## 4 Discussion  
This study provides the in silico modeling and quantification of synergistic effects of multi-combination compounds--phytonutrients--to attenuate joint pain using a computational systems biology method--CytoSolve. The study revealed that four of the five  
Figure 6: Individual (**A**,**B**) and combination effect (**C**) of apigenin and hesperidin on ROS production in chondrocytes over simulations periods of 2 days. Apigenin at 30 mg dose, (**A**) hesperidin at 1000 mg dose, (**B**) and their combination at 1030 mg dose (**C**) reduced the levels of ROS over a period of 2 days.  
\begin{table}
\begin{tabular}{c c} \hline \hline
**In Silico Experiment** & **ROS (nM)** \\ \hline Apigenin First, Hesperidin Second & 1.34 \\ Hesperidin First, Apigenin Second & 12.69 \\ Apigenin and Hesperidin Simultaneously & 1.117 \\ \hline \hline \end{tabular}
\end{table}
Table 5: Estimations for additive ranges of ROS.  
biomarkers--COX-2, TRPV1, CGRP, and ROS--were synergistically affected by the specific combination of apigenin and hesperidin. This study provides an in silico computational systems biology framework to model and quantify the synergistic or additive effects for multi-combination compounds on a particular physiological indication.  
Joint pain is a hallmark symptom of several joint pathologies, such as osteoarthritis, rheumatoid arthritis, etc. [25,26]. In addition, sedentary lifestyles and aging have also been shown to cause joint pain [27]. The first line of treatment for joint pain includes non-steroidal anti-inflammatory drugs (NSAIDs) [28]; however, the chronic use of NSAIDs leads to adverse effects, including ulcers and kidney problems [29]. Increasing evidence suggests that phytonutrients with anti-inflammatory and antioxidant potential lower joint pain and improve inflammatory arthritis [30]. However, the mechanisms of actions of such phytonutrients in reducing joint pain remain poorly understood.  
Results from this study enable the mechanistic understanding of joint pain and the development of a novel plant-based joint pain intervention for joint-related pathologies such as osteoarthritis and rheumatoid arthritis. This study is a first of its kind, to the best of our knowledge, that has not only mathematically modelled mechanisms involved in joint pain using a computational systems biology approach to predict how apigenin and hesperidin modulate the production of pro-inflammatory agents responsible for joint pain but also in providing a quantification of the five specific biomarkers implicated in joint pain to determine synergy versus additive effects of a multi-combination cocktail of compounds.  
Systems biology is becoming a ubiquitous and reliable method to uncover how synthetic small molecules as well as phytonutrients influence a myriad of biological functions and diseases [31]. Leaf green vegetables, an essential part of Mediterranean, fruit, and vegetarian diets, have been shown to have anti-inflammatory effects [32]. Increasing evidences suggests that diets rich in anti-inflammatory and anti-oxidant compounds can modulate pain [33]. In this study, we focused on understanding the effects of apigenin, found in vegetables such as parsley, spinach, and celery, and hesperidin, which is found in citrus fruits, on joint pain. We used a systems biology approach to identify joint-pain molecular mechanisms of action. Both apigenin and hesperidin were found to have molecular targets in these molecular mechanisms of action. A summary of specific effects of apigenin and hesperidin is presented in Table 6 below.