Article pp.173-180 du Vol.26 n°2 (2006)

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NOTE

Chlorogenic acid profile of commercial Brazilian herbal infusions

A. I. da Silva

¹

, C. S. Santana

¹

, S. C. L. Pivato

¹

, C. A. B. De Maria

²

, R. F. A. Moreira

^1,2*

RÉSUMÉ

Composition en acides chlorogéniques des infusions brésiliennes du commerce Ont été établis dans cette étude le profil des acides caffeoylquiniques, feruloylqui- niques et dicaffeoylquiniques dans vingt infusions d’herbes brésiliennes du commerce par l’utilisation d’une méthode de chromatographie liquide à haute efficacité de phase reverse. Les herbes étudiées sont : « Alecrim », « Boldo », « Camomila »,

« Carqueja », « Cavalinha », « Chá preto », « Chá mate tostado », « Chapéu- de-couro », « Cidreira », « Erva-doce », « Espinheira-santa », « Ginkgobiloba »,

« Ginseng », « Guaco », « Hortelã », « Pata-de-vaca », « Porangaba », « Quebra- pedra », « Sálvia » et « Sene ». L’acide 3-caffeoylquinique a été retrouvé dans 75 % des infusions (18 - 5 200 mg.kg^-1), tandis que l’acide 5-caffeoylquinique a été détecté dans 80 % de celles-ci (34 - 9 990 mg.kg^-1). Les plus fortes concentrations de ces deux isomères ont été relevées dans le « Chá mate tostado ». La présence de l’acide 4-caffeoylquinique a été constatée dans 55 % des herbes (23 - 1 600 mg.kg^-1), et ses plus importantes concentrations sont retrouvées dans le

« Chapéu-de-couro » et la « Carqueja ». L’acide 4-feruloylquinique est présent dans 35 % des infusions (51 - 1 800 mg.kg^-1 d’herbe) alors que l’acide 5-feruloylquinique apparaît à concurrence de 45 % (39 - 1 300 mg.kg^-1). Malgré sa concentration peu importante (30 - 560 mg.kg^-1), l’acide 3,5-dicaffeoylquinique est présent dans 50 % des herbes. La distribution des autres isomères dicaffeoylquiniques dans ces infusions s’est révélée faible. L’acide 3,4-dicaffeoylquinique est retrouvé dans 20 % de ces infusions (190 - 1 700 mg.kg^-1), tandis que l’acide 4,5-dicaffeoylquinique n’était présent que dans 15 % de ces herbes. De fortes teneurs de ce dernier isomère n’ont été révélées que dans la « Sálvia » (1 100 ± 270 mg.kg^-1).

Mots clés

chromatographie liquide à haute efficacité, acides chlorogéniques, herbes brésiliennes du commerce.

1. Faculdade de Engenharia de Alimentos – Universidade Estácio de Sá (UNESA).

2. Departamento de Ciências Fisiológicas – Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro (UNIRIO) – Rua Fagundes Varela – 515, 1204, Ingá – Niterói, RJ – Brazil – CEP 24210-520. Fax number: (5521) 25319678.

* Correspondence : ricfelipe@terra.com.br

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SUMMARY

This work established the profile of caffeoylquinic, feruloylquinic and dicaffeoylquinic acids in twenty different Brazilian herbal infusions using a reversed- phase high performance liquid chromatography method. The herbs analysed were: “Alecrim”, “Boldo”, “Camomila”, “Carqueja”, “Cavalinha”, “Chá preto”,

“Chá mate tostado”, “Chapéu-de-couro”, “Cidreira”, “Erva-doce”, “Espinheira- Santa”, “Ginkgobiloba”, “Ginseng”, “Guaco”, “Hortelã”, “Pata-de-vaca”, “Porang- aba”, “Quebra-pedra”, “Sálvia” and “Sene”. The 3-caffeoylquinic acid was found in 75% of the infusions (18 to 5200 mg.^kg^-1), while the 5-caffeoylquinic acid was found in 80% of them (34 to 9990 mg.^kg^-1). The highest concentrations of these isomers were found in “Chá mate tostado”. The 4-caffeoylquinic acid appeared in 55% of the herbs (23 - 1600 mg.^kg^-1), with “Chapéu-de-couro” and “Carqueja”

