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An Enzyme‐Linked Immunosorbant Assay Using Monoclonal Antibody Against Bacoside A 3 for Determination of Jujubogenin Glycosides in Bacopa monnieri (L.) Wettst
Author(s) -
Tothiam Charinrat,
Phrompittayarat Watoo,
Putalun Waraporn,
Tanaka Hiroyuki,
Sakamoto Seiichi,
Khan Ikhlas A.,
Ingkaninan Kornkanok
Publication year - 2011
Publication title -
phytochemical analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 72
eISSN - 1099-1565
pISSN - 0958-0344
DOI - 10.1002/pca.1293
Subject(s) - bacopa monnieri , chemistry , glycoside , salidroside , chromatography , pharmacology , stereochemistry , medicine
ABSTRACT Introduction In Ayurvedic medicines, Bacopa monnieri (L.) Wettst. (brahmi) is known as a medicinal plant used for memory enhancement. Its active compounds are classified as pseudojujubogenin and jujubogenin glycosides. Owing to the lack of chromophore in the saponin glycoside structures, HPLC‐UV–vis gives low sensitivity for determination of such compounds. In the case of the detection of small amounts of saponin glycosides, immunological assay could be a suitable method. Objective To develop and validate a sensitive enzyme‐linked immunosorbant assay (ELISA) using monoclonal antibody (MAb) against bacoside A 3 , the major jujubogenin glycoside found in brahmi. Methodology An immunogen was prepared by conjugating bacoside A 3 with a bovine serum albumin (BSA). To determine its immunogenicity, the ratio of hapten in bacoside A 3 – BSA conjugate was determined by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS). After immunisation in mice, hybridomas secreting MAbs against bacoside A 3 were produced by fusing the immunised splenocytes with SP2/0‐ Ag14 myeloma cells. The antibody was raised specifically against jujubogenin glycosides. The ELISA using anti‐bacoside A 3 MAb was developed. Results Bacoside A 3 in the range of 3.05–97.70 ng mL −1 could be detected by ELISA using anti‐bacoside A 3 MAb. The assay showed a detection limit of 0.48 ng mL −1 (0.517 n m ). The validation study showed that the method was precise, accurate and sensitive. Interestingly, the MAb showed cross‐reactivity with the other jujubogenin glycosides, bacopaside X and IV. However, it did not show cross‐reactivity with any of pseudojujubogenin glycosides. Conclusion The study demonstrated that ELISA using anti‐bacoside A 3 MAb can be used for determination of total jujubogenin glycosides in brahmi. Copyright © 2011 John Wiley & Sons, Ltd.