Pharmacognostic evaluation, and development and validation of a HPLC-DAD technique for gallocatechin and epigallocatechin in rhizomes from <i>Limonium brasiliense</i>

Blainski, Andressa; Antonelli-Ushirobira, Tânia M.; Godoy, Guilherme; Leite-Mello, Eneri V.S.; Mello, João C.P.

doi:10.1016/j.bjp.2016.08.009

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Original articles • Rev. bras. farmacogn. 27 (2) • Mar-Apr 2017 • https://doi.org/10.1016/j.bjp.2016.08.009 copy

Pharmacognostic evaluation, and development and validation of a HPLC-DAD technique for gallocatechin and epigallocatechin in rhizomes from Limonium brasiliense

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Limonium brasiliense (Boiss.) Kuntze, Plumbaginaceae, is a plant from the southern coast of Brazilian that is used for the treatment of premenstrual syndrome, menstrual disorders and genito-urinary infections. The aim of the present study was to determine the quality control parameters for rhizomes collected during different periods by pharmacopoeial and non-pharmacopoeial methods, and to develop and validate a HPLC-DAD method for quantitative control of marker substances. The measured parameters were: granulometric analysis (d₅₀ = 0.21–0.48 mm), loss on drying (11.1–12.4%), total ash (4.9–5.7%), dry residue by extraction with acetone:water (7:3, v/v) (30.6–39.5%), total polyphenol content (8.5–15.8%), and chromatographic fingerprint by HPLC and TLC. Besides, the acetone:water (7:3, v/v) extraction solvent in combination with a turbo-extractor, yielded the crude extract with a significant increase in tannins (F_4,20 = 37.0, p < 0.001). The antioxidant potential of the crude acetone:water (7:3, v/v) extract, as well as the ethyl acetate and water fractions obtained after the partition process was evaluated by DPPH and the results were, respectively: IC₅₀ 6.87, 5.91, and 6.92 µg/ml. The validation parameters for the HPLC-DAD method showed adequate specificity, precision and accuracy. The gallo- and epigallocatechin contents were, respectively, 0.8–2.7% and 1.2–2.2%. These data contribute to analysis of the pharmacognostic quality control of the commonly used part from this species.

Keywords:
Baicuru; Limonium brasiliense; Pharmacognostic evaluation; Quality control; HPLC-DAD

Regression analysis		Calibration curve for GC	Calibration curve for EGC
Slope (SE)		1.44 × 10⁸ (2.29 × 10¹⁴)	1.41 × 10⁸ (4.78 × 10¹⁴)
Intercept (SE)		1.2 × 10⁷ (7.74 × 10⁶)	1.0 × 10⁸ (1.1 × 10⁷)
Regression coefficient (R ²)		0.9980	0.9950
Calculated F-value (critical F-value)		0.30 (3.71)	0.30 (3.71)
Sum of pure error		3.51 × 10¹⁶	5.09 × 10¹⁶
Lack of fit error		3.48 × 10¹⁵	5.04 × 10¹⁵
Analysis of variance		F_1,13 = 6385.7, p < 0.001	F_1,13 = 2610.8, p < 0.001
95% CL Slope		1.40 × 10⁸; 1.48 × 10⁸	1.35 × 10⁸; 1.47 × 10⁸
95% CL Intercept		-4.6 × 10⁶; 2.9 × 10⁷	7.7 × 10⁷; 1.2 × 10⁸
	SS	MS	SS	MS
Regression	1.13 × 10¹⁷	1.13 × 10¹⁷	9.60 × 10¹⁶	9.60 × 10¹⁶
Residual	2.29 × 10¹⁴	1.76 × 10¹³	4.78 × 10¹⁴	3.68 × 10¹³
Total	1.13 × 10¹⁷		9.64 × 10¹⁶

Condition for degradation	GC content (%)/purity peak	EGC content (%)/purity peak
Normal condition	2.8/1.0000	2.3/1.0000
Acid stress	2.3/1.0000	1.1/1.0000
Heat stress	2.3/1.0000	1.8/1.0000
Oxidation stress	2.9/1.0000	2.1/1.0000
Basic stress	0.1/0.8771	0.2/0.9153
Light stress
Amber	2.7/1.0000	2.1/1.0000
Transparent	2.4/1.0000	1.9/1.0000

