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Septama and Panichayupakaranant: Simultaneous HPLC Analysis of Three Flavonoids in the Extracts of Artocarpus heterophyllus Heartwoods
Original Article

Natural Product Sciences 2016;22(2):77-81.

Published online: 17 December 2016

DOI: https://doi.org/10.20307/nps.2016.22.2.77

Simultaneous HPLC Analysis of Three Flavonoids in the Extracts of Artocarpus heterophyllus Heartwoods

Abdi Wira Septama1, Pharkphoom Panichayupakaranant1, 2, ∗

1Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

2Excellent Research Laboratory, Phytomedicine and Pharmaceutical Biotechnology Excellence Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand

∗Author for correspondence Pharkphoom Panichayupakaranant, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand Tel: +66-74-428220; E-mail: pharkphoom.p@psu.ac.th

Received 1 November 2015       Revised 6 December 2015       Accepted 24 December 2015

Copyright © 2016 The Korean Society of Pharmacognosy

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of three antibacterial flavonoids, artocarpanone, artocarpin, and cycloartocarpin in ethyl acetate extracts from Artocarpus heterophyllus heartwoods. Separation was achieved using a TSK-gel ODS-80Tm column (5 µm, 4.6 × 150 mm) at 25oC with a gradient elution system of methanol and water as follows: 0-8 min,60:40; 8-27 min, 80:20; 27-35 min, 60:40, v/v, at a flow rate of 1 mL/min, and a quantitative UV detection at 285 nm. The method was validated by measuring the key parameters, including specificity, linearity, sensitivity, accuracy, repeatability and reproducibility. A high degree of specificity and sensitivity was achieved. The calibration curves for all three flavonoids showed good linearity with a coefficient of determinations (R2) of ≥ 0.9995. The recoveries of the method were from 98-104%, with good reproducibility and repeatability (RSD values of less than 2%) were also achieved. Ethyl acetate was the best solvent for extraction of these three flavonoids using the heat reflux conditions for 1 h. This optimized sample preparation and HPLC method can be practically used for a routine standardization process of the extracts from the A. heterophyllus heartwoods.

Keywords: Artocarpus heterophyllus, Artocarpanone, Artocarpin, Cycloartocarpin, HPLC

REFERENCES

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Fig. 1.
Chemical structures of artocarpanone (1), artocarpin (2), and cycloartocarpin (3).
nps-22-77f1.tif
Fig. 2.
HPLC chromatograms of (A) authentic compounds an (B) heartwood extract of A. heterophyllus. (1: Artocarpanone, 2 Artocarpin, 3: Cycloartocarpin).
nps-22-77f2.tif
Fig. 3.
HPLC chromatograms of hexane, chloroform, ethyl acetate, and methanol extracts from of A. heterophyllus heartwoods.
nps-22-77f3.tif
Table 1.
HPLC calibration data for artocarpanone, artocarpin, and cycloartocarpin
Compounds Linear range (µg/mL) tR (min) Equationa Linearity (R2) LOD (µg/mL) LOQ (µg/mL)
Artocarpanone 6.25–100 6.7 Y = 52290 X − 35882 0.9997 0.06 0.2
Artocarpin 6.25–100 17.8 Y = 47287 X − 44018 0.9998 0.04 0.2
Cycloartocarpin 6.25–100 23.1 Y = 25630 X − 37434 0.9995 0.2 0.4

a Y = a X + b, where Y is a peak area and X is the concentration of the analyzed material

Table 2.
Recovery data for the three flavonoids spiked into A. heterophyllus heartwood extracts
Compounds Spiked level (µg/mL) % Recovery (Mean ± SD)
Artocarpanone 100 101.9 ± 0.83
25 98.2 ± 1.10
6.25 103.4 ± 0.30
Artocarpin 100 103.5 ± 0.75
25 101.7 ± 0.20
6.25 101.6 ± 0.74
Cycloartocarpin 100 102.1 ± 0.63
25 103.8 ± 0.73
6.25 102.4 ± 0.55
Table 3.
Intraday and interday precision data for the quantitative determination of the three flavonoids
Compounds RSD (%)
intra-day (n = 6) interday (n = 3)
Artocarpanone 0.76 1.23
Artocarpin 0.78 1.49
Cycloartocarpin 0.64 1.27

RSD = Relative standard deviation

Table 4.
Content of the three flavonoids in different solvents for extracting A. heterophyllus heartwood
Solvents Yield of dried extracts (% w/w; mean ± SD) Content (%w/w of dried extract; mean ± SD)
Artocarpanone Artocarpin Cycloartocarpin
Hexane 2.2 ± 0.07∗ n.a. 52.84 ± 0.16∗ 2.62 ± 0.02∗
Chloroform 5.4 ± 0.15∗ 1.03 ± 0.03∗ 21.72 ± 0.57 5.51 ± 0.12
Ethyl acetate 7.2 ± 0.20 4.95 ± 0.01 21.84 ± 0.60 5.75 ± 0.20
Methanol 25.6 ± 1.47∗ 1.09 ± 0.05∗ 53.01 ± 0.10∗ 1.09 ± 0.03∗

∗ Significant difference (p < 0.05) when compared with ethyl acetate in the same column. n.a. = not analyzed due to it being lower than the limit of quantification.

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