Effect of Ageratum Conyzoides on Osteoarthritis in an Ageing Adult Population: A Double-Blind Randomized Placebo-Controlled Parallel Study

Abstract

This randomized, double-blind, placebo-controlled study investigated Ageratum conyzoides (A. conyzoides) for alleviating osteoarthritis symptoms and improving quality of life. Conducted in Australia between 2021 and 2024, the study included 70 adults aged ≥45 years with clinically diagnosed osteoarthritis. Participants consumed 250 mg of pyrrolizidine alkaloid-free A. conyzoides extract or a placebo daily for 12 weeks. Pain and function were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) every three weeks. Secondary measures included pain assessed by the Visual Analogue Scale (VAS), the SF-36 quality-of-life questionnaire, inflammatory markers, and safety parameters. A. conyzoides supplementation resulted in significant reductions in total WOMAC scores at weeks 9 and 12 (p < 0.05) compared to placebo. VAS pain scores were significantly lower at weeks 9 and 12 (p < 0.05). SF-36 scores improved significantly in the pain and role limitations due to physical health domains (p < 0.05). Plasma inflammatory markers IL-6 and IL-8 showed significant reductions compared with placebo (p < 0.05). No between-group differences were observed for adverse events. These findings demonstrate that A. conyzoides supplementation is a safe and effective option for reducing osteoarthritis symptoms, with significant improvements observed in pain, function, and inflammatory markers.

1. Introduction

Osteoarthritis (OA) is the most prevalent joint disorder globally, affecting approximately 9.3% of Australians according to the Australian Bureau of Statistics (2017–2018), with prevalence rapidly increasing among individuals over 45 years of age [1]. The prevalence of OA is estimated to range from 45 to 95 percent among affected populations, often resulting in restricted daily activity, work and school absenteeism, and significant personal distress [2,3]. Pain typically begins with joint use and arises from inflammatory processes within the joint structure, persisting throughout daily activities and significantly impacting quality of life [4,5]. Despite its high prevalence and substantial impact on daily life, OA remains inadequately treated, largely due to a limited understanding of disease mechanisms and lack of disease-modifying therapies [6].

The primary mechanisms underlying OA are not yet fully understood. However, painful symptoms are primarily attributed to the excessive production of inflammatory biomarkers, particularly interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8), which peak during disease progression and contribute to cartilage degradation [2,7]. Elevated inflammatory marker levels are directly linked to increased joint inflammation, irregular cartilage matrix degradation through matrix metalloproteinases (MMPs), and synovial tissue changes, all of which contribute to pain perception and heightened sensitivity of nerve endings [8,9].

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, naproxen, and ibuprofen remain the first-line treatment, inhibiting cyclooxygenase enzymes responsible for inflammatory mediator production [10]. While effective for most patients, frequent NSAID use can cause gastrointestinal complications (e.g., ulcers, bleeding) and systemic side effects such as nausea, dizziness, and organ damage [11,12]. Additionally, a large portion of OA patients do not achieve satisfactory symptom control or are unable to tolerate NSAID therapy. In a cross-sectional study, 50% of patients or their physicians expressed dissatisfaction with NSAID treatment, citing poor efficacy, side effects, or tolerance issues [13]. Moreover, real-world data indicate that approximately 10% of participants with moderate-to-severe OA exhibit NSAID intolerance, highlighting the need for safer, more tolerable alternatives [14,15].

Ageratum conyzoides L. (Asteraceae), commonly known as billygoat weed, is an annual herb native to Africa, Asia, and South America [16]. Ageratum has a long tradition of use in ethnomedicine for a wide range of conditions, including fever, rheumatism, headaches, toothaches, skin disorders, leprosy, inflammatory pain, spasm-related ailments, and as a topical dressing for wounds (e.g., as a febrifuge, purgative, anti-inflammatory agent, and wound healer) [17,18]. This therapeutic versatility is attributed to its diverse pharmacological activities, including antioxidant, antimicrobial, analgesic, and anti-inflammatory properties [16,19].

