Isovitexin as marker and bioactive compound in the antinociceptive activity of the Brazilian crude drug extracts of Echinodorus scaber and E. grandiflorus

Abstract

Echinodorus scaber Rataj and Echinodorus grandiflorus (Cham. & Schltdl.) Micheli, Alismataceae, are popularly used to relieve inflammatory complaints and as diuretic. A study on the antinociceptive effect and selected marker compounds in eleven extracts from different locations was undertaken and their antinociceptive effect was assessed. The fingerprints were compared by HPLC-DAD and the content of vitexin, isovitexin, isoorientin and vitexin-2-O-rhamnoside were determined. All samples presented antinociceptive activity reducing the writhes by 36.4-62.5% and 47.4-79.8% at 10 and 50 mg/kg, respectively; indomethacin (5 mg/kg) reduced writhes by 82.6-90.1%. The content of the flavonoids C-glycosides, however, presented a strong variation. Isovitexin and isoorientin were found in all the samples, with content ranging from traces to 14.70 µg/mg and 2.12-84.27 µg/mg extract, respectively, while vitexin-2-O-rhamnoside occurred in quantifiable amounts only in 3 out of 11 samples ranging from 5.43 to 33.13 µg/mg extract; vitexin was not detected at all or detected in trace amounts. According to the fingerprints, the samples could be arranged in four main groups. All eleven extracts showed antinociceptive activity. Isovitexin was the only flavonoid present in all samples and can be regarded, acting in synergy with the other compounds or not, as the responsible for the antinociceptive activity. Therefore, isovitexin is a good choice as chemical marker when the antinociceptive activity of E. scaber and E. grandiflorus is investigated.

Keywords
Chapéu-de-couro; Flavonoids; Isovitexin marker; Fingerprint; Antinociceptive activity

Introduction

The genus Echinodorus, Alismataceae, comprises about 45 species, occurring mainly in tropical areas and about 25 of them have been described in Brazil (Lehtonen, 2008; Matias et al., 2015). The best-known Echinodorus species used as medicinal plant in Brazil is Echinodorus scaber Rataj, heterotypic synonym of Echinodorus macrophyllus subsp. scaber (Rataj) R. R. Haynes and Holm-Niels, but also Echinodorus grandiflorus (Cham. & Schltdl.) Micheli is mistakenly collected and used as a substitute of the official crude drug. Both plants are known as “chapéu-de-couro”, “chá-mineiro”, “erva-de-pântano”, “erva-de-brejo”, among others. A leaf infusion or decoction is used as anti-inflammatory, antinociceptive, diuretic, hypotensive and antirheumatic (Brandão et al., 2009).

Previous chemical studies in E. scaber led to unusual nitrogen-containing clerodane diterpenes (Kobayashi et al., 2000a,b,c). From the methanolic leaf extract several cembrane diterpenes (Manns and Hartmannn, 1993; Tanaka et al., 1997; Shigemori et al., 2002), phenolics (Tanus-Rangel et al., 2010), flavonoids and caffeic acid derivatives (Schnitzler et al., 2007) were reported. A comparative study of five Echinodorus species was undertaken by Tanaka (2000) using hyphenated methods. Diterpenes, steroids and fatty acids were used as chemical markers.

The anti-inflammatory activity of E. scaber (E. macrophyllus) leaf extract in acute and sub-chronic models of inflammation was assessed by Tanus-Rangel et al. (2010) using hydroethanolic extract in mice and rats. Potent acute, systemic and topic anti-inflammatory activity was found. The same authors reported the isolation of isovitexin from the ethyl acetate fraction of the extract and the detection of the close related vitexin in the sample.

The immunosuppressive effect of E. scaber aqueous extract was described by Pinto et al. (2007).

A toxicological study on aqueous extract of E. scaber was undertaken by Lopes et al. (2000). The authors found minor hepatic toxicity but no genotoxic effect at the daily dose recommended in traditional medicine. However, Vidal et al. (2010) using a bacterial assay, found mutagenic effect in the aqueous extract and solvent partition fractions of the plant.

