0.41, respectively. A statistically significant reduction was observed only in the whole fruit group (P = 0.009). Discussion: Based on the results, E. angustifolia L. in the form of whole fruit had beneficial effects on serum TAC and MDA levels in females with knee OA. This may show a possible disease modifying effect of E. angustifolia L. in knee osteoarthritis. ã 2015 Elsevier GmbH. All rights reserved.
Keywords: Female Malondialdehyde Osteoarthritis Total antioxidant capacity
1. Introduction According to worldwide estimates, approximately 10% of men and 18.0% of females aged <60 years have symptomatic osteoarthritis (Woolf and Pfleger, 2003). With increasing life expectancy together with aging populations, it is expected that, by the year
Abbreviations: ANCOVA, analysis of covariance; ANOVA, analysis of variance; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); MDA, malondialdehyde; TAC, total antioxidant capacity; ROS, reactive oxygen species; OA, osteoarthritis; BMI, body mass index. * Corresponding author at: Golgasht St. Attarneishabouri Ave, nutrition faculty, Tabriz University of Medical Sciences,Tabriz, Iran. E-mail address: [email protected] (R. Mahdavi).
2020, osteoarthritis will become the fourth leading cause of disability (Murray and Lopez, 1996). Clinically OA is characterized by joint pain, tenderness, limitation of movement, crepitus, and local inflammation (Woolf and Pfleger, 2003). It can occur in any joint but it is most common in the knee joint (Woolf and Pfleger, 2003). Although little is known about its etiopathology, it is supposed that OA is a consequence of mechanical and biological events that break down articular cartilage, in the synovial joints (Chen et al., 2006). Biological events that may affect articular cartilage are inflammation and a change in antioxidant status. Increasing evidence in both experimental and clinical studies have suggested that oxidative stress is considered to be one of the main causative factors in the pathogenesis of OA (Sarban et al., 2005; Surapaneni and Venkataramana, 2007; Sutipornpalangkul et al., 2009;
http://dx.doi.org/10.1016/j.hermed.2015.09.003 2210-8033/ ã 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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Ostalowska et al., 2006; Karan et al., 2003). Reactive oxygen species (ROS) production has been found to increase in joint diseases such as osteoarthritis (Deberg et al., 2005). Although the precise mechanism remains unclear (Yudoh et al., 2005), some studies have suggested that oxidative stress causes chondrocyte senescence and cartilage aging (Yudoh et al., 2005; Martin et al., 2004). Lipid peroxidation induced by reactive oxygen species (ROS) is considered to be the major mechanism of cell membrane destruction and cell damage (Henrotin et al., 2005). Decomposition of peroxidized lipids leads to production of a wide variety of end-products, including MDA (Gambhir et al., 1997). Elevated levels of MDA as the indicator of lipid peroxidation have been reported in the serum and synovial fluid of OA patients (Lunec et al., 1981). The negative effects of reactive oxygen species are usually neutralized by the total antioxidant capacity (TAC); however, sometimes the TAC cannot cope with the excessive increase of free radicals, and then the TAC will decrease. The decreased levels of serum TAC level in OA patients have been shown in earlier studies (Altindag et al., 2007; McAlindon et al., 1996). Based on the results of previous studies, antioxidant agents such as vitamin C, vitamin E, and b-carotene have recently attracted considerable attention in treating osteoarthritis (McAlindon et al., 1996; Wang et al., 2007). Despite the antioxidant effects of some drugs used in OA treatment (Ozgocmen et al., 2005), they also have potential adverse effects (Silverstein et al., 2002). Thus, considering the sideeffects of these drugs, the use of complementary medicines and therapies are increasing in patients with knee OA. One of the complementary therapies in Iran that has been used as a folk remedy for arthritis patients is Elaeagnus angustifolia L. fruit. This fruit is also used as a folk medicine in Turkey for wound healing (Ersoy et al., 2013), in Pakistan for headache, heartburn and skin infections (Ahmad et al., 2014) and in China (Wang et al., 2012) and Jordan (Lev and Amar 2002) for diarrhea. E. Angustifolia topical gel has also been shown to be effective in the treatment of symptomatic oral lichen planus, with anti-inflammatory and analgesic effects (Taheri et al., 2010). Previous studies have shown that daily supplementation with this fruit had positive effects in improving pain, stiffness and physical function and for decreasing inflammatory cytokines and enhancing anti-inflammatory cytokines (Ebrahimi et al., 2014; Nikniaz et al., 2014). The fruit contains many flavonoids and antioxidant vitamins with significant antioxidant potential (Ersoy et al., 2013). In Iran, this fruit is consumed in two forms; (1) whole fruit powder (the whole fruit powder including seed, medulla and pericarp) (2) only the pulp (medulla) of the fruit which were available in local markets or Iranian traditional medicinal plant stores. Despite its extensive use as a folk remedy (Mirhydar, 1998), to the best of the authors’ knowledge, there are no clinical studies about the effect of this fruit on osteoarthritis patients. As, it was hypothesized that Elaeagnus angustifolia L. as a complementary treatment may have beneficial effects on antioxidant status, the effects of two forms of whole fruit and medulla powders of this fruit on serum TAC and MDA level were assessed in females with knee OA. 2. Materials and methods One hundred and twenty patients with knee osteoarthritis with disease severity from mild to moderate based on the criteria of the American College of Rheumatology (ACR) (Altman et al., 1986) and who had been referred to the Rheumatology Centre of Sina Hospital, Tabriz, Iran, were selected for the study. The patients were included if they were female, aged 40–70 years, had moderate to severe bilateral primary knee osteoarthritis
