Comparative study of the effect of Thymus daenensis gel 5% and diclofenac in patients with knee osteoarthritis

Background: Osteoarthritis is a syndrome characterized by joint pain and reduced performance and efficien- cy in patient. Thymus daenensis has been used since old times for the treatment of bone and joint deformities and pain in traditional medicine. Purpose: This study was conducted to examine traditional usages and pharmacological features of T. daen- ensis with respect to the effect of the plant in patients with osteoarthritis. Methods: 120 patients with osteoarthritis were divided into 3 groups. Patients in each group were treated by 5% Thymus daenensis gel, 1% diclofenac gel, or placebo for 6 weeks, along with oral celecoxib capsules. Patients were assessed in different intervals, based on the VAS score for assessment of pain in the joint and different dimensions of WOMAC questionnaire. Results: Pain level (P < 0.005), stiffness during the day (P < 0.05), morning stiffness (P < 0.05) and physi- cal performance (P < 0.05) were significantly different among the groups. Conclusions: Thymus daenensis gel improves the symptoms in patients equal and without significant difference than diclofenac group. It can be argued that its use can produce a satisfactory effect on patients with osteoarthritis due to its low cost, easy access, the plant’s natively occurring in Iran.

. In this regard, medicinal treatments including NSAIDs are the main choice for osteoarthritis of the knee [10]. However, adverse side effects of medications [11,12] as well as the likelihood of other diseases cause problems in patients especially in the older patients [13,14]. Studies have indicated increased tendency of OA patients to use complementary and alternative treatments [15,16]. These studies show that complementary therapies have wide applications for the treatment of chronic diseases in Iran [17,18]. One of the complementary therapies used in OA is using herbal medicines. Thymus daenensis is one of the effective plants for treatment of osteoarthritis in traditional medicine. Thymus daenensis fresh leaves and stems have a pleasant smell that results from a combination of compounds in oils and extracts of this plant. In vegetative organs of thyme, a compound such as acetate. After 40 minutes, the absorption of samples was read against distilled water (blank) at a wavelength 415 nm. While carrying out the experiment, various concentrations of Rutin were prepared. Absorption of samples was compared with a standard curve and total flavonoid content of each extract was stated as mg of Rutin equivalent/g dried extract [50].

Exclusion criteria
Simultaneous severe diseases such as metabolic and gastrointestinal diseases, fluctuation in the dose of the drug during the study, the presence of severe infection, allergy to drugs, pregnancy, lactation, liver abnormal performance tests, history of fracture or surgery on the knee, rheumatoid arthritis and seronegative and seropositive arthropathies, intra-articular injection during 6 weeks, simultaneous use of any type of tranquilizer that caused disturbance in the result were exclusion criteria of study. In addition, patients who had no willingness in the study were excluded from the study.

Inclusion criteria
Men and women with knee osteoarthritis, diagnosed by an orthopedician according to diagnostic criteria, aged 45-75 years who had referred to the Orthopaedic Clinic of Shahrkord University of Medical Sciences at least 3 months before the start of the study, were included in the study. They signed an informed consent to participate in the study.

Intervention
The first group received 200 mg capsules celecoxib orally and topical gel placebo, the second group received 200 mg capsules celecoxib orally on a daily basis and topical 5% thyme gel and the third group received 200 mg capsules celecoxib orally and topical diclofenac 1% gel 3 times daily for 6 weeks. Patients were instructed that the gel should cover the entire surface of the knee. Topical gel dose for each 5 square centimeters of skin was knuckle. To ensure lack of any additional intervention, patients were asked to not massage the knee.

Effectiveness evaluation
Patient evaluation was performed by means of the Visual Analogue Scale (VAS) to score knee pain at baseline and on the first and third days and first, third, and sixth weeks following the intervention. Besides that, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) that is a general and valid instrument to examine OA was used as another measurement tool. This questionnaire examines three dimensions: pain dimension (scored between 0 representing no pain and 4 representing severe pain), physical performance (scored between 0 representing no problem and 4 representing severe problem) and stiffness (scored between 0 representing without stiffness and 4 representing severe stiffness). The scores of the dimensions were summed and the mean value was calculated. This questionnaire was evaluated before treatment and during the first, third days and the first, third and sixth weeks after the intervention [21].