being the richer ones. The 4-feruloylquinic acid was found in 35% of the infusions (51 - 1800 mg.^kg^-1 of herb) and the 5-feruloylquinic acid was detected in 45% of them (39 - 1300 mg.^kg^-1). In spite of the low concentrations (30 - 560 mg.kg^-1), the 3,5-dicaffeoylquinic acid appeared in 50% of the herbs. The distribution of the other dicaffeoylquinic isomers in the infusions was poor. The 3,4-dicaffeoylquinic acid was detected in 20% of them (190 - 1700 mg.^kg^-1), while only 15% of the herbs comprised the 4,5-dicaffeoylquinic isomer. Considerable amounts of this last isomer were found just in “Sálvia” (1100 ± 270 mg.^kg^-1^).

Keywords

high performance liquid chromatography, chlorogenic acids, commercial Brazilian herbs.

1 – INTRODUCTION

Since herbs infusions have low nutritional value, they have been consumed for pleasure and due to its medicinal properties. In Brazil, the great climate variety and the extremely rich flora assure a vast amount of herbs with singular therapeutic properties.

The majority of these herbs is found in the Atlantic jungle and are used, for instance, in the treatment of kidney affections, rheumatism, diabetes and arteriosclerosis (RODRIGUES et al., 2001; CORTEZ et al., 1999). The part of the population that most uses medicinal plants, has a low degree of education (CORTEZ et al., 1999) and the increased consumption of these herbs is encouraged by its price compared to other medicines and by the sense, not often reasonably explained, that everything that becomes from nature is healthy and always more efficient than synthetic products. So that, herbal infusions may be considered important sources of phenolic acid compounds in the diet of the Brazilian population. These polyphenolic acids show biologi- cal activity like antibacterial, anti-carcinogenic, anti-inflammatory, anti-viral, anti- allergic, estrogenic and immune-stimulating effects (LARSON, 1988). In this class of compounds, the chlorogenic acid (CGA) group deserves attention (figure 1). These acids are naturally occurring phenolic compounds mainly formed by quinic acid esterification with either caffeic (one or two), ferulic or p-coumaric acids (DE MARIAet al., 1999). They have no nutritional value, but in foodstuff they perform an important role in the formation of pigments, taste and flavour and are well known to act as antioxidants due to their o-quinone moity (DE MARIA et al., 2000). The literature also reports that some CGAs isomers could inhibit oxidative damage to both low-density lipoprotein (LARANJINHA et al., 1996) and linoleic acid (OHNISHI et al., 1994) by scavenging reactive species of oxygen (RSO). These RSO are related with some degenerative disorders such as cancer and cardiovascular diseases (AMES et al., 1993). Some CGA isomers

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may preclude the formation of carcinogenic N-nitroso compounds because they are inhibitors of the N-nitrosation reaction in vitro (KONO et al., 1997). In another study, one of the CGA isomers (3,5-dicaffeoylquinic acid) showed activity as a potent inhibitor of the HIV-1 integrase in vivo impairing the HIV-1 replication (ROBINSON et al., 1996).

Based on the information above introduced, it is easy to understand the impor- tance to study the CGA content of the commercial Brazilian herbal infusions. In spite of the large use of these infusions in Brazil, little is known about their chemical composition, particularly about their CGA content, which could explain some of their medicinal properties. The aim of the present work was to determine the content of caffeoylquinic (CQA), feruloylquinic (FQA) and dicaffeoylquinic (di-CQA) acids in 20 medicinal Brazilian herbs.

2 – MATERIALS AND METHODS

2.1 Materials

The study was carried out with twenty different commercial Brazilian herbs (table 1). Each kind of herb was obtained from three different distribution points in the state of Rio de Janeiro. The 5-caffeoylquinic acid standard (5-CQA) was obtained from Sigma Chemical Company (St. Louis, USA). The CQA mixture (3-CQA, 4-CQA and 5CQA) was obtained following a previously described method (TRUGO et al., 1984). A standard misture of diCQAs and FQAs isolated from deffated dried green coffee sam-

R CH OH

CH

O C

O

HO2 C HO

OH OH

6 5

4 2 3 1

R = OH -> 5-caffeoylquinic acid (5-CQA) R = OCH -> 5-feruloylquinic acid (5-FQA)₃

(b) (a)

HO

HO HO

OH

OH O C

Figure 1

General formulas of chlorogenic acids (Moreira et al., 2000). (a) Quinic acid;

(b) R = OH - 5-caffeoylquinic acid and R = OCH₃ - 5-feruloylquinic acid.