(according to ACR classification) (Altman et al., 1986) and body mass index (BMI) of 25–34.9 kg/m2. The patients were excluded if they had secondary osteoarthritis, active synovitis, neurological disorder that affected movement, muscle control and balance, uncontrolled hypertension, diabetes, cardiovascular disorders, chronic kidney disorders or functional liver disorders, were using antioxidants, vitamins and fiber supplements, frosemid, probenecid, anticoagulants, hydantoin, sulfonamides, methotrexate, lithium salts, beta blockers and muscle relaxants, tobacco, or had an allergic reaction to E. Angustifolia L., and were using E. angustifolia L. regularly. The sample size was designed with 90% power and a type I error rate of 1%, which necessitated at least 25 cases in each group. Taking into account the probable withdrawal of patients during the intervention course and those who may not adhere to the study protocol, 30 patients with knee OA were recruited in each group. The volunteer patients were randomly assigned to the two treatment groups or placebo group using a computer-generated random number table. Randomization was performed with the use of sealed envelopes. The investigator and participants were blinded to group allocation. The randomization code was only revealed after the statistical analyses were completed. 2.1. Subjects and study design The present study was a double blind randomized placebocontrolled trial in which the patients were randomly assigned to two interventions or a placebo group. The patients in the first and second intervention groups (n = 30 for each group) were supplemented with 15 g/day whole fruit powder and medulla powder of E. angustifolia L. and patients in the placebo group received 15 g/ day corn starch with isomalte for 8 weeks. All supplements were identically packaged to be indistinguishable. Patients were recommended to consume one packet daily for 8 weeks, preferably with milk after meals to reduce gastrointestinal complications. All patients underwent routine physical examinations and conventional treatments such as acetaminophen (Tylenol) and non-steroidal anti-inflammatory drugs (Celecoxib, Ibuprofen, Naproxen) continued during the course of the study. The use of conventional drugs and treatments was recorded at baseline, at week four, and at the end of week eight. BMI [weight (kilograms)/ height (meters squared)] was calculated from height and weight, which were measured using standardized protocols and calibrated equipment. Twenty four hour dietary recall questionnaires (2 work days and 1 weekend) were filled in by patients at the baseline and end of study for dietary assessment. All subjects were informed at the beginning of the study that they must contact the investigator in the event of any perceived side effects during the period of their participation in the study. The patients were instructed to return unused powders at the end of the study. As a measure of compliance, unused packs were counted by an independent investigator. Patients were excluded from the analysis if they consumed less than 80% of the packets, had changed their medication, or reported severe side effects. A computer-generated random sequence was kept in a remote secure location and administered by an independent third party who was not involved with the clinical conduct of study until all study data was collected and verified. Patients and those involved in enrolling participants, administering interventions, assessing outcomes, and analyzing data were blind to the group assignments. 2.2. Measurement of oxidative stress indices After a 12-h overnight fast a 5 mL blood sample was obtained for the determination of serum TAC and MDA levels. Serum was
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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separated by centrifugation and stored at 80 C until analyzed. Serum TAC was measured using Randox total antioxidant status kit in which ABTS (2,2-azino-di [3-ethyl ben-zthiazolin sulphanate]) is incubated with a peroxidase and H2O2 to produce the radical cation ABTS+. This has a stable blue green color, which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree which is proportional to their concentration. MDA level was determined by thiobarbitoric acid reaction with acid that was extracted with n-butanol and measured spectrophotometerically at 523 nm wavelength and compared with standard curve. 2.3. Elaeagnus angustifolia L. whole fruit and medulla powder preparation E. Angustifolia L. was purchased from the city of Tabriz, Iran in the fall of 2013. It was authorized in the Herbarium of Tabriz University of Medical Sciences, Iran and a voucher specimen was deposited for reference in the Herbarium of Pharmacy Faculty (Voucher No. TBZ-fph721). Before processing, all fruits were thoroughly washed with tap water and any fruit with signs of defect and immaturity and also other plant tissues including leaves sorted out by the research team. Whole fruit and also the medulla (pulp) of this fruit were ground into a powder with a mechanical grinder separately by the research team. The powders were further
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sealed into packs separately, each pack containing 15 g of Elaeagnus Angustifollia L. whole fruit and medulla powder. The packs were stored in a refrigerator until use. 2.4. Statistical analysis Statistical analysis was performed using SPSS version 13.0 and the descriptive data were reported as means and SD’s. The Kolmogrov–Smirnov test was used to analyze the normality of the data distribution. Comparisons within groups were performed using paired sample t-test and ANOVA and ANCOVA were used to assess the differences in baseline characteristics and also the differences in changes of TAC and MDA between groups. The P-value < 0.01 was considered significant. 2.5. Ethics All procedures followed in the present clinical trial were according to the guidelines of the ethics committee of Tabriz University of Medical Sciences and ethics approval has been received from the ethic committee of Tabriz University of medical Sciences (reference number 91171). Written informed consent was signed by all study participants and the study was registered in the Iranian Registry of Clinical Trials (IRCT), available at: http://www. irct.ir (ID: 201208241197N13).
Eligible paents (n=120) 30 paents were not willing to parcipate Included (n=90)
Randomizaon
Medulla powder (n=30)
Whole fruit powder (n=30)
Placebo (n=30)
Follow up for 8 weeks
Follow up for 8 weeks
Follow up for 8 weeks
3 withdrew because of travelling and unrelated illness
3 withdrew because of transportaon difficules and work or family reasons and 1 loss to follow up
3 withdrew due to intending to use other convenonal treatments and traveling and 2 loss to follow up
Completed trial (n=27)
Completed trial (n=26)
Completed trial (n=25)
Fig. 1. Flow chart for patient enrolment, randomization and retention.
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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Table 1 Comparison of the baseline characteristics of the osteoarthritis patients between study groups. Variable
Medulla of fruit (N = 27)
Whole fruit (N = 26)
Placebo (N = 25)
P-valuea
Age (years) Weight (Kg) Height (cm) BMI (kg/m2) Duration of disease (years) Analgesic use (no/week) TAC (mmol/L) MDA (nmol/mL)
54.52 11.19 76.37 10.95 155.87 6.00 32.47 2.67 6.35 4.39 1.70 0.90 1.12 0.34 3.33 0.33
57.47 7.24 74.85 10.24 152.0 5.38 32.50 2.70 6.00 4.66 1.89 0.89 1.29 0.28 3.15 0.51
57 7.77 75.73 9.62 154.21 6.25 32.35 2.77 6.91 5.20 1.66 0.44 1.18 0.19 2.67 0.38
0.49 0.81 0.08 0.82 0.80 0.97 0.97 0.23
BMI, body mass index; TAC, total antioxidant capacity; MDA, malondialdehyde. a P-value of ANOVA.
within group difference was observed in the micronutrient intakes of subjects (Table 2).