Method
This study has three intervention groups (diclofenac group, herbal gel group and placebo group) with the double-blind clinical trial.

Ethical considerations
The study design is in compliance with the ethical principles of Helsinki Declaration. This study protocol was approved at the Behavioral Sciences Ethics Committee of Shahrekord University of Medical Sciences (ethics code IR.SKUMS.REC.1395.54) and then in the Iranian clinical trial registration center with code IRCT2016112231025N1. Informed consent form was taken from patients participating in the study.

Plant material and extraction method
Thymus daenensis aerial parts were collected from mountainous areas of the province in the spring of 2016. Collected Plants were identified by Dr. Shirmardi and a herbarium voucher (no. 248) was deposited at the Herbarium of the Medical Plants Research Center of Shahrekord University of Medical Sciences. The powdered plant was extracted by maceration method with Ethanol 70% for 48 h. The obtained extract was passed through filter paper. To remove solvent, the extract was concentrated by rotary evaporator at 37°C and lyophilized by freeze dryer. Then, Thymus daenensis gel 5% was formulated in gel base from polymer Carbopol-940 as U.S. Pharmacopeia. Diclofenac Gel 1% of Emad-Darman Pars Company and placebo were filled in same aluminum tubes like Thymus daenensis gel.

Standardization of topical gel
The total phenolic content of aqueous Thymus daenensis extract was measured using Folin-Ciocalteu. To this end, 1 mg/ml crude extract reached a volume of 3 ml by addition of distilled water and then combined with 0.5 ml Folin-ciocalteu reagent for 10 minutes. Next, 4 ml sodium carbonate was added to the resulting mixture. The obtained mixture was stored in the dark for half an hour and the optical absorbance was read at 765nm wavelength. The total phenolic content was measured by using calibration curve, and the results were expressed as the total phenolic content (mg of Gallic acid equivalent/g dried extract) [49]. The total flavonoid content Flavonoids were measured according to Liang et al. method with some modifications. In short, 5.0 ml of each extract solution (0.01 g per 10 ml of methanol at 60°C) was mixed with 0.5 ml of 2% aluminum chloride and 3 ml of 5% potassium reactions in follow-up intervals. A checklist including several questions about different organs of the body (such as gastrointestinal problems, nervous system, respiratory system and skin) was used. Potential adverse reactions were investigated by asking the participants open-ended questions.

Sampling, randomization and blinding
Non-probability convenient sampling was used to select samples. To this end, the participants were selected from the patients admitted to the clinic. To enroll minimum number of people by taking dropouts into account, 120 people were enrolled in this study who were assigned to three groups of 40 each. Group 1 received placebo gel, group 2 received Thymus daenensis extract gel, and group 3 received gel containing diclofenac. To collect data, using convenient sampling method, the 120 participants were assigned to tube A, tube B, and tube C (each containing 40) and by using random sampling method; the patient was asked to select one of the Tube A, B, and C. Then, the intervention was performed. Researchers, patients and statistical analysts were blind to the content of three types of tubes.

Statistical Analysis
The sample size was obtained 35 people considering 95% confidence interval, and test power of 90 percent and study power of 85 percent. Considering drop out, 120 were included in the study. Data were analyzed using STATA software. For data analysis, inferential and descriptive statistics and duplicate data analysis were applied. Difference in pretest and post-test scores between the control and intervention groups were compared. Pain level, morning stiffness of knee joint, improvement in physical performance and improvement rate stiffness of knee in studied groups were examined. Repeated measurement and One-way analysis of variance (ANOVA) was used to compare quantitative variables between the three groups. Chi-square was used to compare qualitative variables. Bonferroni test was used for multiple comparisons.

Standardization of Thymus daenensis gel
The total phenolic content of 1 g of Thymus daenensis gel was obtained 5.56 ± 0.98 mg Gallic acid equivalent. Total flavonoid content of 1 g of prepared gel was obtained 0.98 ± 0.06 mg Rutin equivalent.