Therefore, considering the importance of “chapéu-de-couro” to the local folk medicine and the several scientific studies reported above that seems to confirm its traditional use, it is important to develop a simple and cheap method for the quality control of a possible “chapéu-de-couro” phytotherapic medicine. So, the aim of this work is to perform HPLC-DAD fingerprint analyses in “chapéu-de-couro” samples collected in different sites from Mato Grosso and Mato Grosso do Sul states, and to investigate the possible use of vitexin, vitexin-2-O-rhaminoside, isovitexin and isoorientin as chemical markers in the crude drugs presenting antinociceptive activity.

Materials and methods

Chemicals

The solvents used were of ACS grade from Vetec (Rio de Janeiro, Brazil), and for HPLC analysis were of spectroscopic grade from Tedia (Fairfield, OH, USA). Ultrapure water was obtained by the Milli-Q system (Waters, Darmstadt, Germany). Acetic acid from Merck (Darmstadt, Germany) and NaCl Proquimios (Rio de Janeiro, RJ, Brazil). Indomethacin, sodium bicarbonate and standard compounds for the chromatographic analyses from Sigma-Aldrich (St. Louis, MO, USA). The purity of the standards used in the HPLC analysis was as follows: vitexin (>95%), isovitexin (98%), vitexin-2-O-rhamnoside (>98%), catechin (>98%). Isoorientin (≥97%) was isolated from leaves of E. scaber, and its identity was confirmed by ¹H and ¹³C NMR analyses on a Varian Mercury 300 (Palo Alto, CA, USA), in agreement with the literature (Peng et al., 2005).

Plant material

The plants were collected in May 2011 in eleven different locations from the Mato Grosso and Mato Grosso do Sul states, Brazil and were identified by MSc. Vali Joana Pott. Voucher herbarium specimens have been deposited at the Central Herbarium of UFMS (Table 1). The aerial parts of the air-dried plant material were used for the analyses, according to the traditional use. The powdered dry material (20-25 mesh; 5 g) was extracted with EtOH-H₂O (100 ml; 70:30, v/v) at r.t. during 4 h under stirring. After filtration, the solvent was evaporated to dryness under reduced pressure, and the remaining syrup was lyophilized. The extractions were carried out in triplicate.

Table 1
Botanical identification, voucher specimen number, collection place, geographic coordinates and extraction yields of Echinodorus scaber and E. grandiflorus samples investigated.

Plant species and sample	Collection place and state	Geographical coordinates (GPS)	Voucher herbarium number	w/w extraction yield %
E. grandiflorus - L1	Juína, MT	15º56'70''S 56º36'07''W	31645	18.50
E. scaber - L2	Poconé, MT	16º31'40''S 56º43'53''W	33635	12.70
E. grandiflorus - L3	Chapada dos Guimarães, MT	15º36'30''S 56º03'44''W	33637	13.80
E. grandiflorus - L4	Dom Aquino, MT	15º48'40''S 54º55'02''W	33638	20.60
E. scaber - L5	Cuiabá, MT	15º42'80''S 55º53'20''W	33639	18.70
Echinodorus sp. - L6	Comercial sample, Chapada dos Guimarães, MT	-	-	41.50
E. scaber - L7	Campo Grande, MS	20º30'79''S 54º34'87''W	33665	47.38
E. scaber - L8	Campo Grande, MS	20º30'79''S 54º34'87''W	33665	41.31
E. scaber - L9	Campo Grande, MS	20º28'28''S 54º34'06''W	33665	50.21
E. scaber - L10	Campo Grande, MS	20º29'84''S 54º35'52''W	33665	48.95
E. scaber - L11	Campo Grande, MS	20º30'02''S 54º36'52''W	33665	43.32

• States of Mato Grosso (MT) and Mato Grosso do Sul (MS).

Animals

The antinociceptive assays were carried out using Swiss albino mice (20-35 g) from the Central Animal House of UFMT. Animals were kept in polypropylene cages with free access to water and commercial Purina^® (Labina, São Paulo, Brazil) at 25 ± 1 °C and 12 h (light)/12 h (darkness) cycles. Experiments were carried out according to the ethical principles on Animal Experimentation, adopted by the Brazilian College of Animal Experimentation (COBEA). The experiments were allowed by the Animal Use Ethics Committee (CEUA) of the UFMT under authorization number 23.108043016/10-6.

