Abstract
Background
The essential oil from the pericarp (EOP) of Zanthoxylum rhetsa (Roxb.) DC. inhibits prostaglandin E2, which is related to knee osteoarthritis (OA). However, there is no clinical report on its efficacy.
Objectives
To assess the efficacy of EOP in Z. rhetsa (ZR) spray as a novel spray compared to diclofenac (DF) spray in elderly individuals diagnosed with primary knee OA.
Methods
60 patients with unilateral knee pain over three months were randomly assigned to either the ZR spray (experimental) or DF spray (control) group. Each group applied the spray topically 3 times daily (2 mL each time) for 14 consecutive days. Follow-ups occurred after day 7 and after day 14. Primary outcomes included pain score measurements, with secondary outcomes focusing on WOMAC index scores.
Results
The ZR and DF spray groups did not significantly differ at baseline. ZR spray is the first to demonstrate non-inferior efficacy compared to DF spray, with no significant difference in the mean change of pain scores at rest after 10 min (effect size <0.2) and following a 20-m walk test (effect size <0.5), including walking time (effect size <0.2), as well as in WOMAC index scores (effect size <0.3) from baseline to the first and second visits. Additionally, patients treated with ZR spray required less oral medication from the first visit.
Conclusions
Analgesia and improved knee functionality provided by ZR spray are suitable for combined treatment in elderly patients with co-morbidities or limited oral NSAID medication due to increased risk.
Keywords: Zanthoxylum, Osteoarthritis, Analgesia, Pain measurement, Volatile oils, Topical administration
1. Introduction
1.1. Background and rationale
Primary knee osteoarthritis (OA), the most common type, is associated with the degeneration of articular cartilage due to aging and is unrelated to trauma or other disorders [1]. The worldwide prevalence of knee OA occurs in individuals aged 40 and older, with approximately 23% in 2020 [2]. Knee OA is becoming an increasingly significant health concern in East and Southeast Asia [2]. In Thailand, the elderly population diagnosed with knee OA increased to 46.3% in 2020 [2]. Knee pain is the primary complaint among patients [3], with approximately 50% experiencing moderate to severe pain [4,5], and it is significantly associated with worse physical function and joint stiffness [5]. There is a significant association between the presence of knee OA and challenges in undertaking activities of daily living among the elderly, including difficulties in mobility, self-care ability, and performing usual activities [6]. Therefore, effective treatment is urgently needed to improve the quality of life, particularly for the elderly, who are becoming a major segment of the world's population.
The recommended approach for pharmacological management of knee OA is to begin with topical nonsteroidal anti-inflammatory drugs (NSAIDs) before considering oral NSAIDs [7]. The efficacy of both topical NSAIDs and oral NSAIDs indicated no significant difference in relieving pain and improving function during short-term treatment [8]. The primary concern with the use of oral NSAIDs in elderly patients with co-morbidities is the increased risk of gastrointestinal bleeding, as well as cardiovascular and renal diseases [9]. Although topical NSAIDs are generally considered safer than their oral counterparts, data on their long-term safety in older patients is limited. Previous studies have shown that using diclofenac solution for 4 weeks in elderly patients resulted in dry skin (36%), paresthesia (14%), and rash (13%) [10]. Consequently, there has been a notable increase in the utilization of various complementary and alternative medicines (CAM) for managing knee OA, especially among elderly patients. Research indicates that approximately half of these patients exhibit a preference for CAM in the treatment of knee OA, with 30% opting for treatments that incorporate topical herbal agents [11].
According to Thai Traditional Medicine (TTM) and Ayurvedic Medicine (AM), herbs play a role in CAM treatment for chronic pain of primary knee OA. The principles of TTM and AM theories refer to the relationship between Tri Dhatu (TTM theory) or Tri Dosha (AM theory) and the pain of knee OA. These theories also involve the correlation between the medicinal taste of herbs and their ethnopharmacological properties, which show a significant correlation [12]. Pain and degeneration of the knee joint have been considered due to aggravated Vata [13]. Therapeutic heat-based treatments could relieve pain by redistributing aggravated Vata, thereby balancing local Vata and increasing local blood circulation (Kapha) [14]. Therapeutic heat based on the medicinal tastes of herbs includes spicy and pungent tastes, which are predominant tastes in several herbs used to relieve pain and treat musculoskeletal disorders [15].
Zanthoxylum rhetsa (Roxb.) DC. (syn. Zanthoxylum limonella (Dennst.) Alston) [16] is an aromatic plant belonging to the Rutaceae family. It is widely distributed in India, Sri Lanka [16], and locally found in Northern Thailand. Its pericarp and whole fruit were traditionally used according to ancient Thai herbal medicine scriptures due to their medicinal taste, which is spicy with a pungent aroma. Ancient Thai medicinal formulas, such as Bee-Pra-Sen formula ointment and Pa-Ra-Ti-Tri formula oil, include the pericarp and whole fruit of Z. rhetsa to relieve muscle and tendon spasms. Additionally, its pericarp and whole fruit were used as medicaments in ancient Thai medicinal formulas to treat abscesses and as a medicament in Thai traditional household remedies to manage arthritis and myalgia. Our previous study has shown that the pericarp of Z. rhetsa fruits contains the highest content of essential oil at 14.3% (w/w) [17]. This essential oil from the pericarp (EOP) inhibits prostaglandin E2 (PGE2) in RAW264.7 macrophages with an IC50 of 24.13 μg/ml, approximately 1.70 times more potent than the essential oil from the whole fruit [17]. PGE2 is one of the key pain mediators related to knee OA [18]. However, clinical trials have not reported the efficacy of relieving pain using the EOP of Z. rhetsa.
Therefore, scientific evidence from clinical trials is essential to support the use of Z. rhetsa spray (ZR spray) as a combined treatment with modern medicine in elderly individuals with primary knee OA, especially those with comorbidities or limited oral NSAID options due to chronic pain and associated adverse effects. Additionally, the outcomes support and confirm the relationship between the medicinal spicy taste and pungent aroma of Z. rhetsa and its therapeutic effect.