The baseline characteristics of the study
The process of registration of patients was conducted from June to August 2016. A total of 120 registered patients were divided into three groups placebo, diclofenac gel, and herbal gel in a random manner as presented Flow Diagrams (Fig. 1). The average age of the patients was 62.75 ± 8.67 in the range of 45-75 years, and it was 62.6 ±9.04, 63.83 ± 9.12, and 61.82 ± 7.92 in the placebo, herbal gel, and diclofenac groups, respectively. Out of all patients studied, 51 (43%) were male and 69 people (57%) were female. Regarding age and gender, no significant difference was observed between the groups (p = 0.587).

Clinical responses
Pain at six intervals had overall reduction both in herbal gel group and diclofenac group. On the day before the intervention and the first day after the intervention, significant difference was not found among three groups with respect to pain reduction (14.65 ± 0.77 vs. 13.44 ± 0.72, p = 0.628) and pain reduction was not significant, but since the third day, knee pain rate was significantly different between diclofenac and placebo, and between thyme and placebo since the first week (8.05 ± 0.68 vs. 12.35 ± 0.79, 9.02 ± 0.67 vs. 12.35 ± 0.79, p <0.001) and pain in the diclofenac and thyme groups reduced since that time compared to the placebo group. no significant difference was observed between diclofenac and thyme groups with respect to reduced pain up to the third week after the intervention (6.22 ± 0.62 vs. 7.52 ± 0.73, p = 0.796), but in the sixth week, a significant difference was observed between two groups (3.57 ± 0.41 vs. 6.02 ± 3.87, p = 0.02) and rate of reduction in pain was more in diclofenac group (Table 1, Fig.  2). The pain in the right knee since the third day had a significant difference between diclofenac and placebo groups and between placebo and thyme groups since the first week and the reduced pain was observed. In addition, up to the third week, not significant difference was observed between diclofenac and Thymus daenensis groups (1.25 ± 0.13 vs. 1.58 ± 0.13, p = 0.669), but in the sixth week, a significant difference was observed between the two groups (0.67 ± 0.09 vs. 1.25 ± 0.13, p = 0.01) that this reduction was more in the diclofenac group (Table 2, Fig. 3).
The pain in the left knee since the third day had a significant difference between diclofenac and placebo groups and between placebo and thyme groups since the first week and the reduced pain was observed. Besides that, up to the third week, no significant difference was observed between diclofenac and Thymus daenensis groups (1 ± 0.13 vs. 1.45 ± 0.12, p = 0.587), but in the sixth week, a significant difference was noted between the two groups (± 0.09 vs. 1.1 ± 0.12, p = 0.04) that this reduction was more in the diclofenac group (Table 3, Fig. 4). Morning stiffness rate declined both in herbal gel and diclofenac groups. The morn-ing stiffness had no significant difference among the three groups before the intervention and the first day after the intervention (3.02  ± 0.14 vs. 2.83 ± 0.14, p = 0.903), but with reduction in morning stiffness, since the third day, significant difference was observed between diclofenac and placebo groups and between thyme and placebo groups since the first week (  Fig. 5).
Regarding daily stiffness rate, significant difference was not found among three groups before intervention and first day after intervention (3.01 ± 0.15 vs. 2.81 ± 0.14, p > 0.05), but since the third day, significant difference was noted between diclofenac and placebo groups(2.22 ± 0.14 vs. 2.75 ± 0.16, p < 0.01), and since the first week, significant difference was observed between two  In these groups, pain reduction rate in diclofenac and herbal gel groups was more than the placebo group. In addition, no significant difference was observed between diclofenac and Thymus daenensis groups up to the third week after intervention (1.28 ± 0.13 vs. 1.55 ± 0.13, p > 0.05). However, daily stiffness reduction in the diclofenac group was higher in sixth week (0.75 ± 0.09, p < 0.02) ( Table 5, Fig. 6). Both in the herbal gel group and diclofenac group, physical performance improved. Regarding physical performance in one day before the intervention and the first day after the intervention, significant difference was not found among the three groups ( Fig. 7). The knee pain rate based on VAS criterion had no significant difference before intervention and first day after the intervention (7.67 ± 0.33 vs. 7.04 ± 0.31, p > 0.05), but a significant difference was noted between placebo and diclofenac groups since the third day (7.18 ± 0.33 vs. 5.72 ± 0.28, p < 0.05) and between placebo and thyme groups since first week (6.85 ± 0.34 vs. 5.45 ± 0.31, p <0.05) and a reduction in pain was observed. In addition, two groups of diclofenac and Thymus daenensis had no significant different up to a third week (3.9 ± 0.25 vs. 4.75 ± 0.34, p > 0.05), but since the sixth week, the difference was significant between the groups (2.78 ± 0.25 vs. 4.1 ± 0.33, p < 0.05) and pain reduction in the diclofenac group was higher ( Table 7, Fig. 8).
Comparison Means and 95% Confidence Intervals (CIs) of main outcomes in osteoarthritis patients before and after treatment was presented in Table 8.

