Production of polyclonal antibody against madecassoside and development of immunoassay methods for analysis of triterpene glycosides in Centella asiatica.
BACKGROUND
Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory.


OBJECTIVE
To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica.


METHODS
An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy.


RESULTS
The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39-50 µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5-500 ng. The limit of detection for MA and AS was 31.25 ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography.


CONCLUSIONS
These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica.
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Patcharin
P
Tassanawat
Waraporn
W
Putalun
Gorawit
G
Yusakul
Boonchoo
B
Sritularak
Thaweesak
T
Juengwatanatrakul
Hiroyuki
H
Tanaka
D000906:Antibodies / Q000276:immunology*; D028041:Centella / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006027:Glycosides / Q000032:analysis; D006241:Haptens / Q000737:chemistry; D007118:Immunoassay / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014315:Triterpenes / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D006027:Glycosides; D006241:Haptens; D014315:Triterpenes; C093443:madecassoside; C004446:asiaticoside