An HPLC-DAD Method to Quantification of Main Phenolic Compounds from Leaves of Cecropia Species

Um método eficiente e reprodutível por CLAE-DAD foi desenvolvido e validado para quantificação simultânea dos compostos fenólicos majoritários (ácido clorogênico, isoorientina, orientina e isovitexina) presentes nas folhas de duas espécies de Cecropia, C. glaziovii e C. pachystachya. Das folhas de C. glaziovii e C. pachystachya foram isolados os flavonoides Cglicosídeos isoorientina e isovitexina e identificados em ambas as espécies o ácido clorogênico (ácido 3-O-cafeoilquínico) e o flavonoide O-glicosídeo isoquercitrina. O flavonoide C-glicosídeo orientina foi isolado apenas da espécie C. pachystachya. O ácido clorogênico mostrou-se como composto majoritário em ambas as espécies analisadas (11,1 mg g-1 de extrato de C. glaziovii e 27,2 mg g-1 de extrato de C. pachystachya), e em relação aos flavonoides quantificados, isovitexina se apresentou como o flavonoide C-glicosídeo majoritário para C. glaziovii (4,6 mg g-1 extrato) e isoorientina majoritário para C. pachystachya (17,3 mg g-1 de extrato).


Introduction
The genus Cecropia (Urticaceae) comprises around 60 trees species distributed throughout Latin America, some of them occurring in Brazil.Cecropia glaziovii Sneth.and Cecropia pachystachya Trécul.are the two most common species in the Southeast and South of Brazil. 1 These species are both popularly known as "embaúba", and although posses distinct morphologies, such as their height and color of the leaves, both are widely used in Brazilian folk medicine to treat cough, asthma, high blood pressure, inflammation, and as a diuretic. 2,3The main pharmacological activities described in the literature for these species are hypotensive activity [4][5][6] and effects on the central nervous system, including anxiolytic and antidepressant-like activities. 7,8In terms of chemical composition, flavonoids, procyanidins and catechins have already been reported in these plants. 9,10lthough there are some phytopharmaceutical and homeopathic preparations that include species from this genus in some countries, such as Brazil and France, 11 as far as we are aware, there is no comparative evaluation of the chemical fingerprint of C. glaziovii and C. pachystachya leaves in the literature, especially from aqueous extract, which is used in folk medicine.Neither is there any individual quantitative analysis of their major phenolic compounds.Therefore, the development of chromatographic identification and assay methods based on the chemical constituents, particularly the phenolic compounds, may contribute to the standardization of the crude drug and extracts.Vol.22, No. 6, 2011   The aims of this work were to identify and isolate the main C-glycosylflavonoids in aqueous extracts from the leaves of C. glaziovii and C. pachystachya, and to quantify the main phenolic compounds from the leaves of C. glaziovii and C. pachystachya by high performance liquid chromatography (HPLC).

Extraction and isolation
The leaves of both Cecropia species were air dried at 35-40 °C for three days and the aqueous extracts were prepared separately, by infusion.Briefly, powdered leaf material (100 g) was extracted with boiled distilled water (1000 mL, 90 °C) for 30 min, and filtered.An aliquot of 100 mL of this extract was lyophilized and the residue dissolved in methanol:water (1:1 v/v) to obtain sample solutions for HPLC analysis.The remaining extract was evaporated under reduced pressure to a volume of 500 mL.
The crude extracts were stirred with 50 g of Amberlite ® XAD-16 for 1 h.Afterwards, the mixture was filtered and the resin stirred again (1 h) with 500 mL of methanol.This MeOH solution was then filtered and the solvent removed under reduced pressure.This procedure was repeated to yield 5 g of MeOH fraction from each Cecropia species.

