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

Abdi Wira Septama; Pharkphoom Panichayupakaranant

doi:10.20307/nps.2016.22.2.77

Journal List > Nat Prod Sci > v.22(2) > 1060645

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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 Panichayupakaranant^1,^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

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 25^oC 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.

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

REFERENCES

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Fig. 1.

Chemical structures of artocarpanone (1), artocarpin (2), and cycloartocarpin (3).

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Fig. 2.

HPLC chromatograms of (A) authentic compounds an (B) heartwood extract of A. heterophyllus. (1: Artocarpanone, 2 Artocarpin, 3: Cycloartocarpin).

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Fig. 3.

HPLC chromatograms of hexane, chloroform, ethyl acetate, and methanol extracts from of A. heterophyllus heartwoods.

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Table 1.

HPLC calibration data for artocarpanone, artocarpin, and cycloartocarpin

Compounds	Linear range (µg/mL)	t_R (min)	Equation^a	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 (%)
Compounds	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)
Solvents	Yield of dried extracts (% w/w; 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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