Short term tolerance and safety
In the diclofenac group, no topical or systematic adverse effects were reported. In addition, thyme gel was accepted well by patients and no side effects or adverse physical effect was seen in follow-ups.

Discussion
This study examines the effect of Thymus daenensis extract gel on knee pain and performance of patients with osteoarthritis. Our study showed that knee pain of patients in the group consuming the Thymus daenensis gel significantly decreased so that knee pain reduction in thyme group was equal and without significant       [51], which in turn will intensify the pain. As a result, flavonoids reduce the pain and inflammation.
Research has also proven that these compounds can act as ligands for opioid receptors and directly exert their analgesic effect [52].
Since the effects of oxidative stress and the products of oxidative reactions on the creation of osteoarthritis have been proven, antioxidant effects of this plant can be possible causes of the observed effects, especially that effects of topical antioxidants containing vitamin C and E, selenium, zinc, isoflavones and polyphenols in tea in inhibiting oxidative stress processes in the skin have been demonstrated [53]. This suggests that topical application of antioxidants can also be effective. Free radicals of oxygen produced in the progression of this disease in joint, indirectly through causing tissue damage and direct effect on receptors causing pain, stimulate the neuroendocrine system [54]. The results obtained in the case of morning stiffness and stiffness of knee joint of patients during the day also indicated that there is Thymus daenensis gel reduces the stiffness of the knee joint that this reduction was equal to the fifth interval of followup with diclofenac. In the osteoarthritis disease, created inflammation leads to the creation of joint stiffness that our studied plant applies its anti-inflammatory effects through various mechanisms. The substances available in the plant extracts such as phenolic acids, alkaloids, tannins and saponins might be involved in this effect of thyme [20,21,55]. For example, in a study conducted by Cichocki et al. on the effect of several types of phenolic acids on mouse epidermal cells, it was observed that these compounds inhibited COX-2 activation. One of the most important enzymes that is involved in the production of inflammatory mediators such as prostaglandins, prostacyclin and thromboxane is cyclooxygenase whose inhibition inhibits the inflammatory pathways and loss of pain and inflammatory symptoms [56]. Another study by Singh et al. on cancerous cells confirmed the effect of isoquinoline alkaloid on the decreased expression of cyclooxygenase-2 and reduced production of prostaglandin E2 and prostaglandin E2 receptors [57]. In the case of physical performance of patients, physical activity significantly improved in both groups received diclofenac and Thymus daenensis that significant difference was not found between two groups to end of the study. This can be due to a reduction in knee pain of patients during daily activities and improvement in other symptoms of the disease by reducing inflammation.

Limitations of study
Although the effectiveness of the thyme gel was approved in this study, there are some limitations to generalize these results to the entire population, including the small size of the population used in this study. Lack of objective criteria for estimating the performance of the patients, despite validity and reliability of WOMAC questionnaire was another important limitation of the study. In addition, since the osteoarthritis is a disease with chronic nature, the study of variables at an interval after the intervention could be a limitation of our study. With increasing the period of study, we can have a better estimate of the impact and acceptability and safety of the gel. In addition, it determines side effects and topical and general effects resulting from continuous using of the considered drug well.

Conclusion
Based on the results of this study, the medicinal plant thyme could effectively reduce knee pain, morning stiffness and daily stiffness in the knees and improve physical performance of patients. In our study, no side effect for this plant was reported, so it can be argued that its use can produce a satisfactory effect on patients with osteoarthritis due to its low cost, easy access, the plant's natively occurring in Iran and no problem caused by the consumption of chemical drugs. It is suggested that be done further studies with different doses of the plant to be conducted to find the dose that has the highest impact and lowest side effect.