Phytochemical analysis has identified numerous bioactive compounds in A. conyzoides, including benzofurans, phenols, phenolic esters, alkaloids, coumarins, chromenes, terpenoids, steroids, and flavonoids [16,20]. The plant’s anti-inflammatory activity is particularly associated with its flavonoid fraction, which has been extensively documented for its anti-inflammatory properties [21,22].

Several preclinical studies have demonstrated the anti-arthritic properties of pyrrolizidine alkaloid-free A. conyzoides (A. conyzoides). Bahtiar et al. (2017) found that ethanolic extracts of A. conyzoides at doses of 80 mg and 160 mg per 200 g body weight decreased edema volume, increased articular cartilage area and thickness, and elevated proteoglycan levels in monosodium iodoacetate (MIA)-induced OA rats through anti-inflammatory actions, including inhibition of TNF-α and MMP expression [23]. Similarly, Permawati et al. (2019) demonstrated that a nanoemulgel containing A. conyzoides (in combination with Oldenlandia corymbosa) reduced inflammatory biomarkers in MIA-induced OA rats [24].

Given A. conyzoides’ well-documented analgesic and anti-inflammatory properties, particularly those derived from polymethoxyflavone-rich extracts (e.g., SEPAc), and its demonstrated safety during prolonged therapeutic use, it represents a promising alternative for OA management [21,22]. Although preclinical studies have consistently demonstrated the analgesic and anti-inflammatory effects of A. conyzoides, to date no published double-blind, placebo-controlled clinical trials have evaluated its efficacy in OA management. The present study therefore represents the first such clinical trial, addressing this important gap in the literature. This study aimed to evaluate the effectiveness of A. conyzoides supplementation compared with a placebo in alleviating OA symptoms in ageing adults, hypothesizing that A. conyzoides supplementation would significantly reduce pain severity and improve joint function compared with a placebo.

2. Materials and Methods

2.1. Study Design

This was a 12-week, randomized, double-blind, placebo-controlled clinical trial utilizing an active group (A. conyzoides extract) and a placebo group (microcrystalline cellulose). The study was conducted in Australia between November 2021 and December 2024.

2.2. Study Population

Participants were recruited from an existing participant database and via social media outlets from across Australia. Interested individuals were provided with a participant information sheet and screened against the inclusion and exclusion criteria (detailed below) via telehealth consultation.

Seventy (70) adults aged 45 years and older with clinically diagnosed osteoarthritis were enrolled in this study. Participants were included if they provided informed consent, had a clinical diagnosis of osteoarthritis by a qualified health professional with written evidence, reported joint pain ≥ 10 mm on the Visual Analogue Scale (VAS) not associated with acute injury or prescription medication use, had a BMI ≤ 35 kg/m², and agreed not to change their current diet or exercise programs during the trial. Participants were also required to agree not to undertake another clinical trial while enrolled. Participants with clinically diagnosed mild-to-moderate osteoarthritis were included; those with severe OA were excluded.

Exclusion criteria included unstable or serious illness (e.g., serious mood disorders, neurological disorders, kidney disease, liver disease, heart conditions, diabetes, or thyroid dysfunction), current malignancy (excluding basal cell carcinoma) or chemotherapy/radiotherapy treatment within the previous two years, clinically significant acute or chronic inflammation or connective tissue diseases (rheumatoid arthritis, tendinitis, bursitis, fibromyalgia, gout), history of knee or hip surgery or trauma within the previous 12 months, use of anticoagulation therapy, or treatment for joint pain including corticosteroids or supplements (without a required four-week washout period). Other exclusion criteria included active smoking or substance abuse, chronic alcohol use (>14 drinks per week), allergy to study ingredients, pregnancy or breastfeeding, participation in related studies within the previous month, or infection within the month prior to study commencement.

2.3. Chemical Analysis

Both the active and placebo products were manufactured according to Good Manufacturing Practice standards (Pharmaceutical Inspection Co-operation Scheme (PIC/S). Guide to Good Manufacturing Practice for Medicinal Products (PE009-17). Geneva: PIC/S, 2023) in a Therapeutic Goods Administration–approved facility. The active product, supplied by Gencor Pacific Ltd. (Lantau Island, Hong Kong), contained 250 mg of pyrrolizidine alkaloid–free Ageratum conyzoides extract. The final composition of the extract was 6% water, 0.75% palmitic acid, 10% saponin glycosides, 5% tannins, 2% bitters, 10% fatty acids, 15% flavonoids, 10% terpenes, 5% maltodextrin, and 36.25% other compounds co-extracted from the aerial parts of A. conyzoides. The placebo consisted solely of microcrystalline cellulose.

