Development and Validation of Oxyresveratrol Quantification Method in Artocarpus lacucha Roxb. Heartwood by Ultra–High Performance Liquid Chromatography
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Abstract
Introduction and Objective: Oxyresveratrol is an active principle in the heartwood of Artocarpus lacucha Roxb. ex Buch.–Ham. (Ma Hat in Thai) with antioxidative, anti–inflammatory and pain–relieving activities. This heartwood has been used as an herbal ingredient in various traditional medicinal products and herbal health supplements in the markets. However, available analytical methods for the quality control of oxyresveratrol in A lacucha heartwoood are still practically complicated and inconvenient regarding the extraction process and analytical time. Therefore, this study aimed to develop new analytical method for the quantification assessment of oxyresveratrol in A. lacucha heartwood samples using ultra–high performance liquid chromatography (UHPLC) technique.
Methods: The study was divided into three steps, namely, 1) developing UHPLC method for the analysis of oxyresveratrol in A. lacucha heartwood, 2) testing the validity of the newly–developed analytical method, and 3) quantitative analysis of oxyresveratrol in 8 samples of
A. lacucha Roxb. heartwood using new method.
Results: UHPLC method development for the analysis of oxyresveratrol in A. lacucha heartwood: It was found that oxyresveratrol was best extracted by reflux with methanol for
30 minutes. Chromatographic separation was achieved using ARC–18 column (4.6 x 150 mm,
2.7 µm) as the stationary phase, 0.1% ortho–phosphoric acid in water and 0.1% ortho–phosphoric acid in acetonitrile as the isocratic mobile phase, and detection with diode array detector (DAD) at 326 nm. Under these analytical conditions, the average retention time for oxyresveratrol was 4.76 minutes. Method validation: The calibration curve of oxyresveratrol was found to be linear at the concentration range of 14.33 – 229.32 µg/mL with correlation coefficient (r) of 0.9999. The percentage recoveries and % relative standard deviation (%RSD) were in the range of 98.03 – 98.70% and 0.52 – 0.87%, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) of oxyresveratrol was 0.274 and 0.912% w/w of the dried heartwood, respectively. Quantitative analysis of oxyresveratrol in 8 heartwood samples showed that the content of oxyresveratrol was in the range of 1.54 – 26.23% w/w (on a dry weight basis).
Discussion: The developed UHPLC method for determining oxyresveratrol content in A. lacucha heartwood met all acceptance criteria for analytical method validation. The newly developed method is rapid, with a simple, convenient, and efficient mobile phase preparation process, making it suitable for routine analysis of oxyresveratrol in A. lacucha heartwood. The analysis of eight heartwood samples revealed an average oxyresveratrol content of 8.80 ± 8.27% w/w (on a dry weight basis). This wide variation highlights significant quality differences among raw materials currently available in the domestic herbal market. Consequently, further research is needed to determine the optimal harvesting period for A. lacucha heartwood to ensure standardized quality for pharmaceutical and herbal product manufacturing.
Conclusion and Recommendation: The developed method is suitable for the quantitative analysis of oxyresveratrol in A. lacucha heartwood. Furthermore, additional research should be conducted to establish acceptance level of oxyresveratrol content in the monograph of “Kaen Ma Hat” or Artocarpus lacucha heartwood or in the Thai Herbal Pharmacopoeia (THP) in the future.
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