1.2. Objectives
The objectives of this study were to assess the efficacy of the EOP of Z. rhetsa (ZR spray) in a novel topical spray formula enriched with essential oils and to compare it to diclofenac spray (DF spray) for elderly individuals with primary knee OA. Additionally, the study aims to follow up on the advantage of using the spray, which eliminates the need for additional oral medication.
2. Methods
2.1. Plant material preparation and essential oil distillation
Z. rhetsa was collected from Chiang Rai province, Thailand, and authenticated by a botanist who compared it to the voucher specimen (BKF number 193835) preserved at the office of the Forest Herbarium, Bangkok, Thailand. The sun-dried pericarp of Z. rhetsa was ground into a coarse powder and distilled using water distillation in a Clevenger apparatus for 24 h. The percentage yield of the EOP was 13% (v/w).
2.2. Spray preparation
The 9% (v/v) ZR spray, it is currently undergoing a petty patent application process in Thailand, with application number 2303000604 filed on March 3, 2023. This spray contains EOP at a concentration of 9% (v/v) in the base spray (BS). As for the 1% (w/w) DF spray, Uniren® spray, was purchased and manufactured by Unison Laboratories Company, Limited, Thailand. It contains diclofenac sodium at a concentration of 1% (w/w). Both sprays were packaged in 100 ml spray bottles and labeled with white opaque labels.
2.3. Identification of active constituents in ZR spray
The finished product of ZR spray was quality controlled to confirm active constituents before intervention by using Gas Chromatography/Mass Spectrometry (GC/MS). The identification of chemical compounds compared with the mass spectra reference of the National Institute of Standards and Technology (NIST 17, MD, USA) and Wiley 2011 (NJ, USA) databases. Matching scores of compounds more than 90% were selected. Active constituents of the finished product of ZR spray include sabinene, limonene, and terpinen-4-ol, which were compared with the retention times (RT) of reference compounds (Sigma-Aldrich, USA). The GC/MS analysis was conducted to describe the chemical constituents in ZR spray, which were categorized into two groups: 1) active constituents and 2) pharmaceutical ingredients of BS, as presented in Table 1.
Table 1.
Gas Chromatography/Mass Spectrometry (GC/MS) analysis of the chemical constituents of ZR spray.
| Chemical constituents of ZR spray | RT (mins) of compounds | RT (mins) of reference compounds | Matching scores of compounds |
|---|---|---|---|
| Active constituents | |||
| Sabinene | 7.808 | 7.831 | 94.35 |
| Limonene | 9.657 | 9.677 | 96.26 |
| Terpinen-4-ol | 15.572 | 15.634 | 96.21 |
| Pharmaceutical ingredients of base spray | |||
| Camphor | 14.205 | ND | 96.62 |
| Borneol | 15.082 | ND | 90.82 |
| Methyl salicylate | 16.299 | ND | 96.14 |
| Butylated hydroxytoluene | 29.554 | ND | 91.90 |
RT (mins) means retention times (minutes); ND means not detected.
2.4. Trial design
A randomized, double-blinded, and controlled clinical trial study received approval from the Mae Fah Luang University Ethics Committee on Human Research (COA: 022/2022, EC 21166-25) before enrolling patients. It was also registered with the Thai Clinical Trials Registry (Number TCTR20230906004).
2.5. Study population and sample size calculation
The required number of patients was calculated using G∗Power 3.1.9.7, based on either a priori type of power analysis. The calculation was based on previous research [19], focusing on the difference in mean scores on the 0 –10 Visual Analog Scale (VAS) between the two groups. The test's power was set at 90%, and the confidence level was 95%. The effect size d was calculated to be 0.91 for the sample size calculation. A dropout rate of 10% was estimated and adjusted for in the sample size calculation. Therefore, the total number of patients in this research was 60, with 30 individuals in each group.
2.6. Inclusion and exclusion criteria
Patients were enrolled based on the following criteria: 1) Age between 45 and 85 years, 2) Suffering from knee pain on one side for more than 3 months, 3) Having a pain score of at least 3 on the 0–10 VAS, along with joint stiffness and crepitation sound, 4) Diagnosed with primary knee OA by an orthopedic doctor based on clinical and radiographic findings, according to the American College of Rheumatology [20], and classified into grades 1–4 according to the Kellgren and Lawrence (KL) radiographic classification [21], 5) Receiving oral medication treatment with acetaminophen, NSAIDs, muscle relaxants, or symptomatic slow-acting drug of osteoarthritis (SYSADOA) only. Exclusion criteria included: 1) Body mass index (BMI) over 32 kg/m2, 2) Hypersensitivity to essential oils or perfumes, 3) Pregnancy, breastfeeding, cancer, infectious diseases, or disabilities, 4) Undergoing alternative treatments such as massage, acupuncture, or physical therapy, 5) Receiving oral corticosteroids or injectable corticosteroids within 3 months prior to the research or having undergone hyaluronic injections within 6 months prior to the research.
2.7. Study setting
Patients from three sub-districts (Tha Sut, Nang Lae, and Mae Kao Tom) in the Muang district of Chiang Rai province, Thailand, were invited to participate through public announcements. The principal investigator contacted all patients, who then willingly signed the informed consent form, with the option to withdraw from the study at any time.
2.8. Randomization and interventions
A five-block randomization, including gender, age, pain score, BMI, and KL radiographic findings grading, was employed by the clinical assistant for sampling and randomly allocated to either the experimental group (receiving a ZR spray) or the control group (receiving a DF spray). The patients, medical doctor, and principal and co-investigator were blinded to group assignment. Patients received the spray according to their assigned groups with explanations provided by the clinical assistant regarding the procedure for spray application and monitoring patients' compliance during the research period. They began using the spray on day 1, applying it around the area of knee pain 3 times a day in the morning, afternoon, and at bedtime. During each of these intervals, patients were instructed to apply 10 sprays with an estimated total volume of 2 ml and gently rub it in. This spraying regimen was repeated following the same procedure for 14 consecutive days. Patients' compliance with the allocated treatment was monitored by checking a daily compliance record form, and the amount of spray used was observed, typically totaling around 42 ml or approximately half of the spray bottle, during each follow-up visit from the clinical assistant. Throughout the research period, patients were allowed to take oral medication based on their doctor's prescriptions, in accordance with the inclusion criteria of oral medication treatment. The usage of any oral medication was recorded daily on a follow-up medicine usage record form and was monitored by the clinical assistant.