2.4. Study Procedure

The study was conducted remotely, with no face-to-face interaction with participants. All communication between investigators and participants was conducted via phone (primary contact method), email, or text message (for reminders). Eligible participants completed baseline measures [self-reported demographics and questionnaires—International Prostate Symptom Score (IPSS), Aging Males Symptoms (AMS) for males, and Women’s Health Questionnaire (WHQ) for females], after which the trial product was mailed to them.

Compliance was monitored using participant self-reported diaries returned at weeks 6 and 12, as well as by capsule counts of unused product at study completion. Participants received a 6-week supply of capsules at baseline and again at week 6. Weekly phone calls and electronic reminders (email or SMS) were used to reinforce adherence and monitor adverse events.

Participants were randomly allocated (1:1 ratio) to receive either 250 mg of A. conyzoides (active) or a placebo in identical opaque capsules. Randomization was performed online from: https://www.sealedenvelope.com/simple-randomiser/v1/lists (accessed on 13 October 2021) by an individual not involved in the trial to ensure both participants and investigators remained blinded to allocation.

Participants consumed one capsule, once daily in the evening with food throughout the 12-week study period. All participants, investigators, and laboratory personnel remained blinded to treatment allocation throughout the study and analysis phases.

Adverse events (AEs) were monitored throughout the study through spontaneous participant reporting and at scheduled study visits. Investigators recorded the type, duration, severity, and possible relationship of each AE to the investigational product. Severity was graded according to standard criteria (mild, moderate, severe), and any serious adverse events were reported immediately to the ethics committee and trial sponsor in accordance with Good Clinical Practice requirements.

2.5. Outcome Measures

The primary outcome measure for this study was osteoarthritis symptom severity assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC, version 3.1). The WOMAC is a validated self-administered health status measure consisting of 24 questions assessing pain, stiffness, and function in people with osteoarthritis of the hip or knee, completed every three weeks throughout the trial.

Secondary outcome measures included joint pain severity assessed using the VAS where participants scored their morning and evening pain daily for seven days at weeks 3, 6, 9, and 12 on a 100 mm scale (0 = “no pain” to 100 = “worst pain”); the SF-36 Quality of Life Questionnaire completed at baseline, week 6, and week 12; male-specific questionnaires (IPSS and AMS); female-specific questionnaire (WHQ); plasma inflammatory biomarkers (TNF-α, IL-6, IL-8, IL-10, CRP) measured at baseline, week 6, and week 12; safety biomarkers (liver function tests, complete metabolic panel, PSA for males); anthropometric measurements; rescue medication use; and adverse event reporting. Rescue medication use was defined as any self-reported use of analgesics (e.g., ibuprofen, paracetamol) or physiotherapy for OA-related pain, recorded in participants’ study diaries.

2.6. Statistical Analysis

Sample size was calculated to detect a 15-point difference in WOMAC scores between groups with 95% power and α = 0.05. Twenty-seven participants per group were required, with 40 participants per group (80 total) enrolled to account for an expected 30% dropout.

Statistical analyses were conducted using GraphPad Prism 8.0 and SPSS 25. Continuous data were tested for normality using the Shapiro–Wilk test. Changes in outcome measures over time were analyzed using repeated-measures analysis of variance, with time and treatment condition as factors. For post hoc comparisons, paired t-tests evaluated differences between time points within each treatment condition. Between-group differences at each time point were analyzed using unpaired t-tests. Missing data were handled using mixed-effects models. All tests were two-tailed, with statistical significance set at p < 0.05.

2.7. Ethical Considerations

All participants provided written informed consent prior to commencing the study. This study was conducted in compliance with the International Conference on Harmonization Guideline for Good Clinical Practice and the Therapeutic Goods Administration. Ethical approval was obtained from the Bellberry Limited Human Research Ethics Committee (Adelaide, Australia). This clinical trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12621000694819). The trial protocol and statistical plan were not publicly archived.

