Development and Validation of Orientin and Vitexin Quantification Method in Passiflora foetida L. Leaves by Ultra–High Performance Liquid Chromatography

Abstract

Introduction and Objective: “Ka Thok Rok” has the scientific name Passiflora foetida L. belonging to Family Passifloraceae, L., commonly known as Fetid Passionflower, is a medicinal plant whose leaves contain high amounts of phenolic and flavonoid compounds, particularly orientin and vitexin. Ethanol extracts from the leaves exhibit analgesic, antioxidant, and anti–inflammatory activities, leading to their widespread use in pharmaceuticals and herbal health products. However, standardized quality control methods for these active compounds are currently lacking. Therefore, this study aims to develop and validate an Ultra–High Performance Liquid Chromatography (UHPLC) method for the quantitative analysis of orientin and vitexin in P. foetida leaves.

Methods: The study was divided into three steps: developing the UHPLC method for analyzing orientin and vitexin in Ka Thok Rok leaves, testing the validity of the analysis method, and analyzing the quantity of orientin and vitexin in 14 Ka Thok Rok leaf samples.

Results: The method developed for analyzing orientin and vitexin in raw Ka Thok Rok leaves using UHPLC involves the extraction of dried Ka Thok Rok leaves with 50% ethanol as the solvent through the reflux method for 60 minutes, the ARC–18 column, 4.6 x 150 mm, particle size 2.7 μm as the stationary phase, using 0.1% ortho–phosphoric acid in water and 0.1% ortho–phosphoric acid in acetonitrile as the mobile phase, and detecting with diode array detector (DAD) at 340 nm. The analytical system showed average retention times of 3.98 and 4.31 minutes for orientin and vitexin, respectively. This method was validated by establishing the linearity for orientin and vitexin at concentration range of 1.27 – 122.30 and 1.35 – 129.36 μg/mL, respectively, with equal determination correlation coefficient (r) of 0.99998. The % recovery were in the range of 99.03 to 101.74% and 97.70 to 101.05%, respectively. The limit of detection of orientin and vitexin were 0.009 and 0.012% w/w on a dry basis, while limit of quantification were 0.031 and 0.042% w/w, respectively. The average content of orientin and vitexin in the 14 leaf samples were 1.00 and 0.14% w/w, respectively.

Discussion: The method developed for analyzing orientin and vitexin in raw Ka Thok Rok leaves using UHPLC involves the extraction of dried leaves with 50% ethanol as the solvent through the reflux method. Quantitative analysis of orientin and vitexin in 14 Passiflora foetida leaf samples revealed average orientin and vitexin contents of 1.00 and 0.14% w/w, respectively. The results of the validity test of the analytical method were all within acceptable limits. This developed orientin and vitexin quantification method requires less time for analysis and involves a simple, convenient, and rapid preparation of the mobile phase.

Conclusion and Recommendation: The developed analytical method is suitable and reliable for the quantification of orientin and vitexin in Passiflora foetida L. leaves, and serves as a foundation for future research to quantify these compounds in herbal products containing P. foetida leaves. The content of orientin and vitexin found in this study can also provide inputs for the future establishment of standardized content of these active markers in Ka Thok Rok leaves.