Gallocatechin				Epigallocatechin
Theoretical concentration (µg/ml)	Measured concentration (µg/ml)	% Recovery	(x̄±sd) [RSD(%)]	Theoretical concentration (µg/ml)	Measured concentration (µg/ml)	% Recovered	(x̄±sd) [RSD(%)]
LC
3.8266	3.9419	103.0	103.5% ± 0.91 (0.9%)	3.2580	3.1848	97.8	97.8 ± 1.29 (1.3%)
3.7543	3.9246	104.5		3.1998	3.1698	99.1
3.6338	3.7400	102.9		3.1029	2.9938	96.5
IC
4.6284	4.7329	102.2	102.4% ± 0.88 (0.9%)	4.0242	4.0083	99.6	100.2 ± 0.87 (0.9%)
4.7248	4.7996	101.6		4.1018	4.0888	99.7
4.7007	4.8576	103.3		4.0824	4.1293	101.2
HC
5.5929	5.5731	99.6	100.9% ± 1.73 (1.7%)	4.9192	4.8997	99.6	101.2 ± 1.85 (1.8%)
5.5929	5.7554	102.9		4.9192	5.0766	103.2
5.4731	5.4883	100.3		4.9599	4.9921	100.7
Total (x̄±sd) [RSD(%)]		102.3% ± 1.54 (1.5%)				99.7 ± 1.93 (1.9%)

	Sample A	Sample B	Sample C
Granulometry (d₅₀) - MR	0.42 mm	0.21 mm	0.48 mm
Total ash - MR^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	5.6% ± 0.15 [2.7%]	5.7% ± 0.07 [1.4%]	4.9% ± 0.05 [1.0%]
Loss on drying - MR^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	11.1% ± 0.25 [2.3%]	12.4% ± 0.28 [2.4%]	11.9% ± 0.04 [0.4%]
PT - MR^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	15.8% ± 0.12 [0.8%]	8.5% ± 0.36 [4.3%]	9.4% ± 0.11 [1.2%]
Dry residue - MR^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	39.5% ± 0.63 [1.6%]	36.1% ± 0.61 [1.7%]	30.6% ± 0.19 [0.6%]
TP - CE^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	28.9% ± 0.42 [1.5%]	25.0% ± 1.94 [0.8%]	29.1% ± 0.30 [1.1%]
GC - FAE^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	2.6% ± 0.06 [2.3%]	0.8% ± 0.03 [3.7%]	2.7% ± 0.09 [3.2%]
EGC - FAE^a a x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; MR, milled rhizome; CE, crude extract; FAE, ethyl-acetate fractions; TP, total polyphenols; GC, gallocatechin; EGC, epigallocatechin.	1.8% ± 0.03 [1.8%]	1.2% ± 0.02 [1.9%]	2.2% ± 0.13 [5.8%]

	Extract A	Extract B	Extract C	Extract D	Extract E
TP^b b x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; TP, total polyphenols; TT, total tannins.	31.0% ± 0.32 [1.0%]	29.3% ± 0.34 [1.2%]	30.2% ± 1.12 [3.7%]	31.3% ± 0.46 [1.5%]	33.1%% ± 0.64 [2.0%]^a a p < 0.001.
TT^b b x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; TP, total polyphenols; TT, total tannins.	27.6% ± 0.33 [1.2%]	25.3% ± 0.36 [1.4%]	25.9% ± 1.02 [4.0%]	27.7% ± 0.42 [1.5%]	29.5% ± 0.60 [2.0%]^a a p < 0.001.
%TT/TP^b b x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; TP, total polyphenols; TT, total tannins.	88.9% ± 0.27 [0.3%]	86.6% ± 0.34 [0.4%]	85.7% ± 0.50 [0.6%]	88.7% ± 0.21 [0.2%]	89.2% ± 0.49 [0.6%]^a a p < 0.001.
Dry residue^b b x̄±sd [RSD(%)]: x̄, mean; sd, standard deviation; RSD, residual standard deviation; TP, total polyphenols; TT, total tannins.	36.4% ± 0.41 [1.1%]	32.2% ± 0.34 [1.1%]	29.9% ± 0.16 [0.5%]	36.3% ± 0.56 [1.5%]	39.5% ± 0.63 [1.6%]^a a p < 0.001.

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[1] * Corresponding author. E-mail:mello@uem.br (J.C. Mello).