3. Results

Seventy participants (25 male, 45 female) with clinically diagnosed osteoarthritis were randomized (Figure 1). Twelve participants withdrew during the study, leaving 58 who completed the trial and were included in the final analysis (A. conyzoides n = 28 and placebo n = 30). Of the withdrawals, three participants (A. conyzoides n = 2 and placebo n = 1) reported adverse events, none of which were related to the study product. Baseline demographic and anthropometric characteristics were comparable between groups, with no statistical differences observed (

Table 1. Demographics of participants completing the study.

Characteristic	A. conyzoides (n = 28)	Placebo (n = 30)
Age (years)	63.9 (8.4)	64.8 (9.6)
Weight (kg)	75.1 (14.2)	78.7 (15.1)
Height (m)	1.7 (0.1)	1.7 (0.1)
BMI (kg/m2)	26.7 (4.1)	27.8 (4.7)
Waist (cm)	90.4 (11.2)	92.5 (12.2)
Hip (cm)	104.8 (10.8)	106.5 (10.8)

All values are mean (SD).

).

Participants with both knee and hip osteoarthritis recorded data for both joints, resulting in data from 84 joints total. Progressive reductions in WOMAC scores occurred over the 12-week intervention period, with the A. conyzoides group showing greater improvements than placebo (

Table 2. WOMAC total and sub-scores.

	A. conyzoides (n = 28)					Placebo (n = 30)
	Baseline	Week 3	Week 6	Week 9	Week 12	Baseline	Week 3	Week 6	Week 9	Week 12
Joint Pain	6.2 (2.9)	4.9 (2.9)	4.2 (3.0)	4.1 (2.4)	3.6 (2.6) *	6.4 (3.1)	6.4 (3.3)	5.5 (3.0)	5.2 (3.7)	5.4 (3.4)
Joint Stiffness	3.2 (1.6)	2.5 (1.5)	2.1 (1.5)	2.2 (1.3)	2.1 (1.3)	3.1 (1.7)	3.0 (1.4)	2.4 (1.4)	2.6 (1.7)	2.5 (1.4)
Joint Function	21.9 (10.2)	17.0 (8.5)	15.3 (8.7)	14.1 (8.6) *	13.9 (7.6) *	21.0 (12.1)	21.1 (10.9)	17.8 (10.2)	18.5 (12.6)	18.0 (11.5)
WOMAC Total	31.4 (13.9)	24.5 (12.1)	21.6 (12.4)	20.4 (11.5) *	19.6 (10.8) *	30.4 (16.1)	30.5 (14.7)	25.7 (14.1)	26.2 (17.5)	25.9 (15.6)

* Statistically significant difference between active and placebo groups (p < 0.05).

, Figure 2).

Total WOMAC scores were lower in the A. conyzoides group compared to placebo at week 9 (p = 0.031) and week 12 (p = 0.021). Compared to the placebo group, joint pain in the A. conyzoides group decreased significantly at week 12 (Table 2, p < 0.05). Between-group differences were also observed for joint function at weeks 9 and 12, and for pain scores at week 12. No differences were found for joint stiffness at any timepoint.

VAS results were consistent with WOMAC findings, showing significantly lower pain scores in the A. conyzoides group at weeks 9 and 12 compared to placebo (p < 0.05) (Figure 3,

Table 3. VAS Pain scores.

Time	A. conyzoides (n = 28)	Placebo (n = 30)
Baseline	38.4 (17.5)	41.9 (18.9)
Week 3	25.8 (14.8)	31.4 (20.0)
Week 6	21.2 (13.7)	27.4 (18.0)
Week 9	19.0 (13.3) *	27.3 (19.3)
Week 12	16.9 (11.9) *	25.5 (20.1)

* Statistically significant difference between groups (p < 0.05).

).

The A. conyzoides group had significant improvements in SF-36 scores for “pain” and “role limitations due to physical health” domains compared to placebo group (

Table 4. SF-36 Quality of Life Questionnaire.