3. Results The flow chart of the study is presented in Fig. 1. From ninety volunteer patients who were entered in the study, 12 subjects withdrew due to reasons unrelated to the study, i.e. unrelated illnesses, family reasons etc. and lost to follow up. So, the data were reported for 78 patients (27 in medulla powder group, 26 in whole fruit powder and 25 in the placebo group). As stated in the previous study of E. angustifolia L. (Ahmadiani et al., 2000), no side-effect was reported during the study. 3.1. Baseline characteristics As shown in Table 1, the mean SD age and BMI of the study subjects were 56.31 + 8.90 years and 32.11 2.91 kg/m2 respectively. There were not significant between-group differences at baseline in the terms of age, BMI and duration of disease (Table 1). 3.2. Dietary intake Since changes in micronutrient intakes with potential oxidant and antioxidant properties could have confounded the results of the study, their intakes were analyzed. No significant between- or
3.3. Oxidative stress indices The mean SD of serum TAC in different study groups are shown in Fig. 2. The mean SD of serum TAC in medulla, whole fruit and placebo groups before the study were 1.12 0.34, 1.29 0.28 and 1.18 0.19 mmol/L respectively and these values increased after the study to 1.28 0.27, 1.48 0.24 and 1.29 0.22 mmol/L respectively. Although the level of serum TAC increased in all study groups after 8 weeks, the significant increases were only observed in the whole fruit powder supplemented group (P = 0.001). Fig. 3 depicts the mean SD of serum MDA in different study groups. The mean SD of serum MDA in the medulla, whole fruit and placebo groups were 3.33 0.33, 3.15 0.49 and 2.67 0.38 nmol/mL before the study and these values were 3.04 0.41, 2.48 0.41 and 2.41 0.34 nmol/mL, respectively after the study. In contrast to the medulla powder and placebo groups, a significant reduction was only observed in the mean serum MDA level in whole fruit group after intervention (P = 0.009).
Table 2 Micronutrient intakes in the experimental groups at baseline and throughout the study. Variable
Medulla of fruit (N = 27)
Whole fruit (N = 26)
Placebo (N = 25)
Pb
Vitamin A (mg/day) Baseline End of study Pc
750.22 103 683.3 101 0.15
1003 208.5 1166.22 247.6 0.77
599.72 502 610.5 97.6 0.26
0.61 0.53a
Vitamin E (mg/day) Baseline End of study Pc
7.39 2.32 1.64 0.28 0.64
7.04 0.35 1.67 0.3 0.29
7.01 1.31 1.66 0.3 0.68
0.72 0.66a
Vitamin C (mg/day) Baseline End of study Pc
67.80 54.68 35.5 6.4 0.58
40.96 38.5 49.05 11.6 0.52
37.66 32.82 49.71 10.1 0.20
0.06 0.07a
Selenium (mg/day) Baseline End of study Pc
0.06 0.02 0.05 0.03 0.56
0.05 0.03 0.08 0.03 0.14
0.08 0.11 0.09 0.08 0.22
0.40 0.35a
Zinc (mg/day) Baseline End of study Pc
6.51 4.50 9.70 4.2 0.51
4.98 3.20 4.87 1.34 0.89
5.90 7.38 7.18 2.3 0.68
0.71 0.66a
BMI, body mass index; TAC, total antioxidant capacity; MDA, malondialdehyde. a ANCOVA adjusted for baseline measures. b P-value of ANOVA. c Paired t test.
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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Fig. 2. Comparison of serum total antioxidant capacity (TAC) level before and after study in different groups. *Paired t-test; P-value < 0.01.
The results of the ANOVA test showed that there were no significant differences between study groups in the case of changes in serum TAC and MDA level. 4. Discussion It has frequently been reported that oxidative stress is implicated in the pathogenesis of OA (Sarban et al., 2005; Surapaneni and Venkataramana, 2007; Sutipornpalangkul et al., 2009; Ostalowska et al., 2006; Karan et al., 2003). Antioxidant agents such as vitamin C, vitamin E, and b-carotene have recently attracted considerable attention in treating osteoarthritis (McAlindon et al., 1996; Wang et al., 2007). Thus, we hypothesized that supplementing the diet of patients with knee OA with E. angustifolia fruit may positively reduce oxidative stress in the subjects. For the first time, the results of our study showed that E. angustifolia L. powder in the form of whole fruit had a significantly positive effect on serum TAC and MDA level whilst medulla powder had no significant effect on these factors. Since the patient’s antioxidant vitamin and mineral intake did not change significantly in the supplemented and placebo groups after the experimental period (Table 2), it seems that E. angustifolia L. supplementation might be the most important factor affecting the TAC level in this study. The observed insignificant increase in TAC levels and decrease in MDA level in the placebo group may be due to the common treatments of patients.