Total phenolic content

The total phenolic content (TP) was determined by the Folin-Ciocalteu method. All samples and catechin were dissolved in 50% (v/v) aqueous methanol. Samples (50 µl) were placed into test tubes and 250 µl Folin-Ciocalteu reagent were added. The mixture was left to stand for 5 min, and 750 µl of 20% sodium carbonate solution and 5 ml distilled water were added. After 30 min of incubation at room temperature (20 °C) the resulting absorbance was measured at 765 nm. The calibration curve was performed with catechin (concentrations ranging from 4 mg/ml to 15 mg/ml) and the results were expressed as mg of catechin equivalents per 100 g of dry plant material.

Total flavonoids content

Determination of the total flavonoids content (TF) of the hydroalcoholic extract was performed as reported previously, using the AlCl₃ colorimetric method (Simirgiotis et al., 2008). Quantification was expressed by reporting the absorbance in the calibration graph of vitexin used as standard flavonoid (from 1 to 29 µg/µl; R ² = 0.992). Results are expressed as mg vitexin equivalents/g dry mass.

HPLC analyses of marker compounds

HPLC analyses were carried out using Varian Pro Star 5.5 (Palo Alto, CA, USA) equipment with a quaternary pump (model 240), autosampler (model 410) and UV-DAD detector (model 330). The conditions were as follows: column HiChrom C-18 [250 mm × 4.6 mm × 5 µm] (Reading, UK), pre-column Kromasil C-18 [3.0 mm × 4.6 mm] (Bohus, Sweden), mobile phase CH₃CN (solvent A), MeOH (solvent B) and 0.05% (v/v) aqueous trifluoroacetic acid (TFA) solution (solvent C); flow rate, 1 ml/min; injection volume, 4 µl. The gradient program used was as follows: 0-20 min (75% C:15% B:10% A); 20-25 min (65% C:20% B:15% A); 20-25 min (55% C:25% B:20% A); 25-30 min (75% C:15% B:10% A). The analyses were monitored by the ProStar 5.5 software and the spectra were detected using the PoliView 2000 program. The chromatograms were obtained at 270 nm, and spectra from the extract constituents were measured in the range 218-400 nm. Under our experimental conditions, the retention time of the reference compounds were: isoorientin (8.6 min), vitexin-2-O-ramnoside (11.2 min), vitexin (11.6 min) and isovitexin (12.4 min). Calibration curves for the reference compounds were built using the internal standard method (catechin as internal standard), in eight concentrations for vitexin, isovitexin and vitexin-2-O-rhamnoside (0.25-20 µg/ml), and in ten concentrations for isoorientin (7-100 µg/ml), injecting each concentration in triplicate. The internal standard and the analyzed compounds were dissolved (1 mg) separately in CH₃CN:0.05% TFA (10 ml, 1:1, v/v), the injection volume was 4 µl. The linearity of the detector for vitexin, vitexin-2-O-rhamnoside, isoorientin and isovitexin was obtained from linear plots of curves, built-up using the Microsoft Office Excel 2007 program. The concentrations of vitexin, vitexin-2-O-rhamnoside, isovitexin, and isoorientin in leaves of E. scaber and E. grandiflorus were calculated from the peak areas using the equation for linear regression obtained from the calibration curve. The detection limit and quantification limit for the compounds were as follow in µg/µl, respectively: vitexin (0.29 and 0.9), vitexin-2-O-rhamnoside (0.68 and 2.05), isovitexin (0.25 and 0.75) and isoorientin (1.55 and 4.7). The method was validated (specificity, linearity, detection and quantification limits, accuracy and precision), according to the literature (Anvisa, 2003).

Principal Component Analysis

Unsupervised Principal Component Analysis (PCA) was performed with selects peaks obtained by matching from HPLC-DAD date, aligned by Chromaligner platform (Wang et al., 2010). The aligned data were analyzed in MetaboAnalyst platform (Xia et al., 2012). Samples were clustered into two groups based on their geographical location: Mato Grosso (MT) and Mato Grosso do Sul (MS) states. PCA was performed with the log-transformed and auto-scaled data, at UFMS.

