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. 2017 Dec;55(1):1317-1323.
doi: 10.1080/13880209.2017.1300175.

Quantitative analysis of rutin, quercetin, naringenin, and gallic acid by validated RP- and NP-HPTLC methods for quality control of anti-HBV active extract of Guiera senegalensis

Perwez Alam  1 , Mohammad K Parvez  1 , Ahmed H Arbab  1   2 , Mohammed S Al-Dosari  1
Affiliations

Quantitative analysis of rutin, quercetin, naringenin, and gallic acid by validated RP- and NP-HPTLC methods for quality control of anti-HBV active extract of Guiera senegalensis

Perwez Alam et al. Pharm Biol. 2017 Dec.

Abstract

Context: Guiera senegalensis J.F. Gmel (Combretaceae) is a folk medicinal plant used in various metabolic and infectious diseases. In addition to its antiviral activities against herpes and fowlpox, the anti-HBV efficacy is very recently reported.

Objective: To develop and validate simple, sensitive RP-/NP-HPTLC methods for quantitative determination of biomarkers rutin, quercetin, naringenin, and gallic acid in the anti-HBV active G. senegalensis leaves ethanol-extract.

Materials and methods: RP-HPTLC (rutin & quercetin; phase- acetonitrile:water, 4:6) and NP-HPTLC (naringenin & gallic acid; phase- toluene:ethyl acetate:formic acid, 6:4:0.8) were performed on glass-backed silica gel plates 60F254-RP18 and 60F254, respectively. The methods were validated according to the ICH guidelines.

Results: Well-separated and compact spots (Rf) of rutin (0.52 ± 0.006), quercetin (0.23 ± 0.005), naringenin (0.56 ± 0.009) and gallic acid (0.28 ± 0.006) were detected. The regression equations (Y) were 12.434x + 443.49, 10.08x + 216.85, 11.253x + 973.52 and 11.082x + 446.41 whereas the coefficient correlations (r2) were 0.997 ± 0.0004, 0.9982 ± 0.0001, 0.9974 ± 0.0004 and 0.9981 ± 0.0001, respectively. The linearity ranges (ng/spot) were 200-1400 (RP-HPTLC) and 100-1200 (NP-HPTLC). The LOD/LOQ (ng/band) were 33.03/100.1 (rutin), 9.67/29.31 (quercetin), 35.574/107.8 (naringenin), and 12.32/37.35 (gallic acid). Gallic acid (7.01 μg/mg) was the most abundant biomarker compared to rutin (2.42 μg/mg), quercetin (1.53 μg/mg) and naringenin (0.14 μg/mg) in the extract.

Conclusion: The validated NP-/RP-HPTLC methods were simple, accurate, and sensitive for separating and quantifying antiviral biomarkers in G. senegalensis, and endorsed its anti-HBV activity. The developed methods could be further employed in the standardization and quality-control of herbal formulations.

Keywords: Natural products; antiviral; combretaceae; flavonoids; plant extract; polyphenols.

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Figures

Figure 1.
Figure 1.
Pictogram of developed RP-HPTLC plate at λ = 254 nm; mobile phase, acetonitrile:water (4:6, v/v).
Figure 2.
Figure 2.
Pictogram of developed NP-HPTLC plate at λ = 254 nm; mobile phase, toluene:ethyl acetate:formic acid (6:4:0.8, v/v/v).
Figure 3.
Figure 3.
Chromatogram of biomarkers quercetin (Rf = 0.23; 800 ng/spot) and rutin (Rf = 0.52; 800 ng/spot) at λ = 360 nm; mobile phase, acetonitrile:water (4:6, v/v).
Figure 4.
Figure 4.
Chromatogram of biomarkers gallic acid (Rf = 0.28; 600 ng/spot) and naringenin (Rf = 0.55; 600 ng/spot) at 275 nm; mobile phase, toluene:ethyl acetate:formic acid (6:4:0.8, v/v/v).
Figure 5.
Figure 5.
Chromatogram of GSEE (quercetin, spot 4, Rf = 0.23; rutin, spot 7, Rf = 0.52) at 360 nm; mobile phase, acetonitrile:water (4:6, v/v).
Figure 6.
Figure 6.
Chromatogram of GSEE (gallic acid, spot 5, Rf = 0.28; naringenin, spot 9, Rf = 0.56) at λ = 275 nm; mobile phase, toluene:ethyl acetate:formic acid (6:4:0.8, v/v/v).
See this image and copyright information in PMC

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