Previous studies have shown the positive effects of antioxidants in osteoarthritis patients (McAlindon et al., 1996; Wang et al., 2007). However, to the best of our knowledge, there are no published clinical studies investigating the antioxidant effect of E. angustifolia L. This fruit has a high antioxidant activity that may be due to the antioxidant properties of its polyphenol compounds (Bucur et al., 2008). It was shown that the peel, fruit and seed of this fruit contains different polyphenols, flavonoids and terpenoid compounds (Ahmadiani et al., 2000). An analysis of this fruit by Bucur et al. (2009) reported that kaempferol and coumaric acid were the main polyphenol compounds, whilst Ayaz and Bertoft (2001) reported that benzoic and caffeic acids were the main phenolic acids in E. angustifolia L. fruit. In an earlier investigation, Wang et al. (2012) studied the flavonoid glycoside content of E. angustifolia L. pulp and identified 3 new compounds including quercetin 3,4'-O-b-D-diglucoside, quercetin 3-O-b-D-galactopyranoside-4'-O-b-D-glucopyranoside and isorhamnetin 3-O-b-Dgalacto-pyranoside-4'-O-b-D-glucopyranoside. Prior studies of the fruit clarified that the phenolic composition of the whole fruit and medulla were different. Chuan Ling et al. (2011) reported that the peel and seed of E. angustifolia L. contained a higher amount of phenolic compounds than the medulla (fruit pulp). They showed that total flavonoid contents of E. angustifolia L. bark, medulla and seed were around 37.0 mg/g, 1.6 mg/g and 18.6 mg/g, respectively. Thus since the seed and bark of this fruit contains a higher flavonoid content (many flavonoids are potent antioxidants) than the medulla, it can be concluded that the whole fruit of E. angustifolia L. (including bark, medulla and seed) contains
Fig. 3. Comparison of serum malodialdehyde (MDA) levels before and after study in different groups. *Paired t-test; P-value < 0.01.
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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more flavonoids than the medulla alone. In an earlier investigation, Panico et al. (2009) showed that the polyphenol rich extract (including the flavonols quercetin and kaempferol) was effective in reducing the reactive oxygen species in human chondrocyte cultures. Alkaloids are other active components of E. angustifolia L. that may have an important role in its antioxidant activity. Nikolaeva (1970) reported that E. angustifolia L. contains alkaloids mainly harmine and harmaline (b carbolines). Some reports indicate that b-carbolines have effective antioxidant properties. In this respect, harmane, harmaline and harmalol showed antioxidant activity by inhibiting lipid peroxidation in microsomal hepatic preparation and by attenuating oxidative damage of hyaluronic acid, cartilage collagen and immunoglobulin G (Tse et al., 1991; Cho et al., 1995; Kim et al., 1999). Although in comparison with the baseline, the whole fruit supplementation had more positive effects on TAC and MDA levels than medulla powder, no significant differences were found between groups when the changes in the TAC and MDA of serum were compared by ANOVA test. It seems that the probable reason for this observation is the short duration of the study. So, further studies with longer observation periods and also multiple observations are encouraged. Another limitation of this study that should be noted was only measuring TAC and MDA as an oxidative status rather than measuring other antioxidant indicators such as antioxidant enzyme activity. So, additional studies with age, sex, and body composition comparison and also measuring these indicators at different disease stages are warranted. 5. Conclusion Based on the results, E. angustifolia L. in the form of whole fruit had a significantly beneficial effect on serum TAC and MDA levels in females with knee OA. The use of E. angustifolia L. in traditional medicine for the treatment of knee osteoarthritis may be due to its beneficial effects on oxidative stress. However, for any precise conclusions further studies with longer duration are needed. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements The authors wish to thank the Nutrition Research Center at Tabriz University of Medical Sciences for financial support. Special gratitude should go to the subjects who participated in the study. The results of this article are derived from PhD thesis of Zeinab Nikniaz (NO of D/29). References Ahmad, M., Sultana, S., Hadi, S., Hadda, T., Rashid, S., Zafar, M., Khan, A., Khan, M., Yasin, G., 2014. An ethnobotanical study of medicinal plants in high mountainous region of Chail valley (district Swat-Pakistan). J. Ethnobiol. Ethnomed. 10, 36. Ahmadiani, A., Hosseiny, J., Semnanian, S., Javan, M., Saeedi, F., Kamalinejad, M., Saremi, S., 2000. Antinociceptive and anti-inflammatory effects of Elaeagnus angustifolia fruit extract. J. Ethnopharmacol. 72, 287–292. Altindag, O., Erel, O., Aksoy, N., Selek, S., Celik, H., Karaoglanoglu, M., 2007. Increased oxidative stress and its relation with collagen metabolism in knee osteoarthritis. Rheumatol. Int. 27, 339–344. Altman, R., Asch, E., Bloch, D., Bole, G., Borenstein, D., Brandt, K., Christy, W., Cooke, T., Greenwald, R., Hochberg, M., Howell, D., Kaplan, D., Koopman, Longley, S., Mankin, H., McShane, D., Medsger, T., Meenan, R., Mikkelsen, W., Moskowitz, R., Murphy, W., Rothschild, B., Segal, M., Sokoloff, S., Wolfe, F., 1986. The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the knee. Arthritis Rheum. 29, 1039–1049.