2.9. Outcome measurements
The patients underwent assessments at three time points by the principal and co-investigator: baseline (visit 0), after the 7th day (the first visit: 1st visit), and after the 14th day (the second visit: 2nd visit). The primary outcome measure included the use of the 0–10 VAS to assess pain scores at rest [19] and after a 20-m (m) walk test with walking time [22]. These measures were recorded at baseline (visit 0), the 1st visit, and the 2nd visit, with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC index scores) (Likert version 3.1) serving as the secondary outcome measure. Follow-up on oral medication usage with the spray during the 1st and 2nd visits was an additional outcome assessment.
2.10. Statistical analysis
Statistical analysis was carried out based on intention-to-treat (ITT) analysis and SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The results underwent a normality test using the Kolmogorov-Smirnov test. Categorical data were presented as numbers (n) and percentages (%), with statistical analysis performed using tests such as Pearson's Chi-Square or Fisher's Exact test to compare differences between the two groups. Continuous data were presented as either mean ± standard deviation (SD) or mean (95% Confidence Interval: CI), with statistical analysis conducted using methods such as the Independent t-test for parametric data and the Mann-Whitney U test for non-parametric data to assess differences between the two groups. Within each group, statistical analysis involved methods such as the Paired Sample t-test for parametric data and the Wilcoxon Signed Ranks test for non-parametric data. The significance level was set at a p-value of less than 0.05.
Non-inferiority study design: Non-inferiority testing was conducted by calculating the non-inferiority margin [23] based on previous research, focusing on the difference in mean change from baseline to week 4 in either the mean 0–10 VAS scores or mean WOMAC index scores between the two groups [19]. The non-inferiority margin was set at 0 for the pain scores (0–10 VAS) as the primary outcome and at 1 for the WOMAC index scores as the secondary outcome. Cohen's d was calculated to determine the effect size. The effect size was calculated as the difference in mean change between the experimental and control groups, divided by the pooled standard deviation [24]. The mean change in the experimental and control groups was calculated as the difference from baseline to the 1st visit (after the 7th day) and from baseline to the 2nd visit (after the 14th day).
3. Results
The patients were enrolled between April and July 2022. Sixty-eight patients were screened for eligibility, and eight were subsequently excluded. The remaining sixty patients were allocated through block randomization to receive either the ZR spray intervention (n = 30) or the DF spray intervention (n = 30). All patients were assessed for primary and secondary outcomes at baseline (visit 0) and followed up at the 1st visit (after the 7th day). At the 2nd visit (after the 14th day), one patient was lost to follow-up in both the ZR spray and DF spray groups due to Coronavirus (COVID-19) infections. However, all patients were included in the statistical analysis based on intention-to-treat (ITT) analysis (Fig. 1.).
Fig. 1.
CONSORT flow diagram of the study.
3.1. The demographic characteristics of primary knee osteoarthritis patients were obtained through randomization and allocation prior to the spray test
The demographic characteristics of patients in the ZR spray and DF spray groups are presented in Table 2. Most patients were female in both the ZR spray (73.33%) and DF spray (70%) groups. The mean age was 65.17 ± 4.39 years in the ZR spray group and 67.90 ± 8.14 years in the DF spray group. In the ZR spray group, 46.67% of patients were classified as obese, followed by 26.67% as overweight. In the DF spray group, 46.67% were classified as normal weight, with 30% as obese. However, there was no statistically significant difference in BMI classification (p-value = 0.121), similarly, the mean age and BMI also did not differ significantly (p-value = 0.113 and p-value = 0.052, respectively) between the ZR and DF spray groups. The mean pain score was 5.80 ± 2.07 in the ZR spray group and 5.57 ± 1.83 in the DF spray group. In the ZR spray group, 36.67% of patients had KL radiographic grade 3, followed by 26.67% with grade 2. In the DF spray group, 40% had grade 1, with 26.67% at grade 3. No significant differences were found between the groups in mean pain scores (p-value = 0.718) or KL radiographic grading (p-value = 0.257). Acetaminophen was the most commonly used medication, taken by 86.67% of the ZR spray group and 90% of the DF spray group. NSAIDs were used by 13.33% of patients in the ZR spray group, while NSAIDs, muscle relaxants, and SYSADOA were each used by 3.33% of the DF spray group. There was no significant difference in oral medication use for knee OA pain relief between the groups (p-value = 0.282).
Table 2.
Demographic characteristics of primary knee osteoarthritis patients treated with 9% (v/v) Zanthoxylum rhetsa spray (ZR spray) (n = 30) and 1% (w/w) diclofenac spray (DF spray) (n = 30).
