Individuals who are not accustomed to rigorous physical activity, or athletes who are returning to training from a prolonged period of inactivity, often experience a dull, aching pain that usually occurs within 24 hours after activity. This type of soreness is known as delayed onset muscle soreness (DOMS). It usually manifests within the first 24 hours after exercise, reaching its peak within 72 hours, and slowly resolves in 5-7 days post-exercise^1,2. It presents clinically as discomfort, aching pain, swelling, tenderness, stiffness, and temporary loss of muscle strength and function^3,4. Aggregated effects of DOMS could also lead to detrimental injuries.

Etiological factors for DOMS involve both mechanical and biochemical components⁵. Among the multiple causes of DOMS sequential events, the following processes remain vital in the path to understanding this phenomenon: the initial damage, related to the mechanical stress of contraction that produce ruptures of myofibril filaments, and the secondary damage, related to later events of metabolic origin resulting loss of the intracellular calcium homeostasis, oxidative stress, and inflammation⁶. When initial eccentric damage to myofibrils occurs, there is an immediate decrease in muscle strength and membrane damage⁷. Damaged muscle fibers release prostaglandins and proinflammatory cytokines that activate endothelial cells. In turn, these cells express cell surface adhesion molecules and release chemotactic factors and proinflammatory cytokines (e.g., IL-1β, TNF-α, IL-6)⁸. These inflammatory mediators attract phagocytic cells such as neutrophils and monocytes toward the injury site. As a result, in the first few hours after injury, neutrophils and macrophages progressively accumulate in the subjected muscles, helping to remove and degrade damaged tissue by engulfing cellular debris and releasing proteases, inflammatory cytokines, and reactive oxygen species (ROS)⁹. ROS may further lead to activation of NF-κB that can mediate the expression of inflammatory cytokines. This may induce further inflammation and oxidative stress, leading to secondary muscle damage. In these orchestrated sequences of events, some factors seem to play important roles in muscle regeneration and remodeling^8-10. When the excess mechanical and biochemical stress produced at the time of exercise overcome the antioxidant and anti-inflammatory capacity of the body, there will be sustained inflammation and oxidative stress, which ultimately lead to muscle injury, fatigue, and DOMS¹⁰. Given these facts, a natural product with known anti-inflammatory and antioxidant activity with a good record of accomplishment of safety may provide a better intervention to DOMS.

Catechin, the major flavan in Acacia catechu, and the prenylated flavonoids and stilbenoids from the root bark of Morus alba L., possess properties suggestive of benefits in DOMS. These reported activities include: (i) inhibition of the activities of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), platelets phospholipase A2, and proinflammatory cytokines such as TNF-α, ILs 1, 2, 6, 8, and 12^11,12 as a result of catechin; (ii) inhibition of inflammation activity¹³; (iii) suppression effect of T-cell migration and inflammation induction¹⁴; (iv) inhibition of nitrogen oxide (NO), inducible NO synthase expression, prostaglandin E2 production, and activation of NF-κB¹⁵; (v) inhibition of proinflammatory mediators such as IL-1β, and IL-6 and COX-2¹⁶; and (vi) activation of total antioxidant ability^17,18 as a result of prenylated flavonoids and stilbenoids from M. alba root bark extract.

Furthermore, the standardized blend AmLexin has showed anti-inflammatory and antioxidant properties when evaluated in our laboratory using multiple models. In the carrageenan-induced rat paw edema model, AmLexin resulted in 46.3-53.3% reductions in paw edema and 43.6-54.8% reductions in pain sensitivity (common cardinal signs of inflammation) compared to the vehicle-treated control¹⁹. IC₅₀ (concentration causing 50% inhibition) values for major inflammation-mediating enzymes, such as cyclooxygenase 1 (COX-1), cyclooxygenase 2 (COX-2), and 5-lipoxygenase (5-LOX), were found to be 20.9 μg/mL, 49.2 μg/mL, and 11.1 μg/mL, respectively¹⁹. Administered at 50 mg/kg, AmLexin also showed statistically significant inhibition in key inflammatory cytokines such as IL-1β, TNF-α, and IL-6 in a collagen-induced rat arthritis model. AmLexin also showed higher capacity in neutralizing superoxide anions when tested for its antioxidant ability in an Oxygen Radical Absorption Capacity (ORAC) assay (manuscript submitted to Nutrients journal).