Antinociceptive activity

The antinociceptive activity was determined in mice as described by Koster et al. (1959). The experiments were carried out employing 6-8 animals per treatment group. The extracts were administered orally at doses of 10 or 50 mg/kg body weight, 60 min before intraperitoneal injection of 0.6% acetic acid in 0.9% normal saline (0.1 ml/10 g body weight) to induce the characteristic writhings. Vehicle (10 ml/kg body weight, p.o.) and indomethacin (5 mg/kg body weight, p.o.) were dissolved in 2% NaHCO₃ solution and were given to mice in the control and reference groups, respectively. The mice were observed and counted for the number of abdominal writhes and stretchings in a period of 0-30 min. The responses in the treated groups were compared with those of animals in the control group and reference compound. The percentage of inhibition of the number of writhings was calculated and results are presented as percent inhibition.

Statistical analyses

The results are expressed as means ± S.E.M. One way analysis of variance (ANOVA) was used for the comparison of more than two means followed by Student-Newman-Keuls test; p values <0.05 were considered significant. Graph Pad InStat^®version 2.01 was used for data analysis.

Results

Taxonomic authentication

Eleven samples (L₁-L₁₁) from “chapéu-de-couro” were collected or purchased in different locations of Mato Grosso (MT) and Mato Grosso do Sul (MS) states in 2011, during the flowering time. From the eleven samples, seven were confirmed as E. scaber, while three turned out to be the close related species E. grandiflorus and a commercial sample remains as Echinodorus sp. The w/w extraction yields for the E. scaber samples ranged between 12.70 and 50.21% while for E. grandiflorus the soluble content was 13.80 and 20.60%, respectively and 41.50% for the Echinodorus sp. sample. The best extraction yields (41.31-50.21%) were observed from the samples collected in MS sites (Table 1).

The total phenolic, flavonoid and C-glycoside flavones quantification

The total phenols and flavonoid content, as well as the C-glycosyl flavonoid content of hydroethanolic extracts from “chapéu-de-couro” leaves are given in Table 2. Table 3 provides calibration curve data, obtained using standard C-glycoside flavones. The total phenolic concentration did not vary appreciably among the eleven analyzed samples, ranging from 283.25 mg/g (Echinodorus sp., from MT) to 330.00 mg/g (E. scaber from MS). However, great variation was observed regarding the total flavonoid concentration, ranging from 3 mg/g (E. scaber, from MS) to 233.75 mg/g (E. scaber from MT).

Table 2
Total phenolic (TP), total flavonoid (TF) (mg of catechin and vitexin equivalents per g of dry plant material, respectively), isovitexin, vitexin-2-O-rhamnoside and isoorientin (µg per mg of hydroethanolic extracts of Echinodorus scaber and E. grandiflorus).

Plant species and sample	TP (mg/g)	TF (mg/g)	Isovitexin (µg/mg)	Vitexin-2-O-rhamnoside (µg/mg)	Isoorientin (µg/mg)
E. grandiflorus - L1	290.00	25.50	nq	nd	2.12
E. scaber - L2	292.50	198.00	0.72	13.46	21.82
E. grandiflorus - L3	296.75	39.25	1.06	nd	22.24
E. grandiflorus - L4	296.00	145.5	0.62	nq	21.54
E. scaber - L5	324.00	233.75	nq	5.43	14.34
Echinodorus sp. - L6	283.25	95.00	2.44	33.13	84.27
E. scaber - L7	308.75	141.75	6.75	nq	12.22
E. scaber - L8	298.50	61.25	14.70	nq	34.27
E. scaber - L9	287.50	100.50	9.22	nq	18.10
E. scaber - L10	330.00	123.00	6.99	nq	35.54
E. scaber - L11	308.75	3.00	5.71	nq	16.89

• Collection places L₁-L₅ and L₇-L₁₁ are from the Mato Grosso (MT) and Mato Grosso do Sul (MS) states, respectively; L₆ refers to a commercial sample (please see Table 1); nq, detected, but not quantified (below quantification limit); nd, not detected.

Table 3
Linear regression equation, correlation coefficient, detection and quantification limits for vitexin-2-O-rhamnoside, vitexin, isovitexin and isoorientin.