| General characteristic | Results |
p-value | |
|---|---|---|---|
| ZR spray (n = 30) | DF spray (n = 30) | ||
| Gender (male/female), n (%) | 8/22(26.67/73.33%) | 9/21 (30.00/70.00%) | 0.774a |
| Age (Year), mean ± SD | 65.17 ± 4.39 | 67.90 ± 8.14 | 0.113b |
| Body mass index (kg/m2) | |||
| Underweight (<18.5 kg/m2), n (%) | 2 (6.67) | 3 (10.00) | 0.121a |
| Normal (18.5–22.9 kg/m2), n (%) | 6 (20.00) | 14 (46.67) | |
| Overweight (23–24.9 kg/m2), n (%) | 8 (26.67) | 4 (13.33) | |
| Obese (≥25 kg/m2), n (%) | 14 (46.67) | 9 (30.00) | |
| Mean ± SD | 24.96 ± 3.75 | 22.99 ± 3.93 | 0.052b |
| Pain score (0–10 Visual analog scale) | |||
| Mean ± SD | 5.80 ± 2.07 | 5.57 ± 1.83 | 0.718b |
| Kellgren and Lawrence radiographic grading, n (%) | |||
| Grade 1 | 5 (16.67) | 12 (40.00) | 0.257a |
| Grade 2 | 8 (26.67) | 6 (20.00) | |
| Grade 3 | 11 (36.67) | 8 (26.67) | |
| Grade 4 | 6 (20.00) | 4 (13.33) | |
| History of oral medications usage, n (%) | |||
| Acetaminophen | 26 (86.67) | 27 (90.00) | 0.282a |
| NSAIDs | 4 (13.33) | 1 (3.33) | |
| Muscle relaxant | 0 (0.00) | 1 (3.33) | |
| SYSADOA | 0 (0.00) | 1 (3.33) | |
Statistical analysis was conducted using methods such as.
Pearson Chi-Square or Fisher's Exact test, and.
Independent t-test or Mann-Whitney U test, as appropriate.
3.2. The effectiveness of spray in alleviating pain in patients with primary knee osteoarthritis
At baseline, both the ZR spray and DF spray groups did not show any significant difference in pain scores after a 10-min (min) rest period (p-value = 0.372, effect size = 0.24) and a 20-m walk test (p-value = 0.319, effect size = 0.19). The effectiveness of the ZR spray and DF spray treatment interventions in reducing pain scores after a 10-min rest period and a 20-m walk test led to improvements in pain scores among patients from baseline to the 1st visit and from baseline to the 2nd visit (Table 3). Between baseline and the 1st visit, the ZR spray showed improvements in pain scores after a 10-min rest, with a mean change of 1.07 ± 2.29 (21.10% improvement) from a baseline of 5.07 ± 2.53, and after the 20-m walk test, with a mean change of 1.47 ± 1.80 (26.25% improvement) from a baseline of 5.60 ± 2.11. In contrast, the DF spray showed a greater improvement in pain scores after the 20-m walk test, with a mean change of 2.33 ± 1.86 (38.83% improvement) from a baseline of 6.00 ± 2.15, but only a slight improvement after a 10-min rest, with a mean change of 0.70 ± 2.49 (15.66% improvement) from a baseline of 4.47 ± 2.45. Between baseline and the 2nd visit, both sprays showed further improvements in pain scores. After a 10-min rest, the ZR spray had a mean change of 2.23 ± 2.75 (43.98% improvement) and the DF spray 2.20 ± 2.63 (49.22% improvement). After the 20-m walk test, the ZR spray had a mean change of 2.27 ± 2.02 (40.54% improvement) and the DF spray 3.17 ± 2.44 (52.83% improvement).
Table 3.
Comparison of the mean change in pain scores from baseline to each subsequent visit (after the 7th and the 14th day) in 9% (v/v) Zanthoxylum rhetsa spray (ZR spray) (n = 30) and 1% (w/w) diclofenac spray (DF spray) (n = 30).
| Outcome variable | Baseline |
Mean change ±SD |
p-valueb | |
|---|---|---|---|---|
| Mean ± SD | The 1st visit | The 2nd visit | ||
| Rest time of 10 min | ||||
| Pain score | ||||
| ZR spray | 5.07 ± 2.53 | 1.07 ± 2.29 | 2.23 ± 2.75 | 0.002 |
| DF spray | 4.47 ± 2.45 | 0.70 ± 2.49 | 2.20 ± 2.63 | <0.001 |
| p-valuea | 0.372 | 0.390 | 0.863 | |
| Effect size | 0.24 | 0.15 | 0.01 | |
| A 20-m walk test | ||||
| Pain score | ||||
| ZR spray | 5.60 ± 2.11 | 1.47 ± 1.80 | 2.27 ± 2.02 | 0.002 |
| DF spray | 6.00 ± 2.15 | 2.33 ± 1.86 | 3.17 ± 2.44 | 0.011 |
| p-valuea | 0.319 | 0.103 | 0.177 | |
| Effect size | 0.19 | 0.47 | 0.40 | |
| Walking time (seconds) | ||||
| ZR spray | 17.57 ± 5.34 | 1.83 ± 4.29 | 2.87 ± 4.06 | 0.116 |
| DF spray | 17.53 ± 2.89 | 2.67 ± 4.11 | 3.10 ± 3.97 | 0.388 |
| p-valuea | 0.575 | 0.409 | 0.823 | |
| Effect size | 0.01 | 0.20 | 0.06 | |
Statistical analysis used.
The Independent t-test or Mann-Whitney U test to compare differences in mean or mean change between the ZR and DF spray groups, and.
The Paired t-test or Wilcoxon Signed Ranks test to compare mean changes between the 1st and 2nd visits from baseline.
This improvement is evidenced by the significant differences observed in the mean changes of pain scores from baseline to the 1st visit and from baseline to the 2nd visit (Table 3). The ZR spray exhibited a significant improvement in the mean change of pain scores for both the 10-min rest period and the 20-m walk test (p-value = 0.002). The DF spray exhibited a significant improvement in the mean change of pain scores after a 10-min rest period and a 20-m walk test (p-value <0.001 and p-value = 0.011, respectively). These results suggest that the potency of the ZR spray is similar to that of the DF spray. Both the ZR spray and the DF spray did not show a significant difference in the mean change of pain scores after a 10-min rest period from baseline to the 1st visit (p-value = 0.390 with an effect size of 0.15) and from baseline to the 2nd visit (p-value = 0.863 with an effect size of 0.01). Additionally, both ZR and DF sprays also did not show a significant difference in the mean change of pain scores after a 20-m walk test from baseline to the 1st visit (p-value = 0.103 with an effect size of 0.47) and from baseline to the 2nd visit (p-value = 0.177 with an effect size of 0.40) (Table 3).