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Panico, A., Cardile, V., Santagati, N.A., Messina, R., 2009. Antioxidant and protective effects of sumac leaves on chondrocytes. J. Med. Plants Res. 3 (11), 855–861. Sarban, S., Kocyigit, A., Yazar, M., Isikan, U.E., 2005. Plasma total antioxidant capacity, lipid peroxidation, and erythrocyte antioxidant enzyme activities in patients with rheumatoid arthritis and osteoarthritis. Clin. Biochem. 38, 981–986. Silverstein, F., Faich, G., Goldstein, J., Simon, L., Pincus, T., Whelton, A., Makuch, R., Eisen, G., Agrawal, N.M., Stenson, W.F., Burr, A.M., Zhao, W.W., Kent, J.D., Lefkowith, J.B., Verburg, K.M., Geis, G.S., 2002. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: a randomised controlled trial. J. Am. Med. Assoc. 284, 1247–1255. Surapaneni, K.M., Venkataramana, G., 2007. 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Journal of Herbal Medicine xxx (2015) xxx–xxx
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Research paper
Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis Zeinab Nikniaza , Reza Mahdavib,* , Alireza Ostadrahimib , Aliasghar Ebrahimic , Leila Nikniazd, Amirmansour Vatankhahe a
Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Nutrition Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran c Connective Tissue Diseases Research Centre, Tabriz University of Medical Sciences, Imam Reza Hospital, Iran d Tabriz Health Services Management Research Center, Tabriz University of Medical Sciences, Tabriz, Iran e Drug Applied Research Center, Tabriz University of Medical Science, Tabriz, Iran b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 June 2015 Received in revised form 14 July 2015 Accepted 1 September 2015 Available online xxx
Aim: The present study aimed to determine the effects of Elaeagnus angustifolia L. supplement on serum antioxidant status in females with knee osteoarthritis. Methods: In this 8-week randomized clinical trial, 90 females with knee osteoarthritis were randomly assigned to one of three groups—medulla powder (15 g medulla powder), whole fruit powder (15 g whole fruit powder) or placebo. Information on dietary intake was collected using 24-h recall method for 3 days (one weekend day included). The serum levels of total antioxidant capacity (TAC) and malondialdehyde (MDA) were measured using the Randox1 assay and the fluorometry method respectively. Paired sample t-test, ANOVA and ANCOVA were used for statistical analysis. Results: The mean serum TAC level in the medulla, whole fruit and placebo groups increased from 1.12 0.34 to 1.28 0.27, 1.30 0.28 to 1.50 0.28 and 1.18 0.19 to 1.30 0.22 mmol/L respectively. In terms of the TAC level, the significant increase was only observed in the whole fruit group (P = 0.001). The mean serum MDA level in the medulla, whole fruit and placebo groups decreased from 3.80 0.33 to 3.30 0.41, 3.25 0.51 to 2.45 0.43 and 3.38 0.63 to 2.56 0.41, respectively. A statistically significant reduction was observed only in the whole fruit group (P = 0.009). Discussion: Based on the results, E. angustifolia L. in the form of whole fruit had beneficial effects on serum TAC and MDA levels in females with knee OA. This may show a possible disease modifying effect of E. angustifolia L. in knee osteoarthritis. ã 2015 Elsevier GmbH. All rights reserved.
Keywords: Female Malondialdehyde Osteoarthritis Total antioxidant capacity
1. Introduction According to worldwide estimates, approximately 10% of men and 18.0% of females aged <60 years have symptomatic osteoarthritis (Woolf and Pfleger, 2003). With increasing life expectancy together with aging populations, it is expected that, by the year
Abbreviations: ANCOVA, analysis of covariance; ANOVA, analysis of variance; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); MDA, malondialdehyde; TAC, total antioxidant capacity; ROS, reactive oxygen species; OA, osteoarthritis; BMI, body mass index. * Corresponding author at: Golgasht St. Attarneishabouri Ave, nutrition faculty, Tabriz University of Medical Sciences,Tabriz, Iran. E-mail address: [email protected] (R. Mahdavi).
2020, osteoarthritis will become the fourth leading cause of disability (Murray and Lopez, 1996). Clinically OA is characterized by joint pain, tenderness, limitation of movement, crepitus, and local inflammation (Woolf and Pfleger, 2003). It can occur in any joint but it is most common in the knee joint (Woolf and Pfleger, 2003). Although little is known about its etiopathology, it is supposed that OA is a consequence of mechanical and biological events that break down articular cartilage, in the synovial joints (Chen et al., 2006). Biological events that may affect articular cartilage are inflammation and a change in antioxidant status. Increasing evidence in both experimental and clinical studies have suggested that oxidative stress is considered to be one of the main causative factors in the pathogenesis of OA (Sarban et al., 2005; Surapaneni and Venkataramana, 2007; Sutipornpalangkul et al., 2009;
http://dx.doi.org/10.1016/j.hermed.2015.09.003 2210-8033/ ã 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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Ostalowska et al., 2006; Karan et al., 2003). Reactive oxygen species (ROS) production has been found to increase in joint diseases such as osteoarthritis (Deberg et al., 2005). Although the precise mechanism remains unclear (Yudoh et al., 2005), some studies have suggested that oxidative stress causes chondrocyte senescence and cartilage aging (Yudoh et al., 2005; Martin et al., 2004). Lipid peroxidation induced by reactive oxygen species (ROS) is considered to be the major mechanism of cell membrane destruction and cell damage (Henrotin et al., 2005). Decomposition of peroxidized lipids leads to production of a wide variety of end-products, including MDA (Gambhir et al., 1997). Elevated levels of MDA as the indicator of lipid peroxidation have been reported in the serum and synovial fluid of OA patients (Lunec et al., 1981). The negative effects of reactive oxygen species are usually neutralized by the total antioxidant capacity (TAC); however, sometimes the TAC cannot cope with the excessive increase of free radicals, and then the TAC will decrease. The decreased levels of serum TAC level in OA patients have been shown in earlier studies (Altindag et al., 2007; McAlindon et al., 1996). Based on the results of previous studies, antioxidant agents such as vitamin C, vitamin E, and b-carotene have recently attracted considerable attention in treating osteoarthritis (McAlindon et al., 1996; Wang et al., 2007). Despite the antioxidant effects of some drugs used in OA treatment (Ozgocmen et al., 2005), they also have potential adverse effects (Silverstein et al., 2002). Thus, considering the sideeffects of these drugs, the use of complementary medicines and therapies are increasing in patients with knee OA. One of the complementary therapies in Iran that has been used as a folk remedy for arthritis patients is Elaeagnus angustifolia L. fruit. This fruit is also used as a folk medicine in Turkey for wound healing (Ersoy et al., 2013), in Pakistan for headache, heartburn and skin infections (Ahmad et al., 2014) and in China (Wang et al., 2012) and Jordan (Lev and Amar 2002) for diarrhea. E. Angustifolia topical gel has also been shown to be effective in the treatment of symptomatic oral lichen planus, with anti-inflammatory and analgesic effects (Taheri et al., 2010). Previous studies have shown that daily supplementation with this fruit had positive effects in improving pain, stiffness and physical function and for decreasing inflammatory cytokines and enhancing anti-inflammatory