Standard	Conc. range (µg/ml)	Linear regression (y = a + bx)	Correlation coefficient (r 2)	Detection limit (µg/ml)	Quantification limit (µg/ml)
Vitexin-2-O-rhamnoside	0.50-8.00	y = 0.0280 + 0.1051x	0.9967	0.68	2.05
	8.00-20.00	y = 0.1651 + 0.0877x	1.0000
Vitexin	0.25-8.00	y = 0.0136 + 0.2242x	0.9994	0.29	0.90
	8.00-20.00	y = 0.574 + 0.1562x	1.0000
Isovitexin	0.25-8.00	y = 0.0072 + 0.2512x	0.9996	0.25	0.75
	8.00-20.00	y = 0.3473 + 0.2073x	0.9997
Isoorientin	7.00-40.00	y = 8.3363 + 6.1229x	0.9982	1.55	4.70
	40.00-100.00	y = 7.109x - 36,274	0.9997

The content of the C-glycoside flavones also presented great variation (Table 2). Vitexin was not detected in any but one of the eleven analyzed samples, whereas vitexin-2-O-rhamnoside was quantified in only three sites (all from MT), ranging from 5.43 to 33.13 µg/mg, and isovitexin and isoorientin were detected in all samples with concentration ranging from traces 14.70 µg/mg for the former and 2.12-84.27 µg/mg for the latter. An interesting picture, however, arises from the flavone content found in the Echinodurus sp. analyzed; while E. grandiflorus samples presented only trace amounts or none of vitexin-2-O-rhamnoside and very small amounts of isovitexin (traces - 1.06 µg/mg) but appreciable content of isoorientin (2.12-22.24 µg/mg), a different pattern can be observed to E. scaber, also presenting trace amounts of vitexin-2-O-rhaminoside (except sites 2 and 5; 13.46 and 5.43 µg/mg respectively) along with higher concentrations of isovitexin (traces - 14.70 µg/mg) and high concentrations of isoorientin (12.22-35.54 µg/mg). The commercial Echinodorus sp. sample presented very high concentration of isoorientin and vitexin-2-O-rhaminoside (84.27 and 33.13 µg/mg respectively) and a moderate amount of isovitexin (2.44 µg/mg).

Antinociceptive activity

Oral administration of the extracts elicited antinociception in the animals at both selected doses of 10 and 50 mg/kg. In vehicle treated groups, injection of acetic acid caused an intense nociceptive response in mice, ranging from 38.9 ± 2.5 to 40.4 ± 4.0 writhings (Table 4).

Table 4
Effect of oral administration of Echinodorus scaber and E. grandiflorus hydroethanolic extracts on acetic acid-induced writhes in mice.

Extracts	Dose (mg/kg)	No. of writhes	% inhibition
Vehicle	-	38.9 ± 2.5	-
E. grandiflorus - L1	10	21.0 ± 2.2b	46.0
	50	20.4 ± 2.6b	47.4
E. grandiflorus - L3	10	15.9 ± 1.7b	59.2
	50	13.0 ± 3.6b	66.5
E. scaber - L10	10	14.6 ± 2.6b	62.5
	50	9.4 ± 3.3b	75.7
Indomethacin	5	5.3 ± 0.9b	86.3
Vehicle	-	40.1 ± 3.0b	-
E. scaber - L2	10	19.0 ± 2.7b	52.7
	50	14.1 ± 2.1b	64.8
Echinodorus sp. - L6	10	21.3 ± 4.2b	46.9
	50	18.1 ± 3.6b	54.8
Indomethacin	5	4.8 ± 1.1b	88.0
Vehicle	-	40.4 ± 4.0	-
E. scaber - L7	10	23.4 ± 3.9a	42.1
	50	8.2 ± 1.7b	79.8
E. scaber - L8	10	18.7 ± 4.7b	53.8
	50	8.6 ± 2.5b	78.6
E. scaber - L9	10	19.8 ± 2.3b	51.0
	50	12.6 ± 1.6b	68.9
Indomethacin	5	4.0 ± 1.2b	90.1
Vehicle	-	40.3 ± 1.7b	-
E. grandiflorus - L4	10	19.0 ± 2.8b	52.9
	50	15.7 ± 3.0b	61.1
E. scaber - L5	10	25.7 ± 4.1b	36.4
	50	15.0 ± 2.0b	62.8
E. scaber - L11	10	22.8 ± 2.5b	43.4
	50	10.3 ± 1.8b	74.5
Indomethacin	5	7.0 ± 1.1b	82.6