In the 20-m walk test, at baseline, both the ZR spray and DF spray groups did not show any significant difference in walking time (p-value = 0.575, effect size = 0.01). Both treatments demonstrated a notable reduction in walking time from baseline to the 1st visit, with mean changes of 1.83 ± 4.29 (10.42% improvement) for ZR spray and 2.67 ± 4.11 (15.23% improvement) for DF spray, from baseline values of 17.57 ± 5.34 and 17.53 ± 2.89, respectively (Table 3). At the 2nd visit, both groups exhibited slight improvements, with mean changes of 2.87 ± 4.06 (16.33% improvement) for ZR spray and 3.10 ± 3.97 (17.68% improvement) for DF spray (Table 3). Therefore, there was no significant difference in the mean change of walking time from baseline to the 1st visit and from baseline to the 2nd visit for both ZR spray and DF spray (p-value = 0.116 and p-value = 0.388, respectively) (Table 3). The efficacy of the ZR spray compared to the DF spray was also evaluated, showing no significant difference in mean changes of walking time from baseline to the 1st visit (p-value = 0.409, effect size = 0.20) or from baseline to the 2nd visit (p-value = 0.823, effect size = 0.06) (Table 3).
3.3. The effectiveness of spray in alleviating symptoms and functional limitations in patients with primary knee osteoarthritis
The effectiveness of ZR spray and DF spray treatments in reducing pain, stiffness, and improving physical function, as measured by WOMAC index scores, led to improvements in the mean change of the total score and 3 subscale scores (pain, stiffness and physical function) from baseline to the 1st visit and from baseline to the 2nd visit (Table 4). At baseline, there was no significant difference in total scores between the ZR spray and DF spray groups (p-value = 0.920, effect size = 0.03). Both treatments showed effective improvements in symptoms and functional limitations by the 1st visit, with mean changes in total scores of 11.93 ± 14.19 (30.59% improvement) for ZR spray and 16.53 ± 16.19 (41.88% improvement) for DF spray, compared to baseline scores of 39.00 ± 18.61 and 39.47 ± 17.29, respectively. These improvements continued to the 2nd visit, with mean changes of 21.77 ± 16.45 (55.82% improvement) for ZR spray and 24.87 ± 15.58 (63.01% improvement) for DF spray, compared to baseline. For the pain and physical function subscale scores, there was no significant difference between the ZR spray and DF spray groups at baseline (p-value = 0.976, effect size = 0.01 for pain; p-value = 0.938, effect size = 0.02 for physical function). Both treatments demonstrated effective improvement in pain subscale scores by the 1st visit, with mean changes of 3.30 ± 3.13 (36.38% improvement) for ZR spray and 4.20 ± 3.86 (46.15% improvement) for DF spray, compared to baseline scores of 9.07 ± 4.70 and 9.10 ± 3.67, respectively. These improvements increased by the 2nd visit, with mean changes of 5.43 ± 3.62 (59.87% improvement) for ZR spray and 5.80 ± 3.52 (63.74% improvement) for DF spray, respectively, compared to baseline. Similarly, both groups showed improvements in the physical function subscale scores. At the 1st visit, the mean change for ZR spray was 7.93 ± 11.06 (28.80% improvement), and for DF spray, it was 11.83 ± 12.35 (42.55% improvement), from baseline scores of 27.53 ± 13.13 and 27.80 ± 13.13, respectively. By the 2nd visit, mean changes increased to 15.00 ± 12.52 (54.49% improvement) for ZR spray and 18.03 ± 12.29 (64.86% improvement) for DF spray, compared to baseline.
Table 4.
Comparison of the mean change in WOMAC index scores from baseline to each subsequent visit (after the 7th and the 14th day) in 9% (v/v) Zanthoxylum rhetsa spray (ZR spray) (n = 30) and 1% (w/w) diclofenac spray (DF spray) (n = 30).
| Outcome variable | Baseline |
Mean change ±SD |
p-valueb | |
|---|---|---|---|---|
| Mean ± SD | The 1st visit | The 2nd visit | ||
| WOMAC index scores | ||||
| A total score | ||||
| ZR spray | 39.00 ± 18.61 | 11.93 ± 14.19 | 21.77 ± 16.45 | <0.001 |
| DF spray | 39.47 ± 17.29 | 16.53 ± 16.19 | 24.87 ± 15.58 | <0.001 |
| p-valuea | 0.920 | 0.247 | 0.457 | |
| Effect size | 0.03 | 0.30 | 0.19 | |
| Pain subscale score | ||||
| ZR spray | 9.07 ± 4.70 | 3.30 ± 3.13 | 5.43 ± 3.62 | <0.001 |
| DF spray | 9.10 ± 3.67 | 4.20 ± 3.86 | 5.80 ± 3.52 | 0.009 |
| p-valuea | 0.976 | 0.326 | 0.692 | |
| Effect size | 0.01 | 0.26 | 0.10 | |
| Stiffness subscale score | ||||
| ZR spray | 2.43 ± 2.36 | 0.50 ± 2.47 | 1.33 ± 2.17 | 0.017 |
| DF spray | 2.57 ± 1.79 | 0.50 ± 2.19 | 1.03 ± 1.85 | 0.069 |
| p-valuea | 0.621 | 1.000 | 0.686 | |
| Effect size | 0.07 | 0.00 | 0.15 | |
| Physical function subscale score | ||||
| ZR spray | 27.53 ± 13.13 | 7.93 ± 11.06 | 15.00 ± 12.52 | <0.001 |
| DF spray | 27.80 ± 13.13 | 11.83 ± 12.35 | 18.03 ± 12.29 | <0.001 |
| p-valuea | 0.938 | 0.203 | 0.347 | |
| Effect size | 0.02 | 0.33 | 0.24 | |
Statistical analysis used.
The Independent t-test or Mann-Whitney U test to compare differences in mean or mean change between the ZR and DF spray groups, and.
The Paired t-test or Wilcoxon Signed Ranks test to compare mean changes between the 1st and 2nd visits from baseline.