Based on these anti-inflammatory and antioxidant properties of AmLexin, we hypothesized that AmLexin could be a safe and effective botanical alternative for DOMS. The current double-blind placebo-controlled clinical trial was therefore designed to evaluate this hypothesis. AmLexin is a proprietary combination of extracts from the heartwood of Acacia catechu and root bark of Morus alba.

In the present study, a standardized blend from the heartwood of Acacia catechu and root bark of Morus alba (referred to as AmLexin) has been tested for its effect on delayed onset muscle soreness. This was done in a randomized double-blind placebo-controlled clinical trial on healthy subjects, following 13.1 miles of running. The active product was administered twice a day orally at 200 mg for a total daily dose of 400 mg/day over 9 weeks. The placebo group received a look-alike at the same dosage for the same duration. Subjects were trained for 8 weeks before the half marathon and were monitored for 1 week after the running. WOMAC was assessed for discomfort, stiffness, and activity of daily living, and VAS for major body muscles such as quadriceps, hamstrings, calves, and knees. Antioxidant activity of the products was also evaluated upon completion of the study. The study lasted for a total of 9 weeks. Data depicted here showed that subjects in the AmLexin group experienced statistically significant improvement in discomfort, ADL, and total WOMAC compared to the placebo group. There were significant reductions in muscle soreness for the AmLexin group following the half-marathon, with reduced oxidative stress.

Delayed onset muscle soreness is characterized as a grade 1 muscle strain injury that occurs as a result of unaccustomed exercise, strenuous physical activity, or high-intensity exercise involving eccentric muscle contraction. This includes resistance training, downhill running, cycling, or stepping⁴. It is known to decrease muscle strength, causing a profound impact on the ability to conduct subsequent sessions of exercise. This affects the overall performance of athletes and hinders individuals’ normal routine activities of daily living²³. A marathon is considered an endurance-demanding exercise that could cause a significant increase in oxidative stress, both in elite athletes and individuals new to running. This suggests that this is a good model for exercise-induced oxidative stress. In this class of intense physical exercise, there is always sustained inflammation and oxidative stress, which ultimately lead to muscle injury, fatigue, and DOMS.

Currently there are unmet demands for DOMS interventions. Through the years, many DOMS management modalities including stretching, massage, cryotherapy, nonsteroidal anti-inflammatory drugs (e.g. ibuprofen), or supplements rich in phytochemicals (e.g. catechin, quercetin, resveratrol, curcumin, gingerol, etc.) have been suggested. While some have shown future potential, many have been found to be ineffective^24-26. For example, when healthy subjects (n = 7) were supplemented with resveratrol for 7 days at an oral dose of 600 mg/day, no changes were observed in blood C-reactive protein and VAS pain and discomfort scale 24 hours after the marathon, in comparison with the placebo group²⁴. Similar outcomes were observed when healthy subjects were supplemented with Omega-3 fatty acids and isoflavons from soy for 5 weeks. These were administered for 30 days before and 1 week during induction of DOMS through eccentric elbow flexion contractions. The authors reported that there were no significant treatment effects between groups for the physical parameters or biomarkers CK, IL-6, TNF-α, and MDA at 48, 72, and 168 hours post-exercise²⁵. Donnelly et al. have also reported that ibuprofen is not an appropriate treatment for delayed onset muscle soreness and damage²⁶. One possible reason for this could be the drug’s lack of antioxidant properties. Therefore, an appropriate selection of interventions for DOMS should encompass both antioxidant and anti-inflammatory properties.