Domain	A. conyzoides (n = 28)			Placebo (n = 30)
	Baseline	Week 6	Week 12	Baseline	Week 6	Week 12
Physical Functioning	53.3 (20.8)	62.2 (22.5)	65.2 (18.7)	56.3 (24.4)	62.2 (22.0)	60.3 (22.2)
Role limitations (physical)	54.2 (43.1)	61.7 (40.3)	84.2 (29.0) *	64.7 (37.5)	72.1 (36.3)	67.6 (42.4)
Role limitations (emotional)	73.3 (39.5)	88.9 (26.7)	88.9 (25.3)	75.5 (37.9)	82.4 (32.0)	81.4 (36.0)
Energy/fatigue	59.0 (17.1)	63.7 (14.9)	65.8 (17.1)	58.8 (16.4)	61.2 (15.4)	60.1 (17.1)
Emotional well-being	80.3 (12.4)	82.7 (11.0)	81.9 (14.2)	77.5 (10.6)	80.9 (10.9)	79.3 (14.3)
Social functioning	84.2 (18.0)	87.5 (13.9)	90.4 (13.0)	84.9 (20.1)	90.4 (11.5)	87.5 (16.6)
Pain	58.1 (17.9)	70.3 (12.5)	73.5 (13.9) *	56.1 (16.1)	67.1 (18.3)	66.6 (15.1)
General Health	75.7 (12.7)	76.7 (12.0)	76.0 (11.7)	74.7 (14.4)	76.2 (14.1)	73.7 (17.7)

* Statistically significant difference between groups (p < 0.05).

). No significant changes were observed in male-specific questionnaires (IPSS and AMS) or female-specific questionnaires (WHQ).

Significant reductions from baseline were observed in the A. conyzoides group for plasma IL-6 (−2.40 ± 5.47 pg/mL, p < 0.05) and IL-8 (−6.35 ± 16.37 pg/mL, p < 0.05) compared to placebo (

Table 5. Plasma biomarker changes from baseline.

Marker	A. conyzoides (n = 28)		Placebo (n = 30)
	Baseline	Change at Week 12	Baseline	Change at Week 12
CRP (mg/L)	3.72 (4.59)	−0.26 (6.06)	3.84 (3.41)	0.77 (4.13)
IL-10 (pg/mL)	33.13 (56.73)	−5.39 (18.27)	19.33 (32.64)	0.71 (9.20)
IL-6 (pg/mL)	17.84 (26.78)	−2.40 (5.47) *	10.79 (20.14)	0.45 (4.56)
IL-8 (pg/mL)	29.78 (41.93)	−6.35 (16.37) *	22.62 (38.83)	1.80 (9.71)
TNF-α (pg/mL)	14.29 (13.57)	0.65 (4.93)	11.94 (11.07)	−0.10 (3.93)
PSA (ng/mL) a	2.25 (2.38)	0.04 (0.22)	1.22 (1.20)	0.04 (0.36)

^a measured in males only; * Significant difference between groups (p < 0.05).

). No significant changes were observed for other inflammatory markers.

A significant reduction in rescue medication use over the 12 weeks was seen in the A. conyzoides group (25 to 16 participants), while the placebo group showed no change (21 to 19 participants) (χ² = 4.97, p = 0.026).

Five adverse events were recorded during the study. Two events in the A. conyzoides group were deemed unrelated to the study product. Three events occurred in the placebo group: one unrelated to the product and two gastrointestinal events (flatulence and nausea) possibly related to treatment.

4. Discussion

This randomized, double-blind, placebo-controlled trial is the first to demonstrate the clinical efficacy of A. conyzoides extract in managing OA symptoms among ageing adults. The daily dose of 250 mg administered over 12 weeks resulted in statistically significant and clinically meaningful improvements in pain, functional capacity, quality of life, and reductions in inflammatory biomarkers.

Progressive reductions in WOMAC scores, particularly within the pain and function domains, suggest the ability of A. conyzoides to target central features of OA symptoms. Improvements increased over the intervention period, reflecting a potential cumulative therapeutic benefit. While the observed effect did not reach the postulated 15-point difference, consistency across secondary measures, including VAS pain scores and SF-36 quality of life assessments, provides strong support for the clinical relevance of these findings [10,25].