This improvement is evidenced by the significant differences observed in the mean changes of the total score and the 2 subscale scores (pain and physical function) from baseline to the 1st visit and from baseline to the 2nd visit (Table 4). Both ZR spray and DF spray demonstrated effectiveness in significantly improving the mean change in the total score (p-value <0.001) and the pain subscale score (p-value <0.001 and p-value = 0.009, respectively), as well as in the physical function subscale score (p-value <0.001). ZR spray was also observed to have efficacy comparable to DF spray, with no significant difference between the two sprays in the mean change of the total score and 2 subscale scores (pain and physical function) from baseline to the 1st visit or from baseline to the 2nd visit (Table 4). For the total score: baseline to the 1st visit had a p-value of 0.247 with an effect size of 0.30, and baseline to the 2nd visit had a p-value of 0.457 with an effect size of 0.19. For the pain subscale score: baseline to the 1st visit had a p-value of 0.326 with an effect size of 0.26, and baseline to the 2nd visit had a p-value of 0.692 with an effect size of 0.10. For the physical function subscale score: baseline to the 1st visit had a p-value of 0.203 with an effect size of 0.33, and baseline to the 2nd visit had a p-value of 0.347 with an effect size of 0.24.
At baseline, there was no significant difference in stiffness subscale scores between the ZR spray and DF spray groups (p-value = 0.621, effect size = 0.07). Both treatments showed improvement in stiffness subscale scores by the 1st visit compared to baseline, with further increases observed by the 2nd visit. For the ZR spray, the mean change increased from 0.50 ± 2.47 (20.58% improvement) at the 1st visit to 1.33 ± 2.17 (54.73% improvement) at the 2nd visit, compared to a baseline score of 2.43 ± 2.36. For the DF spray, the mean change increased from 0.50 ± 2.19 (19.46% improvement) at the 1st visit to 1.03 ± 1.85 (40.08% improvement) at the 2nd visit, compared to a baseline score of 2.57 ± 1.79. Although the ZR spray significantly improved the mean change in the stiffness subscale score from baseline to the 1st visit and from baseline to the 2nd visit (p-value = 0.017), the DF spray did not show a significant difference in the mean change in the stiffness subscale score from baseline to the 1st visit compared to baseline to the 2nd visit (p-value = 0.069). However, these results support the potency of the ZR spray compared to the DF spray, showing no significant difference in the stiffness subscale score between the two sprays from baseline to the 1st visit (p-value = 1.000, effect size = 0.00) or from baseline to the 2nd visit (p-value = 0.686, effect size = 0.15) (Table 4).
3.4. The non-inferiority efficacy of ZR spray compared to the standard treatment of diclofenac spray in patients with primary knee osteoarthritis
The ZR spray demonstrated efficacy comparable to the DF spray, a positive control for alleviating symptoms and enhancing physical function in patients with primary knee OA. The investigation of the non-inferiority efficacy of the ZR spray compared to the DF spray was conducted by comparing the lower bound of the 95% CI for the ZR spray to the non-inferiority margin (Table 5). The non-inferiority margin was set at 0 for the pain scores after both the 10-min rest period and the 20-m walk test, and at 1 for the WOMAC index scores. The 95% CI for the mean change in pain score after a 10-min rest period was (0.21, 1.92) from baseline to the 1st visit and (1.21, 3.26) from baseline to the 2nd visit, indicating that the lower bounds of the 95% CI for the ZR spray were greater than the non-inferiority margin. Similarly, the 95% CI for the mean change in pain score after a 20-m walk test was (0.80, 2.14) for the 1st visit and (1.51, 3.02) for the 2nd visit, with corresponding walking times of (0.23, 3.44) and (1.35, 4.38), respectively, also showing that the lower bounds of the 95% CI for the ZR spray were greater than the non-inferiority margin. Additionally, the 95% CI for the mean change in the WOMAC index scores from baseline to the 1st visit was as follows: total score (6.64, 17.23) and subscale scores for pain (2.13, 4.47) and physical function (3.81, 12.06). For the 2nd visit, the 95% CI was: total score (15.62, 27.91) and subscale scores for pain (4.08, 6.78) and physical function (10.33, 19.67). These results indicate that the lower bounds of the 95% CI for the ZR spray were greater than the non-inferiority margin at both visits. In contrast, the stiffness subscale scores indicated that the 95% CI for the mean change was (−0.42, 1.42) from baseline to the 1st visit and (0.52, 2.14) from baseline to the 2nd visit. This indicates that the lower bounds of the 95% CI for the ZR spray were less than the non-inferiority margin, while the upper bounds were greater than the non-inferiority margin at both visits.
Table 5.
Comparison of the non-inferiority efficacy of ZR spray with the non-inferiority margin, as observed from the lower bound of the 95% confidence interval (CI) of the mean change in pain scores and WOMAC index scores from baseline to each subsequent visit (after the 7th and the 14th day).
| Outcome variable | Mean change (95% CI) |
Non-inferiority margin | |
|---|---|---|---|
| The 1st visit | The 2nd visit | ||
| Rest time of 10 min | |||
| Pain score | 1.07 (0.21, 1.92) | 2.23 (1.21, 3.26) | 0 |
| A 20-m walk test | |||
| Pain score | 1.47 (0.80, 2.14) | 2.27 (1.51, 3.02) | 0 |
| Walking time (seconds) | 1.83 (0.23, 3.44) | 2.87 (1.35, 4.38) | 0 |
| WOMAC index scores | |||
| A total score | 11.93 (6.64, 17.23) | 21.77 (15.62, 27.91) | 1 |
| Pain subscale score | 3.30 (2.13, 4.47) | 5.43 (4.08, 6.78) | 1 |
| Stiffness subscale score | 0.50 (−0.42, 1.42) | 1.33 (0.52, 2.14) | 1 |
| Physical function subscale score | 7.93 (3.81, 12.06) | 15.00 (10.33, 19.67) | 1 |
3.5. The advantage of using the spray is that it eliminates the need for additional oral medication in patients with primary knee osteoarthritis
During the 1st visit, the number of patients using only the ZR spray, without additional oral medications (including acetaminophen, NSAIDs, muscle relaxants, or SYSADOA), was 23.33%, which was approximately 2.60 times lower than the number of patients using only the DF spray without oral medication (60%). This difference was statistically significant (p-value = 0.004). However, the number of patients using only the ZR spray without oral medication increased at the 2nd visit to 66.67%, and there was no significant difference (p-value = 1.000) (Table 6).