The anti-inflammatory and antioxidant activity of AmLexin has been reported both separately and in combination. Previously, AmLexin was tested in subjects with osteoarthritis in the knee, in comparison to active and placebo-controlled randomized clinical trials. Kalman and Hewlings have reported improvements in discomfort, stiffness, and activities of daily living measured by the WOMAC questionnaire and VAS (pain/discomfort) within all groups. There was a significant difference between the changes of uCTX-II for AmLexin and placebo groups after 12 weeks of study. Their data showed that early intervention with AmLexin, aimed at reducing bone and cartilage degradation through reported inhibition of catabolic proinflammatory pathways, might assist in preventing joint cartilage damage²⁷. Similarly, Yimam et al. have also reported the anti-inflammatory activities of AmLexin in in vivo and in vitro models with significant implication in DOMS¹⁹. Recently, we tested the effect of AmLexin on key proinflammatory cytokines such as IL-1β, Il-6 and TNF-α, and documented significant anti-inflammatory activity (manuscript submitted to Nutrients journal). The improved symptom relief observed in this clinical trial could be due to the direct benefits of the anti-inflammatory properties of AmLexin.

Oxidative stress (reflected in excessive production of ROS) is an imbalance between the production of free radicals and the inherent capacity of the body to counteract or neutralize their harmful effects. Unchecked, it will have detrimental consequences in performance or wellness. For example, excessive exercise (e.g. a marathon) is known to induce production of ROS, resulting in oxidative stress that may cause muscle damage, fatigue, oxidative damage, production of proinflammatory cytokines, and immune dysfunction. Previously, it has been shown that exercise utilizing large muscle groups, such as running, induces oxidative stress in correlation with increases in plasma levels of IL-6²⁸. AmLexin has been shown to reduce these factors significantly. The significant reductions in discomfort observed in the major muscles and body parts such as the calves, quadriceps, hamstrings, and knees could be true reflections of the antioxidant and anti-inflammatory activity of AmLexin.

Substantiating our findings, there is evidence that supports the ability of catechin to prevent exercise-induced muscle damage that could affect physical performances of athletes and/or daily activities of living. For instance, senescence-accelerated animals given daily treatment of catechin for 8 weeks have showed decreased loss of muscle force, exercise-induced muscle damage, and oxidative stress biomarkers such as creatine kinase, acetate dehydrogenase and malondialdehyde, while increasing activity of glutathione reductase. It was also found that long-term use of catechin reduced mRNA expression of TNF-α and monocyte chemoattractant protein-1 from the gastrocnemius muscle, possibly through the inhibition of NF-κB activation²⁹. Clinically, chronic consumption of a polyphenolic blend containing catechins and theaflavins for 13 weeks improved antioxidant status. This was measured by the ferric-reducing ability of plasma, reduced markers of muscle stress (such as cortisol and creatine phosphokinase), and the promotion of strength recovery post-exercise³⁰. Additionally, increase in the daily energy expenditure³¹, increased VO2 max ³², and reduced plasma creatinine kinase³³ are some of the major activities of catechin, with beneficial applications in exercise and DOMS management. Hence, the significant clinical improvements observed in the current clinical trial could be partially due to the contribution of catechin in AmLexin. Both medicinal plants standardized in AmLexin have traditionally been used for various human ailments with a long history of safe human consumption, making them viable natural alternative for DOMS supplementation.

In summary, exercise can trigger a cascade of inflammatory and oxidative stress-related events, leading to delayed onset muscle soreness. Data depicted here provides noteworthy evidence for the use of antioxidant and anti-inflammatory dietary supplementations such as AmLexin) before or during exercise, in order to minimize exercise-induced oxidative stress and inflammation that could cause discomfort, muscle damage, fatigue, aching pain, inflammation, and ultimately, cellular damage.