Marked decreases in circulating plasma IL-6 and IL-8 concentrations are noteworthy, given their established associations with OA pathology. IL-6 plays a central role in synovial inflammation and the astimulation of matrix metalloproteinases (MMPs) leading to cartilage degradation, while IL-8 contributes to leukocyte infiltration and sustained joint inflammation [7,8]. Attenuation of these cytokines suggests that A. conyzoides may act through anti-inflammatory pathways to reduce structural and symptomatic disease burden. The results align with preclinical studies in animal models showing inhibition of TNF-α and MMP expression following treatment with A. conyzoides extracts [23,24]. In the present study, no between-group differences were observed in TNF-α, CRP or IL-10. This may indicate a mechanism of action targeting a specific anti-inflammatory pathway or a discrete stage within one. Differences from preclinical studies may reflect dose or exposure variation, model design or species context. Further investigations focusing on biomarkers isolated from synovial fluid may provide a clearer delineation of the mechanism.

Osteoarthritis is a multifactorial disease involving multiple cell types, including chondrocytes, synoviocytes, and infiltrating immune cells, with NF-κB signaling, IL-6/IL-8 release, and leukocyte infiltration identified as central drivers of inflammation and joint damage [26,27]. Preclinical studies of A. conyzoides provide mechanistic support for our findings. Flavonoid-rich extracts of A. conyzoides have been shown to suppress NF-κB activation, reduce pro-inflammatory cytokine release (including IL-6 and IL-8), and attenuate leukocyte infiltration in animal models [17,21,22]. These actions align with our observed reductions in circulating IL-6 and IL-8, suggesting that A. conyzoides may exert targeted anti-inflammatory effects relevant to OA pathophysiology.

Reduced use of rescue medication in the A. conyzoides group provides an important indicator of meaningful symptom relief in daily life. Findings also confirmed a favourable safety profile, with no significant alterations in liver function tests or clinical biochemistry, and minimal adverse events, comparable with placebo. As such, A. conyzoides may offer a safer alternative or help reduce reliance on NSAIDs—a clinically significant outcome given the cardiovascular, renal, and gastrointestinal risks associated with long-term NSAID consumption in older adults [3].

The symptomatic benefits observed may derive from the plant’s diverse phytochemical composition. Flavonoids and other bioactive compounds present in A. conyzoides have been reported to modulate inflammatory signalling cascades, inhibit prostaglandin synthesis, and decrease the release of pro-inflammatory mediators [17,21,22].

However, several potential limitations must be acknowledged. The modest sample size may limit statistical power for secondary outcomes and subgroup comparisons. The 12-week intervention period, while sufficient to establish efficacy, does not inform long-term effects, durability of benefit, or optimal dosing schedules. Additionally, participants presented with mild-to-moderate OA; thus, extrapolation to individuals with severe disease may not be appropriate [1,2]. Another limitation is the absence of imaging (X-ray or MRI) to assess structural joint changes such as cartilage thickness or synovial inflammation. Future studies should incorporate imaging analyses to complement symptomatic and biomarker outcomes.

Further investigation is warranted to determine the long-term impact of A. conyzoides on disease progression and potential adverse effects. Trials of extended duration with larger cohorts could evaluate sustained symptom relief, radiographic or MRI-based structural changes, and possible disease-modifying effects. Exploration of synergistic effects with conventional therapies and optimal dosing regimens would also enhance translational value.

5. Conclusions

Daily supplementation with 250 mg of A. conyzoides extract over 12 weeks was associated with significant improvements in pain, physical function, and quality of life in individuals with osteoarthritis, accompanied by reductions in key inflammatory cytokines. Consistent improvements across patient-reported outcomes, functional measures, and biomarker assessments underscores the therapeutic potential of A. conyzoides in OA management. Importantly, the favourable safety profile and reduced reliance on rescue medication further strengthens its suitability in the long-term management of osteoarthritis.

Collectively, the results of this study position A. conyzoides as a promising, safe, and well-tolerated adjunct or alternative to current pharmacological approaches. Further investigations of longer duration and larger sample sizes, incorporating imaging measures, are warranted to confirm efficacy.