Table 6.
Comparison of oral medication usage at each subsequent visit (after the 7th and 14th day) in 9% (v/v) Zanthoxylum rhetsa spray (ZR spray) (n = 30) and 1% (w/w) diclofenac spray (DF spray) (n = 30).
| Using medication | Number of patients using oral medication, n (%) |
|||
|---|---|---|---|---|
| The 1st visit |
The 2nd visit |
|||
| ZR spray | DF spray | ZR spray | DF spray | |
| Non-usage | 7 (23.33) | 18 (60.00) | 20 (66.67) | 20 (66.67) |
| Usage | 23 (76.67) | 12 (40.00) | 10 (33.33) | 10 (33.33) |
| p-value | 0.004 | 1.000 | ||
Statistical analysis was conducted using the Pearson Chi-Square test.
4. Discussion
Our findings demonstrate that ZR spray alleviates pain and functional limitations to a similar extent as DF spray, a topical NSAID, in standard treating patients with primary knee OA [7]. Our study conducted quality control testing on ZR spray, which is a finished product. Identifying components in a finished herbal product is crucial for quality control to confirm active constituents and pharmaceutical ingredients. Our analysis of ZR spray revealed that its active constituents are sabinene, limonene, and terpinen-4-ol (Table 1). These active constituents were reported to inhibit inflammatory mediators and cytokines implicated in pain and the degenerative mechanism of knee OA, thereby reducing the breakdown of chondrocytes and extracellular matrix components (ECM) such as collagen II and proteoglycans [25]. Several factors are associated with pain and the progression of knee OA, including: 1) upregulated inducible nitric oxide synthase (iNOS) in chondrocytes leads to high nitric oxide (NO) levels, inducing chondrocyte apoptosis and inhibiting collagen II and proteoglycan synthesis in the ECM [26]. NO has been utilized as one of the inflammatory markers in elderly patients with knee OA and causes pain [25,27], and 2) PGE2 and cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) also contribute to knee OA progression and causes pain. TNF-α and IL-1β induce the expression of cyclooxygenase-2 (COX-2) and microsomal PGE2 synthase, leading to the induction of PGE2 synthesis [28]. Elevated PGE2 levels degrade knee OA cartilage through collagen II breakdown and decreased proteoglycan synthesis in the ECM, thereby causing pain [18]. Limonene is one of the major active constituents in ZR spray, reported to inhibit iNOS and NO production in human chondrocytes [29]. It also inhibits PGE2 and COX-2 production, and TNF-α and IL-1β cytokines in RAW264.7 macrophages [30]. Other active constituents in ZR spray include sabinene and terpinen-4-ol. Sabinene inhibits NO production in RAW264.7 macrophages [31], while terpinen-4-ol inhibits PGE2 production and TNF-α and IL-1β cytokines in human monocytes [32]. Therefore, the active constituents in ZR spray include sabinene, limonene, and terpinen-4-ol, which are supported by strong scientific reports for their effectiveness in preventing cartilage breakdown by protecting chondrocytes and preserving ECM tissues. This results in the alleviation of pain in knee OA and slows its progression.
Additionally, limonene, sabinene, and terpinen-4-ol were the phytochemicals identified as spices [33]. The medicinal properties of spices and aromatic spices are extensively used to relieve aches and pain [34]. Similar to the TTM and AM theories, the properties of spices and aromatic spices give the medicinal taste of herbs a spicy and pungent flavor, which helps relieve pain by redistributing aggravated Vata [14,15]. Therefore, our ZR spray revealed that its active constituents: sabinene, limonene, and terpinen-4-ol, provide scientific evidence supporting the medicinal taste of herbs as spicy and pungent flavors with their therapeutic properties to relieve pain.
Influential factors significantly associated with pain and physical function in primary knee OA include age, BMI, and KL radiographic grading, with gender showing no significant association [35]. Our study controlled for these factors affecting pain as the primary outcome and physical function as the secondary outcome through block randomization. Therefore, before testing the spray-assigned groups, there were no significant differences in demographic characteristics between the ZR spray and DF spray groups (Table 2). Additionally, the majority of patients in both groups were using acetaminophen for pain relief, as it is recommended as a first-line analgesic drug for early OA pain management [36]. However, the usage of oral medications for pain relief did not differ significantly between the ZR spray and DF spray groups before testing (Table 2).
Our results demonstrated that ZR spray significantly improved the mean changes in pain scores after a 10-min rest period and a 20-m walk test from baseline to the 1st and 2nd visits (Table 3). Additionally, the efficacy of the ZR spray was comparable to that of the DF spray, with no significant difference in the mean changes of pain scores after a 10-min rest period and a 20-m walk test from baseline to the 1st and 2nd visits, with effect sizes between 0.2 and 0.5 (Table 3) indicating small effects [24]. The analgesic effect and anti-inflammatory properties of ZR spray are supported by essential oil extracted from the whole fruit composed of the pericarp of Z. rhetsa. The in vivo study demonstrated pain reduction by observing the licking behavior of rats after treatment with a topical gel containing 6% (w/w) essential oil obtained from whole fruits, which reduced pain in the tail-flick test in rats [37]. As expected, diclofenac sodium at 1% (w/w) in diclofenac gel reported a notable 57% reduction in mean VAS knee pain over a 4-week period and also resulted in a 21.5% decrease in the time for a 3-m walk, as measured by a timed up-and-go test [38]. Similar to our study, both ZR spray and DF spray showed efficacy in pain relief, resulting in a significant improvement in walking time since the 1st visit (Table 3). There was no significant difference between ZR spray and DF spray in the mean changes of walking time from baseline to the 1st and 2nd visits, with an effect size of less than 0.2 (Table 3), indicating a small effect [24].