• Results are presented as mean values ± S.E.M. for n = 6-8 animals/group. One way ANOVA followed by the Student-Newman-Keuls test.
• ^a
  p < 0.01 vs vehicle.
• ^b
  p < 0.001 vs vehicle.

Pretreatment of the animals with hydroethanolic extracts of “chapéu-de-couro” (10 mg/kg), resulted in inhibition of writhings, attaining the lowest value (36.4%, p < 0.001) with E. scaber - L₅ and highest value (62.5%, p < 0.001) with E. scaber - L₁₀, when compared to the vehicle group. When a 50 mg/kg extract dose was employed, reduction in number of writhings was greater than at the lower dose, reaching the lowest value (47.4%, p < 0.001) with E. scaber - L₁, and the highest value (79.8%; p < 0.001) with E. scaber - L₇, when compared to the vehicle group.

Animals that received 5 mg/kg indomethacin, the standard drug for this test, had the highest percentage inhibition of nociceptive response, with values ranging from 82.6% to 90.1% (p < 0.001).

Discussion

This work performed fingerprint analyses, as well as flavonoid quantification on samples of the popular phytotherapic drug named as “chapéu-de-couro”. The samples were collected in different places from Mato Grosso and Mato Grosso do Sul States in Brazil, to search for phytochemical differences, and for chemical markers in the crude drugs presenting antinociceptive activity.

The doses of E. scaber and E. grandiflorus extracts in antinociceptive tests models were chosen based on murine pilot experiments of our lab with the acetic acid test, and from previous literature reports on other inflammatory or antinociceptive models. The classical method of Whittle (1964), using acetic acid, was chosen because it is a simple, rapid and cheap test, being a non-selective model that serves for both peripheric and central analgesia, without altering consciousness. Indomethacin was used as a positive control since it is a classical standard non-steroidal drug that assures its specificity as an inhibitor of cyclooxygenase (Whittle, 1964; Cabral-Silva, 2013).

Despite the distinct profile of the two studied species and Echinodorus sp. (commercial sample) regarding flavonoid content, the amount of the C-glycosylated flavonoids can be successfully used in the quality control if a “chapéu-de-couro” drug is to be commercialized. The PCA (Fig. 1) shows that the geographical location of the species is the main differentiating factor in the polar secondary metabolism. In the last years several authors showed that edaphoclimatic factors may substantially change metabolomics composition (Gobbo-Neto and Lopes, 2007).

Fig. 1
Distribution of Echinodorus scaber, E. grandiflorus and E. sp. on the plan of the first two principal components PC1 and PC2 (64.8% variance explained).

Leaf extracts from “chapéu-de-couro”, corresponding to the crude drug were orally administered at the doses of 10 and 50 mg/kg using for comparative purposes the acetic acid writhing assay. The best effect (Table 4) was associated with the sample origin, with a clear dose-response effect for the MS plants. Taken together, higher extraction yields with good antinociceptive effect suggest that the crude drug from MS present a better profile regarding the traditional indications of use. The related species, E. grandiflorus, with the same common name and use, showed similar antinociceptive effect than that observed for the MT collections of E. scaber.

The analgesic and anti-inflammatory activity of extracts obtained from the rhizomes of E. grandiflorus was reported by Dutra et al. (2006) and the hypotensive effect of the leaf ethanol extract on spontaneous hypertensive rats was also described (Lessa et al., 2008).

Garcia et al. (2010) published a combined phytochemical and anti-inflammatory study of E. grandiflorus leaves extract and found activity at the highest dose of 1 g/kg, too high to explain the traditional indications of use.

The main differences of the present work with previous studies is the ethnopharmacological approach, using the crude drug following the traditional indications of use and the doses used which are lower than in the study of Dutra et al. (2006). In addition, our work was undertaken using a larger number of plant samples, including material from different locations and purchased at local markets. In this way, a closer picture of the crude drug “chapéu-de-couro” is obtained.