The esterification between quinic and cafeic or ferulic acids can also occur in the carbon atoms 3 and 4 of the quinic acid. Two caffeic acids can be esterified

with the quinic acid for the formation of the di-CQA isomers.

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ples in our laboratory was also used in this study. HPLC grade solvents were obtained from Carlo Erba (Milano, Italy). All other reagents were of laboratory grade. Carrez reagent corresponds to two different solutions. The first one (Carrez 1) is an aqueous solution of bi-hydrated zinc acetate (219 mg.mL^-1) containing 3% (v/v) of glacial acetic acid, while the other is an aqueous solution of potassium ferrocyanide (106 mg.mL^-1).

Table 1

Contents of individual CGAs in different herbs.

Herbs

3-CQA (Avg ± SD)

mg.kg^-1 4-CQA (Avg ± SD)

mg.kg^-1

5-CQA (Avg ± SD)

mg.kg^-1

4-FQA (Avg ± SD)

mg.kg^-1 5-FQA (Avg ± SD)

mg .kg^-1

3.4-diCQA (Avg ± SD)

mg.kg^-1

3.5-diCQA (Avg ± SD)

mg.kg^-1

4.5-diCQA (Avg ± SD)

mg.kg^-1

« Alecrim »

(Rosmarinus officinallis S.) 18 ± 9 27 ± 13 34 ± 15 ND ND ND ND ND

« Boldo »

(Pneumus boldus) 820 ± 30 36 ± 4 82 ± 9 ND ND 190 ± 110 30 ± 3 ND

« Camomila »

(Matricaria chamomila) 370 ± 50 640 ± 110 930 ± 60 1800 ± 400 ND 1700 ± 400 73 ± 23 ND

« Carqueja »

(Baccharis trimera) 400 ± 130 1500 ± 200 2100 ± 120 350 ± 50 180 ± 20 280 ± 80 460 ± 20 ND

« Cavalinha »

(Equisetum aruense) ND ND ND ND ND ND ND ND

“Chá preto”

(Camellia sinensis) ND ND 1600 ± 50 1200 ± 50 ND ND ND ND

“Chá-mate tostado”

(Illex paraguariensis) 5200 ± 170 ND 9990 ± 410 ND ND ND 560 ± 25 ND

“Chapéu-de-couro”

(Echinodorus

macrophyllus Mich.) 950 ± 330 1600 ± 390 2500 ± 230 ND 1300 ± 420 1200 ± 400 ND ND

“Cidreira”

(Cymbopogon citratus) 170 ± 60 23 ± 2 210 ± 70 51 ± 4 210 ± 20 ND 150 ± 10 25 ± 2

“Erva doce”

(Pimpinella anisum) 280 ± 120 150 ± 30 590 ± 310 ND 150 ± 1 ND ND ND

“Espinheira Santa”

(Maytenus ilicifolia) 410 ± 170 250 ± 130 340 ± 180 520 ± 10 39 ± 10 ND 280 ± 50 ND

“Ginkgobiloba”

(Ginkgo biloba) ND ND 96 ± 52 ND ND ND 39 ± 16 ND

“Ginseng”

(Pfaffia paniculata) ND ND ND ND ND ND ND ND

“Guaco”

(Mikania glomerata S.) 670 ± 220 ND 430 ± 210 ND ND ND ND ND

“Hortelã”

(Mentha piperita) 290 ± 130 180 ± 30 ND ND 67 ± 10 ND 75 ± 6 ND

“Pata de vaca”

(Bauhinia forficata) 290 ± 80 280 ± 170 180 ± 60 66 ± 34 76 ± 55 ND 35 ± 5 ND

“Porangaba”

(Cordia ecalyculata) 4300 ± 260 560 ± 200 400 ± 280 ND 170 ± 40 ND 60 ± 4 ND

“Quebra-pedra”