cytokines (Ebrahimi et al., 2014; Nikniaz et al., 2014). The fruit contains many flavonoids and antioxidant vitamins with significant antioxidant potential (Ersoy et al., 2013). In Iran, this fruit is consumed in two forms; (1) whole fruit powder (the whole fruit powder including seed, medulla and pericarp) (2) only the pulp (medulla) of the fruit which were available in local markets or Iranian traditional medicinal plant stores. Despite its extensive use as a folk remedy (Mirhydar, 1998), to the best of the authors’ knowledge, there are no clinical studies about the effect of this fruit on osteoarthritis patients. As, it was hypothesized that Elaeagnus angustifolia L. as a complementary treatment may have beneficial effects on antioxidant status, the effects of two forms of whole fruit and medulla powders of this fruit on serum TAC and MDA level were assessed in females with knee OA. 2. Materials and methods One hundred and twenty patients with knee osteoarthritis with disease severity from mild to moderate based on the criteria of the American College of Rheumatology (ACR) (Altman et al., 1986) and who had been referred to the Rheumatology Centre of Sina Hospital, Tabriz, Iran, were selected for the study. The patients were included if they were female, aged 40–70 years, had moderate to severe bilateral primary knee osteoarthritis
(according to ACR classification) (Altman et al., 1986) and body mass index (BMI) of 25–34.9 kg/m2. The patients were excluded if they had secondary osteoarthritis, active synovitis, neurological disorder that affected movement, muscle control and balance, uncontrolled hypertension, diabetes, cardiovascular disorders, chronic kidney disorders or functional liver disorders, were using antioxidants, vitamins and fiber supplements, frosemid, probenecid, anticoagulants, hydantoin, sulfonamides, methotrexate, lithium salts, beta blockers and muscle relaxants, tobacco, or had an allergic reaction to E. Angustifolia L., and were using E. angustifolia L. regularly. The sample size was designed with 90% power and a type I error rate of 1%, which necessitated at least 25 cases in each group. Taking into account the probable withdrawal of patients during the intervention course and those who may not adhere to the study protocol, 30 patients with knee OA were recruited in each group. The volunteer patients were randomly assigned to the two treatment groups or placebo group using a computer-generated random number table. Randomization was performed with the use of sealed envelopes. The investigator and participants were blinded to group allocation. The randomization code was only revealed after the statistical analyses were completed. 2.1. Subjects and study design The present study was a double blind randomized placebocontrolled trial in which the patients were randomly assigned to two interventions or a placebo group. The patients in the first and second intervention groups (n = 30 for each group) were supplemented with 15 g/day whole fruit powder and medulla powder of E. angustifolia L. and patients in the placebo group received 15 g/ day corn starch with isomalte for 8 weeks. All supplements were identically packaged to be indistinguishable. Patients were recommended to consume one packet daily for 8 weeks, preferably with milk after meals to reduce gastrointestinal complications. All patients underwent routine physical examinations and conventional treatments such as acetaminophen (Tylenol) and non-steroidal anti-inflammatory drugs (Celecoxib, Ibuprofen, Naproxen) continued during the course of the study. The use of conventional drugs and treatments was recorded at baseline, at week four, and at the end of week eight. BMI [weight (kilograms)/ height (meters squared)] was calculated from height and weight, which were measured using standardized protocols and calibrated equipment. Twenty four hour dietary recall questionnaires (2 work days and 1 weekend) were filled in by patients at the baseline and end of study for dietary assessment. All subjects were informed at the beginning of the study that they must contact the investigator in the event of any perceived side effects during the period of their participation in the study. The patients were instructed to return unused powders at the end of the study. As a measure of compliance, unused packs were counted by an independent investigator. Patients were excluded from the analysis if they consumed less than 80% of the packets, had changed their medication, or reported severe side effects. A computer-generated random sequence was kept in a remote secure location and administered by an independent third party who was not involved with the clinical conduct of study until all study data was collected and verified. Patients and those involved in enrolling participants, administering interventions, assessing outcomes, and analyzing data were blind to the group assignments. 2.2. Measurement of oxidative stress indices After a 12-h overnight fast a 5 mL blood sample was obtained for the determination of serum TAC and MDA levels. Serum was
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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separated by centrifugation and stored at 80 C until analyzed. Serum TAC was measured using Randox total antioxidant status kit in which ABTS (2,2-azino-di [3-ethyl ben-zthiazolin sulphanate]) is incubated with a peroxidase and H2O2 to produce the radical cation ABTS+. This has a stable blue green color, which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree which is proportional to their concentration. MDA level was determined by thiobarbitoric acid reaction with acid that was extracted with n-butanol and measured spectrophotometerically at 523 nm wavelength and compared with standard curve. 2.3. Elaeagnus angustifolia L. whole fruit and medulla powder preparation E. Angustifolia L. was purchased from the city of Tabriz, Iran in the fall of 2013. It was authorized in the Herbarium of Tabriz University of Medical Sciences, Iran and a voucher specimen was deposited for reference in the Herbarium of Pharmacy Faculty (Voucher No. TBZ-fph721). Before processing, all fruits were thoroughly washed with tap water and any fruit with signs of defect and immaturity and also other plant tissues including leaves sorted out by the research team. Whole fruit and also the medulla (pulp) of this fruit were ground into a powder with a mechanical grinder separately by the research team. The powders were further
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sealed into packs separately, each pack containing 15 g of Elaeagnus Angustifollia L. whole fruit and medulla powder. The packs were stored in a refrigerator until use. 2.4. Statistical analysis Statistical analysis was performed using SPSS version 13.0 and the descriptive data were reported as means and SD’s. The Kolmogrov–Smirnov test was used to analyze the normality of the data distribution. Comparisons within groups were performed using paired sample t-test and ANOVA and ANCOVA were used to assess the differences in baseline characteristics and also the differences in changes of TAC and MDA between groups. The P-value < 0.01 was considered significant. 2.5. Ethics All procedures followed in the present clinical trial were according to the guidelines of the ethics committee of Tabriz University of Medical Sciences and ethics approval has been received from the ethic committee of Tabriz University of medical Sciences (reference number 91171). Written informed consent was signed by all study participants and the study was registered in the Iranian Registry of Clinical Trials (IRCT), available at: http://www. irct.ir (ID: 201208241197N13).