All extracts were compared by HPLC looking for characteristic compounds in the polar extracts. According to the fingerprints, our samples can be arranged in four main groups, two of them representative of E. scaber from MT and MS and a third set in agreement with E. grandiflorus, being the fourth sample the commercial Echinodorus sp. The chromatograms representing the different profiles are presented in Fig. 2. Table 5 presents the retention time, the UV data and the tentative identification of the main phenolic compounds found in the analyzed “chapéu-de-couro” leaf extracts.

Fig. 2
Representative HPLC chromatograms of the Echinodorus scaber and E. grandiflorus samples from the Brazilian Pantanal. (A) E. grandifloras: L₄ (representative from collection places L₁, L₃, and L₄ from MT state); (B) E. scaber: L₅ (representative from places L₂ and L₅ - MT state); (C) Echinodorus sp. - L₆ (commercial sample); (D) E. scaber: L₈ (representative from collection places L₇ to L₁₁ from MS state). Peak identities: (1) catechin (internal standard), retention time: 5 min, λ _max 324 and 279 nm; (2) isoorientin*, retention time: 8.6 min, λ _max 348 and 269 nm; (3) swertiajaponin**, retention time: 9.2 min, λ _max 344 and 269 nm; (4) vitexin-2-O-rhamnoside, retention time: 11.2 min, λ _max 334 and 269 nm; (5) vitexin*, retention time: 11.6 min, λ _max 335 and 269 nm; (6) isovitexin*, retention time: 12.4 min, λ _max 335 and 269 nm; (7) caffeoyl/feruloyl acid derivatives, retention time: 18.2 min, λ _max 327, 290sh and 245 nm; (8) caffeoyl/feruloyl acid derivative, retention time: 25.1 min, λ _max 328, 290sh and 243 nm. *Identity confirmed by co-injection of standards. **Identity confirmed by NMR data, UV spectrum and comparison with literature (Schnitzler et al., 2007; Peng et al., 2005).

Table 5
Identification of phenolic compounds in Echinodorus scaber and E. grandiflorus hydroethanolic” extracts by HPLC-DAD.

Peak	Rt (min)	λmax (nm)	Tentative identificaction
1	5.0	324, 279	Catechin (internal standard)
2	8.6	348, 269	Isoorientina
3	9.2	344, 269	Swertiajaponinb
4	11.2	334, 269	Vitexin-2-O-rhamnosidea
5	11.6	335, 269	Vitexina
6	12.4	335, 269	Isovitexina
7	18.2	327, 290sh, 245	Caffeoyl/feruloyl acid derivativeb
8	25.1	328, 290sh, 243	Caffeoyl/feruloyl acid derivativeb

• Rt: Retention time; HPLC-DAD: High Performance Liquid Chromatography with Diode Array Detector.
• ^a
  Identity confirmed by coinjection of standards.
• ^b
  Identity confirmed by UV spectrum and comparison to the literature (Schnitzler et al., 2007).

The HPLC chromatograms representative of all of our E. scaber samples from MS shows two main compounds identified as isoorientin and isovitexin (Fig. 2, trace D; peaks 2 and 6, respectively). However, a subset from the samples can be recognized by comparing the occurrence of two compounds eluting at retention time (Rt) of 18.2 and 25.1 min (peaks 7 and 8), respectively, both presenting UV spectra with maxima at 327, 290sh and 245 nm, in agreement with caffeoyl/feruloyl acid derivatives (Schnitzler et al., 2007). Fig. 2, trace B, representing samples L₂ and L₅ from MT shows isoorientin (peak 2) as the main constituent and vitexin-2-O-rhamnoside (peak 4) as secondary compound, differently from the MS plants, which show very low content of this compound. The unidentified crude drug sample of Echinodorus sp. purchased in MT (L₆ in Fig. 2, trace C) is close to E. scaber from MT according to the phenolic compounds pattern and showed the highest content in vitexin-2-O-rhamnoside from all the samples investigated.

