A leaf-disc punching assay for rapid phytochemical screening of Centella asiatica: A complementary tool for on-farm and industrial quality monitoring

Centella asiatica (L.) Urban is a medicinal industrial crop whose phytochemical content varies widely with genotype and environment, complicating raw-material standardisation. Conventional weighing–grinding methods are accurate but process only 10–50 samples per day, and existing leaf-disc protocols target single endpoints in other species and have never been deployed on C. asiatica phytochemicals. To address this gap, we developed what is, to our knowledge, the first leaf-disc-based multi-class phytochemical-quantification workflow for C. asiatica , an area-normalised punching protocol that removes weighing and grinding. Uniform 5-mm discs (19.6 mm²) were extracted in 96-deep-well plates with methanol for 24 h, delivering 92–99% recovery relative to the 48-h kinetic plateau and to conventional grinding for total phenolic, flavonoid, and triterpenoid pools. Across eight genetically diverse Thai accessions, punching agreed closely with grinding (Pearson r = 0.955, 0.969, and 0.989, respectively; Bland–Altman bias ≈ 0 µg/mm²; 95% limits ±10–15%). A low-cost (< USD 500) camera + ImageJ readout correlated strongly with plate-reader spectrophotometry across 40 field samples ( r = 0.930, 0.988, 0.823); Passing–Bablok and Deming regression confirmed the OLS conversion equations and showed the phenolic slope to be statistically indistinguishable from unity. Under salinity and osmotic stress the workflow resolved up to seven treatment groups and detected 2.0–2.5-fold increases. A formal green-chemistry assessment (AGREE 0.68 vs 0.44; GAPI 28/45 vs 34/45) confirmed the workflow is substantially greener than grinding. Potential throughput is up to ∼1000 samples per technician per day under optimised conditions, supporting on-farm cultivation feedback, germplasm breeding, and pre-transaction processor-intake screening.