HPTLC Stability Indicating Analytical Method of Andrographolide and 5-fluorouracil with Network Pharmacology Analysis against Cancer

Background: Herbal drugs when used in combination with chemotherapeutic drugs can reduce the side effects and increase the efficacy by acting on multiple targets. Andrographolide (AG), a diterpene lactone isolated from Andrographis paniculata Nees, is a bioactive compound with anticancer potential, and 5-fluorouracil (FU), a pyrimidine analogue, is used in the treatment of cancer. Both drugs are used to formulate combination nanoformulation to increase absorption, thereby increasing their oral bioavailability. Objective: The study aimed to develop and validate stability indicating simultaneous HPTLC method for quantification of FU and AG in combination nanoformulation along with in silico docking and network pharmacology analysis to understand the interaction between the drugs and cancer targets. Methods: Chromatographic separation was performed using mobile phase chloroform: methanol: formic acid (9: 0.5: 0.5, v/v/v) on HPTLC silica plates 60 F254 as a stationary phase using UV-Vis detector and HPTLC scanner at 254 nm. Further, in silico docking analysis was performed to predict the binding affinity of AG and FU with different proteins and network pharmacology to find out the exact biomolecular relationship of AG and FU in alleviating cancer. Results: The data from the calibration curve showed a good linear regression relationship with r² = 0.9981 (FU) and r² = 0.9977 (AG) in the concentration range of 0.1-2.0 μg/mL. The developed method was validated according to the ICH guidelines. Stability studies showed changes in peak patterns and areas. Bioinformatic and network pharmacology analyses of AG and FU with target proteins and genes associated with cancer play a multimechanistic role in alleviating cancer. Conclusion: The developed method has been concluded to be robust, simple, precise, reproducible, accurate, and stability indicating for simultaneous quantification of AG and FU, and the molecular interaction studies have further indicated that the combination nanoformulation of AG and FU could be effective against cancer.