The HPLC profile of the E. grandiflorus extracts corresponding to “chapéu-de-couro” (L₁, L₃ and L₄ - Fig. 2, trace A) were similar to those reported by Garcia et al. (2010) for a sample from the same species collected in the Paraná state in southern Brazil. Peaks 2 and 3 from our sample show the same UV spectrum suggesting that they are flavonoids from the same structural group, namely isoorientin and apparently swertiajaponin.

In a study of the phenolic constituents from E. grandiflorus ssp. aureus, Schnitzler et al. (2007) carried out a fractionation of the plant extract and unambiguously identified their main compounds by spectroscopic and spectrometric means. The sample reported, however, correspond to a variety of E. grandiflorus that can be easily recognized by the main peak of swertiajaponin (peak 3). The plant was collected in the Santa Catarina state, southern Brazil.

The main flavonoids occurring in the polar hydroethanolic extracts of “chapéu-de-couro” are C-glycosyl flavonoids differing in the number of free hydroxy and/or methoxy groups in the flavonoid ring, namely isoorientin, swertiajaponin, vitexin-2-O-rhamnoside and isovitexin.

Vitexin is a C-glycosyl flavone used as a chemical marker for Passiflora species (Dhawan et al., 2004). The C-glycosyl flavonoids found in the plant have been associated with antiinflammatory (Sridhar et al., 2006) and antinociceptive effect (Kumar et al., 2012).

Isoorientin is present in high concentrations in all extracts of “chapéu-de-couro” (Table 2); this flavone is also found in species of Passiflora and is frequently associated with anti-inflammatory and antinociceptive activities. However, this compound cannot be considered the main responsible for the antinociceptive activity of “chapéu-de-couro”, since the antinociceptive tests responded randomly to isoorientin concentrations. Swertiajaponin was tentatively identified (Table 5) only in E. grandiflorus extracts and, therefore, cannot be the main responsible for the antinociceptive activity.

Vitexin-2-O-rhaminoside is often found in Passiflora species. In the present work this compound was quantified only in extracts of E. scaber from MT as well as the commercial sample (Table 2). In extracts where this flavone was not detected, however, there was considerable percentage of pain inhibition, indicating that vitexin-2-O-rhaminoside cannot be the main responsible by the analgesic activity presented by “chapéu-de-couro” samples analyzed in this work.

Isovitexin was detected in all extracts from E. scaber, in concentration ranging from traces to 14.60 µg/mg; higher amounts were detected in samples from MS (Table 2). A dose-dependent antinociceptive activity could be observed, when comparing isovitexin concentration and the percentage of inhibition of E. scaber extracts (Table 4). The same trend was observed when analyzing E. grandiflorus samples. As commercial Echinodorus and Passiflora species share the same C-glycosylflavonoid constituents, new studies on the anxiolytic effect of “chapéu-de-couro” are of interest. Recent reports on the effect of Passiflora extracts on the central nervous system include the isoorientin and isovitexin (Li et al., 2011).

Overall the HPLC method presented allowed a clear and rapid differentiation of E. scaber and its substitute E. grandiflorus and is suitable for comparative analyses of Echinodorus samples.

The collections displaying better antinociceptive effect shows the higher isovitexin content and also better w/w yields, making the MS samples the better source for the crude drug. Further studies should be carried out, including the harvesting sources of the crude drug for commercial purposes to provide recommendations on quality control based on the active constituents and extraction yields. Thus, isovitexin can be regarded, at least in part, as responsible for the antinociceptive activity and seems to be a suitable chemical marker for “chapéu-de-couro” crude drug.

Ethical disclosures

Protection of human and animal subjects

The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).

Confidentiality of data

The authors declare that no patient data appear in this article.

Right to privacy and informed consent

The authors declare that no patient data appear in this article.

Acknowledgements

We are grateful to INCT-Áreas Úmidas (INAU)/CNPq, Centro de Pesquisas do Pantanal (CPP-MCTI) and CAPES for scholarships and financial support. We are also grateful to M.Sc. Isanete Geraldini Costa Bieski for valuable help in plant collection.

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Publication Dates

• Publication in this collection
  Sep-Oct 2017

History

• Received
  6 Feb 2017
• Accepted
  9 May 2017