Quantitative high-performance liquid chromatography analysis
The quantitative analysis of phenolic compounds was carried out in a PerkinElmer Series 200 high-performance liquid chromatography (HPLC) system, equipped with diode array detection (DAD), quaternary pump, online degasser and autosampler.The data were processed using the TotalChrom ® Workstation software.The injection volume was 10 mL.The baseline resolution was obtained at room temperature (24 ± 2 °C) using a PerkinElmer Brownlee Choice C 18 column (150 × 4.6 mm i.d.; 5 mm) and a gradient combining solvent A (acetonitrile) and solvent B (acetic acid 1%, adjusted to pH 3.0) as follows: 0-30 min, linear change from A-B (5:95 v/v) to A-B (20:80 v/v); 30-40 min, isocratic A-B (20:80 v/v).The mobile phase was prepared daily and degassed by sonication before use.The flow rate was kept constant at 1.0 mL min -1 and the chromatograms were recorded at 340 nm while the UV spectra were monitored over a range of 450 to 200 nm.The peaks were characterized by comparing the retention time and UV spectra with the reference standards, and by the coinjection of the sample and authentic samples.The standard solutions were prepared in different concentrations, as follows: chlorogenic acid (3-O-caffeoylquinic acid), 2.5, 5.0, 10.0, 15.0, 25.0, 30.0 mg mL -1 ; isoorientin, 0.8, 1.0, 2.0, 5.0, 7.0, 15.0, 20.0 mg mL -1 ; orientin, 0.7, 1.0, 3.0, 7.0, 10.0, 20.0 mg mL -1 and isovitexin, 0.5, 1.0, 2.0, 5.0, 7.0, 10.0, 15.0 mg mL -1 .The concentration of the extracts analyzed were 1,000 mg mL -1 and fractions were 500 mg mL -1 , excepted for accuracy assay, which employed crude extracts at 1,500 mg mL -1 and 700 mg mL -1 for C. glaziovii and C. pachystachya, respectively.Quantification of the individual compounds was performed using a six-point regression curve (r 2 > 0.995).All standard solutions were analyzed in triplicate and the peak average areas measured.

Validation of analytical procedures
The validation of analytical procedures was performed according to Cass and Degani and the ICH guidelines. 14,15he validated parameters were specificity, linearity, accuracy, precision (repeatability and intermediate precision), limit of quantification (LOQ) and limit of detection optical (LOD).

Results and Discussion
Isolation and identification C. glaziovii has about 16 to 20 meters tall in their adult stage and leaves with green color on both sides.On the other hand, C. pachystachya reaches a maximum of 12 meters and its leaves have dense arachnoid indumentum on the lower surface, yielding a white-gray color.Although these species posses distinct morphologies, both are popular known in Brazil as "embaúba", fact that could lead to mistakes at collections.It is well accepted that medicinal plants can be distinguished from each other by comparing their chemical fingerprints.Analysis of both crude aqueous extract and its phenolic compounds enriched fraction by TLC and HPLC-DAD showed different profiles between C. glaziovii and C. pachystachya, based on their flavonoid fingerprint (Figure 1 and 2).Although previous phytochemical analyses report the presence of flavonoids in these species, 9,10,16 there are no comparative studies concerning the chemical profiles between Cecropia species, which could increase the knowledge of their differentiation.
Both extracts were fractionated, and three flavonoids were isolated from C. pachystachya, codified as 2 (36.4 mg), 3 (40.8mg), 4 (3.3 mg). 2 was identified as isoorientin, 3 as orientin and 4 as isovitexin, by co-chromatographic analysis with standard C-glycosylflavonoids and UV spectra/shift reagents.Additionally, these compounds were submitted to 1 H NMR spectrometry analysis in order to confirm their identity.
From C. glaziovii, two flavonoids were isolated, codified as 4 (9.0 mg) and 2 (9.0 mg).TLC and HPLC-DAD co-chromatography with compounds previously isolated from C. pachystachya, together with UV spectra/ shift reagents, led to the identification of 4 as isovitexin and 2 as isoorientin.
These flavonoids were already reported for these species. 9,10,17Besides these compounds, two other previously reported phenolic compounds 9,10 were identified for these species: chlorogenic acid (3-O-caffeoylquinic acid) and isoquercitrin.Another significant substance was observed in C. glaziovii extract (HPLC Rt > 35 min), which was present just in traces in the C. pachystachya extract.Data about the UV profile of this peak suggests a flavones skeleton, although this compound was not isolated, identified or quantified.

HPLC analysis
Our preliminary investigations testing HPLC systems previously reported in the literature for Cecropia species 9,10,17 resulted in poor resolution of the compounds analyzed, or a long run time.The chromatographic system that showed the most promising results was achieved using a reverse-phase column (C 18 ), with acetonitrile and acidified water (with acetic acid, 1% v/v, pH 3.0) as the mobile phase.The use of acetonitrile instead of methanol improved the resolution, resulting in sharp and symmetrical peaks, with a good baseline level and minimal tailing, thus facilitating the accurate measurement of the peak area ratio.

