Aims:The oriental-based herbs Acalypha indica (AI), Centella asiatica (CA), and Sesbania grandiflora (SG) possess a broad range of undisclosed therapeutic activities which are edible and easily available throughout the year. To convert the herb extracts into a potential drug form, aqueous (A) and methanol (M) extracts of herbs were assessed alone and in combination for their antifungal-demelanising activity and nitric oxide (NO) immunomodulatory responses. A new bioactive synergistic and antagonistic assessments approach was made on these herbs to identify which extract combination qualifies as a natural drug candidate. Methodology and results: Via micro-dilution technique, methanol extract of A. indica (AI-M) showed the strongest antifungal activity against Aspergillus niger, with a minimum inhibitory concentration (MIC) of 50 mg/mL and a minimum fungicidal concentration (MFC) of 100 mg/mL. Sublethal (50 mg/mL) and subinhibitory (25 mg/mL) doses of AI-M produced the optimal black pigmentation reduction to demelanise A. niger. The combinations AI-M+CA-M, AI-M+SG-M, and CA-M+SG-M showed similar antifungal activities (MIC = 100 mg/mL). At 500 µg/mL, CA-A and the combination CA-A+SG-A successfully induced RAW264.7 cells to produce NO at 17.85 µM and 40.84 µM, respectively. The combination of herbs extract showed synergistic interaction towards stimulation of NO production. In contrast, they demonstrated antagonism towards antifungal-demelanising properties. Compound identification of AI-M, SG-M, and SG-A were performed using a UHPLC-QTrap-MS/MS system, which detected phenolic compounds from various groups (cinnamic acids, benzoic acids, and flavonoids). Conclusion, significance and impact of study: The combination of herb extracts showed better stimulation of NO production while the single herb extracts demonstrated good antifungal-demelanising activity. These findings help in the selection of herbs combination for potential natural drug discovery. A good combination of herbs demonstrated synergism to execute better bioactivities compared to individual herb extracts.

Aims: Acalypha indica (AI), Centella asiatica (CA), and Sesbania grandiflora (SG) are vegetables commonly used in traditional medicine in Asian countries to treat skin problems. In this study, we investigated their pharmacological activities relevant to wound healing and synergistic actions to provide an insight into a promising vegetable combination as a candidate treatment for wounds. Methodology and results: The stimulatory, antioxidant, and antibacterial activities of aqueous (A) and methanol (M) extracts of all the three vegetables were assessed alone and in combination in normal human dermal fibroblast (NHDF) cells in vitro. CA-A (89.52%) and the combination of AI-A+CA-A (90.76%) produced the highest percentage of wound closure. AI-A exhibited the highest total phenolic content (TPC) (82.94 mg GAE/g) and moderate reducing activity (61.63 mM Fe (II)/mg) when assessed by ferric reducing antioxidant power (FRAP) assay. Free radical scavenging activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), and the combination of AI-A+CA-A exhibited scavenging activity at IC50 = 379.75 µg/mL and IC50 = 578.7 µg/mL, respectively. Pre-treatment of NHDF cells with CA-M at 100 µg/mL offered the highest protection against hydrogen peroxide. All single and combined vegetable extracts showed poor antibacterial properties against Gram negative and Gram positive bacterial species implicated in wound infection. Only AI-A+CA-A executed synergism in fibroblast migration when assessed via the combination index (CI). Furthermore, screening and identification of AI-A, CA-A, and CA-M via UHPLC (LC-MS/MS) system revealed that the major components responsible for all the tested bioactivities were phenolic groups such as simple polyphenols, flavonoids, polysaccharides, and triterpenes (asiaticoside and madecassosides). Conclusion, significance and impact of study: The vegetable extracts of A. indica, C. asiatica, and S. grandiflora exhibited good bioactivities independently. However, only AI-A+CA-A showed synergism in-combination to accelerate the migration of fibroblast and antioxidant activities. These findings demonstrate the potential formulation of combined vegetable extracts from the two species of A. indica and C. asiatica for optimum wound healing properties.