Eligible paents (n=120) 30 paents were not willing to parcipate Included (n=90)
Randomizaon
Medulla powder (n=30)
Whole fruit powder (n=30)
Placebo (n=30)
Follow up for 8 weeks
Follow up for 8 weeks
Follow up for 8 weeks
3 withdrew because of travelling and unrelated illness
3 withdrew because of transportaon difficules and work or family reasons and 1 loss to follow up
3 withdrew due to intending to use other convenonal treatments and traveling and 2 loss to follow up
Completed trial (n=27)
Completed trial (n=26)
Completed trial (n=25)
Fig. 1. Flow chart for patient enrolment, randomization and retention.
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Table 1 Comparison of the baseline characteristics of the osteoarthritis patients between study groups. Variable
Medulla of fruit (N = 27)
Whole fruit (N = 26)
Placebo (N = 25)
P-valuea
Age (years) Weight (Kg) Height (cm) BMI (kg/m2) Duration of disease (years) Analgesic use (no/week) TAC (mmol/L) MDA (nmol/mL)
54.52 11.19 76.37 10.95 155.87 6.00 32.47 2.67 6.35 4.39 1.70 0.90 1.12 0.34 3.33 0.33
57.47 7.24 74.85 10.24 152.0 5.38 32.50 2.70 6.00 4.66 1.89 0.89 1.29 0.28 3.15 0.51
57 7.77 75.73 9.62 154.21 6.25 32.35 2.77 6.91 5.20 1.66 0.44 1.18 0.19 2.67 0.38
0.49 0.81 0.08 0.82 0.80 0.97 0.97 0.23
BMI, body mass index; TAC, total antioxidant capacity; MDA, malondialdehyde. a P-value of ANOVA.
within group difference was observed in the micronutrient intakes of subjects (Table 2).
3. Results The flow chart of the study is presented in Fig. 1. From ninety volunteer patients who were entered in the study, 12 subjects withdrew due to reasons unrelated to the study, i.e. unrelated illnesses, family reasons etc. and lost to follow up. So, the data were reported for 78 patients (27 in medulla powder group, 26 in whole fruit powder and 25 in the placebo group). As stated in the previous study of E. angustifolia L. (Ahmadiani et al., 2000), no side-effect was reported during the study. 3.1. Baseline characteristics As shown in Table 1, the mean SD age and BMI of the study subjects were 56.31 + 8.90 years and 32.11 2.91 kg/m2 respectively. There were not significant between-group differences at baseline in the terms of age, BMI and duration of disease (Table 1). 3.2. Dietary intake Since changes in micronutrient intakes with potential oxidant and antioxidant properties could have confounded the results of the study, their intakes were analyzed. No significant between- or
3.3. Oxidative stress indices The mean SD of serum TAC in different study groups are shown in Fig. 2. The mean SD of serum TAC in medulla, whole fruit and placebo groups before the study were 1.12 0.34, 1.29 0.28 and 1.18 0.19 mmol/L respectively and these values increased after the study to 1.28 0.27, 1.48 0.24 and 1.29 0.22 mmol/L respectively. Although the level of serum TAC increased in all study groups after 8 weeks, the significant increases were only observed in the whole fruit powder supplemented group (P = 0.001). Fig. 3 depicts the mean SD of serum MDA in different study groups. The mean SD of serum MDA in the medulla, whole fruit and placebo groups were 3.33 0.33, 3.15 0.49 and 2.67 0.38 nmol/mL before the study and these values were 3.04 0.41, 2.48 0.41 and 2.41 0.34 nmol/mL, respectively after the study. In contrast to the medulla powder and placebo groups, a significant reduction was only observed in the mean serum MDA level in whole fruit group after intervention (P = 0.009).
Table 2 Micronutrient intakes in the experimental groups at baseline and throughout the study. Variable
Medulla of fruit (N = 27)
Whole fruit (N = 26)
Placebo (N = 25)
Pb
Vitamin A (mg/day) Baseline End of study Pc
750.22 103 683.3 101 0.15
1003 208.5 1166.22 247.6 0.77
599.72 502 610.5 97.6 0.26
0.61 0.53a
Vitamin E (mg/day) Baseline End of study Pc
7.39 2.32 1.64 0.28 0.64
7.04 0.35 1.67 0.3 0.29
7.01 1.31 1.66 0.3 0.68
0.72 0.66a
Vitamin C (mg/day) Baseline End of study Pc
67.80 54.68 35.5 6.4 0.58
40.96 38.5 49.05 11.6 0.52
37.66 32.82 49.71 10.1 0.20
0.06 0.07a
Selenium (mg/day) Baseline End of study Pc
0.06 0.02 0.05 0.03 0.56
0.05 0.03 0.08 0.03 0.14
0.08 0.11 0.09 0.08 0.22
0.40 0.35a
Zinc (mg/day) Baseline End of study Pc
6.51 4.50 9.70 4.2 0.51
4.98 3.20 4.87 1.34 0.89
5.90 7.38 7.18 2.3 0.68
0.71 0.66a
BMI, body mass index; TAC, total antioxidant capacity; MDA, malondialdehyde. a ANCOVA adjusted for baseline measures. b P-value of ANOVA. c Paired t test.
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Fig. 2. Comparison of serum total antioxidant capacity (TAC) level before and after study in different groups. *Paired t-test; P-value < 0.01.
The results of the ANOVA test showed that there were no significant differences between study groups in the case of changes in serum TAC and MDA level. 4. Discussion It has frequently been reported that oxidative stress is implicated in the pathogenesis of OA (Sarban et al., 2005; Surapaneni and Venkataramana, 2007; Sutipornpalangkul et al., 2009; Ostalowska et al., 2006; Karan et al., 2003). Antioxidant agents such as vitamin C, vitamin E, and b-carotene have recently attracted considerable attention in treating osteoarthritis (McAlindon et al., 1996; Wang et al., 2007). Thus, we hypothesized that supplementing the diet of patients with knee OA with E. angustifolia fruit may positively reduce oxidative stress in the subjects. For the first time, the results of our study showed that E. angustifolia L. powder in the form of whole fruit had a significantly positive effect on serum TAC and MDA level whilst medulla powder had no significant effect on these factors. Since the patient’s antioxidant vitamin and mineral intake did not change significantly in the supplemented and placebo groups after the experimental period (Table 2), it seems that E. angustifolia L. supplementation might be the most important factor affecting the TAC level in this study. The observed insignificant increase in TAC levels and decrease in MDA level in the placebo group may be due to the common treatments of patients.