Quantification and validation procedures
Standard solutions of chlorogenic acid, isoorientin, orientin and isovitexin were prepared, at a concentration range of 0.5-30 mg mL -1 , and the quantification showed a good linear relationship between peak area and concentration (r 2 > 0.995) for all standard solutions (Table 1).The limit of quantification (LOQ) and limit of detection (LOD) were defined by relative standard deviation (RSD > 5%) and by a signal:noise ratio of 3:1, respectively.The contents of the four phenolic compounds in these two species are shown in Table 2.The chromatographic analysis showed a distinct HPLC profile for these two Cecropia species, in which isovitexin is the major C-glycosylflavonoid for C. glaziovii, while isoorientin and orientin are the main C-glycosylflavonoids for C. pachystachya.
There are no reports in the literature concerning the quantification of C-glycosylflavonoids in Cecropia species, in spite of several studies concerning the quantification of these compounds in other species. 18,19,20In the Cecropia genus, quantitative analysis of chlorogenic acid has been reported only for C. obtusifolia.The contents of  this compound in the leaves of this species are in the range of 3.0-13.2mg g -1 of dry weight. 21,22Other works report a content of 0.2 mg g -1 aqueous extract. 23,24In our analysis, higher chlorogenic acid contents were found for C. glaziovii (11.1 ± 0.42 mg g -1 extract) and C. pachystachya (27.2 ± 0.94 mg g -1 extract) than those reported for C. obtusifolia, according to Revilla-Monsalve and coworkers. 23t is important to emphasize that the methodology developed herein allowed the analysis of two Cecropia species and the quantification of four substances from two classes of phenolic compounds (phenolic acids and C-glycosylflavonoids) in a single run.On the other hand, the analysis showed a relative long run time (40 min) considering only C-glycosylflavonoids, but bearing in mind that different classes of secondary metabolites could be analyzed with good separation and baseline resolution between all peaks, the methodology represents an improvement on the simultaneous quantitative assay of these phenolic compounds, compared with other reports in the literature. 25,26,27Besides the fact that C-glycosylflavonoids isomer pairs, especially orientin/ isoorientin, are usually difficult to separate with good resolution, 28,29 the 40 min analysis is acceptable, since our method is suitable for this separation.
The precision was determined by repeatability (intraday assay) and intermediate precision (inter-day assay) (Table 3). 30The intra-day assay was performed by triplicate analysis of three different concentrations of standard solutions, and expressed as relative standard deviation.Good repeatability was obtained from lower, medium and higher concentrations of the curve, with an RSD < 3.5% for all standard analyses.The inter-day assay was determined by the analysis of a medium concentration in the curve, three times a day, on three different days.Like the other parameters of precision, the RSD value did not exceeded the limits recommended in the literature. 14,15In relation to accuracy, good recovery was observed for all the standards in both extracts, which was determined by spiking samples with the standard solutions of chlorogenic acid, isoorientin, orientin or isovitexin (1:1 v/v).The concentrations of samples and standard solutions, as well as the average recovery values, are shown in Table 4.

Conclusions
Five phenolic compounds were identified in C. glaziovii and C. pachystachya and three of them (orientin, isoorientin a Six data points (n = 3).b LOD = limit of detection; LOQ = limit of quantification.and isovitexin) were isolated.Furthermore, a precise, accurate and reproducible HPLC-DAD method has been developed.The C-glycosylflavonoids contents reported here suggest that it is possible to use isovitexin as a phytochemical marker for C. glaziovii, while isoorientin could be used as a phytochemical marker for C. pachystachya.

Figure 3 .
Figure 3. Compounds identified in the leaves of C. glaziovii and C. pachystachya.
Aerial parts of C. glaziovii Sneth.were collected in Florianópolis, in the State of Santa Catarina, Brazil, in September 2007, and a voucher specimen (FLOR 37143) was deposited in the Herbarium of the Universidade Federal de Santa Catarina, Florianópolis, Brazil.Aerial parts of C. pachystachya Trécul.were collected in Viamão, in the State of Rio Grande do Sul, Brazil, in March 2007 and a voucher specimen (ICN 150025) was deposited in the Herbarium of the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

Table 1 .
Calibration data of phenolic standards

Table 2 .
Phenolic acid and C-glycosylflavonoids content in Cecropia extracts a a Expressed as mg g -1 of extract ± SD (n = 3).

Table 4 .
Accuracy data of phenolic compounds Recovery was determined by injection of spiked samples, in triplicate, with standard solution. a