The WOMAC index score was used to assess symptoms of pain, stiffness, and functional limitation in knee OA patients during daily activities. A significant correlation between the pain subscale scores of WOMAC and the stiffness and physical function subscale scores [39] indicated that the reduction of pain in knee OA was improving functional performance in activities. The analgesic effects of both ZR spray and DF spray demonstrated significant enhancement in the mean changes of the total score and 3 subscale scores in WOMAC index scores (Table 4). Additionally, the efficacy of ZR spray was also comparable to that of the DF spray for the mean changes in total and 3 subscale scores, with no significant difference from baseline to the 1st and 2nd visits, as indicated by an effect size of less than 0.3 (Table 4), suggesting a small effect [24]. In a similar manner, previous findings from a systematic review of herbal Chinese medicine analgesic plaster, which includes Chuan Jiao (Z. bungeanum Maxim.), have indicated that topical treatment for knee OA could also reduce WOMAC index scores in patients compared to NSAIDs [40]. These findings align with a meta-analysis of topical diclofenac solution for knee OA, demonstrating its efficacy in reducing mean scores of pain, stiffness, and physical function subscales, while also indicating a notable difference from the vehicle control [41]. While there have not been any reports on the efficacy of Z. rhetsa in improving WOMAC index scores, our study is the first to demonstrate its efficacy.
Additionally, our study investigated the non-inferiority efficacy of ZR spray compared to DF spray. Our results indicated that the lower bound of the 95% CI for the mean change in pain scores after a 10-min rest period and after a 20-m walk test, with walking time as the primary outcome, and for the mean change in WOMAC index scores as the secondary outcome, was greater than the non-inferiority margin (Table 5). This suggests that ZR spray demonstrated non-inferior efficacy to DF spray [23], a topical NSAID used as a positive control. While clinical trials assessing the non-inferiority efficacy of ZR spray containing the EOP of Z. rhetsa in improving pain and WOMAC index scores have not been reported, our study is the first to demonstrate its efficacy in clinical trials.
Several clinical trials on diclofenac sodium in knee OA allowed acetaminophen as rescue medication [[42], [43], [44]]. In our study, patients used oral medication as prescribed, with almost 90% of patients in both the ZR spray and DF spray groups using acetaminophen (Table 2). Treatment with ZR spray and DF spray reduced the number of patients needing oral medication since the 1st visit, continuing to the 2nd visit, with no significant difference between the two groups (Table 6). These findings suggest the superior potency of ZR spray and DF spray as topical NSAIDs compared to acetaminophen, leading to reduced acetaminophen consumption. Similar conclusions were drawn in a systematic review indicating that various herbal preparations could alleviate knee OA symptoms, potentially reducing NSAIDs consumption [45]. Additionally, a meta-analysis showed that topical NSAIDs are more effective in reducing pain and improving function in knee OA patients compared to acetaminophen [8]. Another study found that knee OA patients using topical 1% (w/w) diclofenac sodium did not require acetaminophen significantly more weeks than patients in the vehicle group [42].
Although our findings demonstrate the non-inferiority efficacy of ZR spray containing the EOP of Z. rhetsa compared to DF spray, ZR spray also has a few limitations, which include: 1) the stability of active constituents and the shelf-life of the product, and 2) The measurement of the range of motion of the knee joint helps support the efficacy of ZR spray for the stiffness subscale score in the WOMAC index.
5. Conclusion
Our study is the first to demonstrate the analgesic properties of the EOP of Z. rhetsa in a clinical trial, independent of essential oil from the whole fruit, and to confirm active constituents in ZR spray. ZR spray demonstrates analgesic effects and improves knee functionality in aging individuals with chronic pain in primary knee OA, comparable to DF spray as topical NSAIDs. This scientific evidence supports ZR spray's use for combined treatment with modern medicine for aging individuals with co-morbidities, reducing the need for additional oral medicines to manage chronic pain and avoiding associated adverse effects. It also supports its application in relieving pain from chronic inflammation associated with arthritis and myalgia, and also confirms the relationship between the medicinal spicy taste and pungent aroma of Z. rhetsa and its therapeutic effect.
Ethics approval with registration and consent to participate
The research obtained approval from the Mae Fah Luang University Ethics Committee on Human Research on February 11, 2022 (COA: 022/2022, EC 21166-25). It was also registered with the Thai Clinical Trials Registry (Number TCTR20230906004). Patients willingly signed the informed consent form and had the option to withdraw from the study at any time.
Funding sources
This research was funded (grant number: 641B10019, 2021) and received a publication fee from Mae Fah Luang University.
Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data statement
The data utilized to substantiate the findings within this research article are accessible from the corresponding author upon request.
Author's contributions
C.I.: Conceptualization, Methodology/Study design, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Writing – review and editing, Visualization, Supervision, Project administration, Funding acquisition. N.W.: Methodology/Study design, Investigation, Resources, Writing – original draft, Writing – review and editing, Project administration. W.C.: Methodology/Study design, Investigation, Resources, Writing – original draft, Writing – review and editing.
Declaration of generative AI in scientific writing
The authors declare that they did not use any artificial intelligence for writing this paper.
Acknowledgements
This research received support from Mae Fah Luang University and its affiliated units, namely the Medicinal Plant Innovation Center of Mae Fah Luang University and the Scientific and Technological Instruments Center of Mae Fah Luang University. The authors extend their gratitude to Mae Fah Luang University Medical Center for their assistance with clinical investigation. Additionally, the authors express their appreciation to the botanists at the Department of National Parks, Wildlife and Plant Conservation in Bangkok, Thailand, for plant authentication, and to the Forest Herbarium in Bangkok, Thailand, for preserving voucher specimens.
Footnotes
Peer review under responsibility of Transdisciplinary University, Bangalore.
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