Previous studies have shown the positive effects of antioxidants in osteoarthritis patients (McAlindon et al., 1996; Wang et al., 2007). However, to the best of our knowledge, there are no published clinical studies investigating the antioxidant effect of E. angustifolia L. This fruit has a high antioxidant activity that may be due to the antioxidant properties of its polyphenol compounds (Bucur et al., 2008). It was shown that the peel, fruit and seed of this fruit contains different polyphenols, flavonoids and terpenoid compounds (Ahmadiani et al., 2000). An analysis of this fruit by Bucur et al. (2009) reported that kaempferol and coumaric acid were the main polyphenol compounds, whilst Ayaz and Bertoft (2001) reported that benzoic and caffeic acids were the main phenolic acids in E. angustifolia L. fruit. In an earlier investigation, Wang et al. (2012) studied the flavonoid glycoside content of E. angustifolia L. pulp and identified 3 new compounds including quercetin 3,4'-O-b-D-diglucoside, quercetin 3-O-b-D-galactopyranoside-4'-O-b-D-glucopyranoside and isorhamnetin 3-O-b-Dgalacto-pyranoside-4'-O-b-D-glucopyranoside. Prior studies of the fruit clarified that the phenolic composition of the whole fruit and medulla were different. Chuan Ling et al. (2011) reported that the peel and seed of E. angustifolia L. contained a higher amount of phenolic compounds than the medulla (fruit pulp). They showed that total flavonoid contents of E. angustifolia L. bark, medulla and seed were around 37.0 mg/g, 1.6 mg/g and 18.6 mg/g, respectively. Thus since the seed and bark of this fruit contains a higher flavonoid content (many flavonoids are potent antioxidants) than the medulla, it can be concluded that the whole fruit of E. angustifolia L. (including bark, medulla and seed) contains
Fig. 3. Comparison of serum malodialdehyde (MDA) levels before and after study in different groups. *Paired t-test; P-value < 0.01.
Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003
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more flavonoids than the medulla alone. In an earlier investigation, Panico et al. (2009) showed that the polyphenol rich extract (including the flavonols quercetin and kaempferol) was effective in reducing the reactive oxygen species in human chondrocyte cultures. Alkaloids are other active components of E. angustifolia L. that may have an important role in its antioxidant activity. Nikolaeva (1970) reported that E. angustifolia L. contains alkaloids mainly harmine and harmaline (b carbolines). Some reports indicate that b-carbolines have effective antioxidant properties. In this respect, harmane, harmaline and harmalol showed antioxidant activity by inhibiting lipid peroxidation in microsomal hepatic preparation and by attenuating oxidative damage of hyaluronic acid, cartilage collagen and immunoglobulin G (Tse et al., 1991; Cho et al., 1995; Kim et al., 1999). Although in comparison with the baseline, the whole fruit supplementation had more positive effects on TAC and MDA levels than medulla powder, no significant differences were found between groups when the changes in the TAC and MDA of serum were compared by ANOVA test. It seems that the probable reason for this observation is the short duration of the study. So, further studies with longer observation periods and also multiple observations are encouraged. Another limitation of this study that should be noted was only measuring TAC and MDA as an oxidative status rather than measuring other antioxidant indicators such as antioxidant enzyme activity. So, additional studies with age, sex, and body composition comparison and also measuring these indicators at different disease stages are warranted. 5. Conclusion Based on the results, E. angustifolia L. in the form of whole fruit had a significantly beneficial effect on serum TAC and MDA levels in females with knee OA. The use of E. angustifolia L. in traditional medicine for the treatment of knee osteoarthritis may be due to its beneficial effects on oxidative stress. However, for any precise conclusions further studies with longer duration are needed. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements The authors wish to thank the Nutrition Research Center at Tabriz University of Medical Sciences for financial support. Special gratitude should go to the subjects who participated in the study. The results of this article are derived from PhD thesis of Zeinab Nikniaz (NO of D/29). References Ahmad, M., Sultana, S., Hadi, S., Hadda, T., Rashid, S., Zafar, M., Khan, A., Khan, M., Yasin, G., 2014. An ethnobotanical study of medicinal plants in high mountainous region of Chail valley (district Swat-Pakistan). J. Ethnobiol. Ethnomed. 10, 36. Ahmadiani, A., Hosseiny, J., Semnanian, S., Javan, M., Saeedi, F., Kamalinejad, M., Saremi, S., 2000. Antinociceptive and anti-inflammatory effects of Elaeagnus angustifolia fruit extract. J. Ethnopharmacol. 72, 287–292. Altindag, O., Erel, O., Aksoy, N., Selek, S., Celik, H., Karaoglanoglu, M., 2007. Increased oxidative stress and its relation with collagen metabolism in knee osteoarthritis. Rheumatol. Int. 27, 339–344. Altman, R., Asch, E., Bloch, D., Bole, G., Borenstein, D., Brandt, K., Christy, W., Cooke, T., Greenwald, R., Hochberg, M., Howell, D., Kaplan, D., Koopman, Longley, S., Mankin, H., McShane, D., Medsger, T., Meenan, R., Mikkelsen, W., Moskowitz, R., Murphy, W., Rothschild, B., Segal, M., Sokoloff, S., Wolfe, F., 1986. The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the knee. Arthritis Rheum. 29, 1039–1049.
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Please cite this article in press as: Z. Nikniaz, et al., Effects of Elaeagnus angustifolia L. powder supplementation on serum total antioxidant capacity and malondialdehyde levels in females with knee osteoarthritis, J. Herbal Med. (2015), http://dx.doi.org/10.1016/j. hermed.2015.09.003