(Phyllanthus niruri) ND ND ND ND 52 ± 14 ND ND 29 ± 2

“Sálvia”

(Salvia officinalis) 200 ± 19 ND 360 ± 45 390 ± 110 ND ND ND 1100 ± 270

“Sene”

(Cassia augustifolia Vahl.)4300 ± 260 ND 1500 ± 290 ND ND ND ND ND Scientific names were obtained from the specialised literature (Joly, 1979; Atoui et al., 2005). Data are given as mean values of three independent experiments with three replicates of each kind of herb. The results are expressed as mg of individual CGA / kg of dry herb. ND – not detected. Avg – average concentration. SD – standard deviation.

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2.2 Extraction method

First of all, the dried herbs samples were ground in a mill to pass 0.75 mm.

About two grams of each dried sample undergo extraction with boiling water during 10 minutes, after the material has been mixed by the quarters sequencial division method. The extracts were treated with 1 ml of each Carrez reagent solution to pre- cipitate coloidal material. After that, the extracts were diluted to 100 ml with bidis- tilled water, filtered through a Whatman number 1 filter paper and, to conclude, were passed through a Millipore membrane (0.45 µm).

2.3 HPLC analysis

The reversed-phase HPLC-UV analysis was carried out based on a previous method described in the literature (PONTES et al., 2002) using a Varian system (Plus model, USA) with a 20 µL loop injector and an UV detector at 325 nm. The separa- tion of the CGA isomers was achieved using a 5 µm Lichrosorb RP-18 column, 4.0 mm i.d. x 250 mm (Lichrocart^®, Merck, Germany). A mobile phase composed by trisodium citrate (0.01 M, pH 2.5) / methanol (60:40, v/v) was used at a flow rate of 1 mL.min^-1. The peaks had tentatively been identified by comparing the retention times in the sample chromatograms with those of the available standards chromatograms. To aid the assignment of the peaks, some samples were spiked with the standard mixtures obtained from 5-CQA or from the green coffee extracts. Quantifi- cation was based on area measurement and comparison with the 5-CQA standard.

Individual isomers were calculated using their molar extinction coefficients according to the equation below (RUBACH, 1969)

C = (RF x ε1 x MR₂ x A) / (ε2 x MR₁)

where C is the concentration of the isomer in g.L^-1, RF is the response factor of the 5-CQA standard (concentration in g.L^-1 / area unit), ε1 is the molar extinction coefficient of 5-CQA, ε2 is the molar extinction coefficient of the isomer in question, MR₁ is the relative molecular mass of 5-CQA, MR₂ is the relative molecular mass of the isomer in question and A is the area of the peak corresponding to the isomer in question. Molar extinction coefficients (M^-1.cm^-1.10⁴) are as follows (RUBACH, 1969): at λmax 330 nm, 5-CQA = 1.95, 4-CQA = 1.80, 3-CQA = 1.84, 3,4-diCQA = 3.18, 3,5- diCQA = 3.16 and 4,5-diCQA = 3.32; at λmax 325 nm, 5-FQA = 1.93, 4-FQA = 1.95 and 3-FQA = 1.90.

2.4 Statistical analysis

Mean and standard deviation calculation and one-way analysis of variance (at the 0.05 significance level) were carried out for statistical evaluation of the diffe- rences between the individual CGA contents of the analysed herbs.

3 – RESULTS, DISCUSSION AND CONCLUSIONS

Different systems to extract CGAs from foodstuff have been described in the literature, using either organic solvents or hot water (KY et al., 1997). We adopted the boiling water extraction system since it is cheaper, safer and also because the

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heating inhibits the action of the polyphenoloxidase, reducing the possibility of CGA oxidation. In addition, boiling water is the solvent normally used for herbal infusions prepared by the consumers and we assumed that the chemical results could then be more representative of the CGA content actually consumed by people. Besides, to represent with greater fidelity the infusion preparation process carried out at home, the herbs didn´t have their lipid content removed before hot water extraction.

The analysis of individual CGAs (CQAs, FQAs and di-CQAs) in herbal infusions was based on the comparision of peak areas of the individual isomers with that of the 5-CQA standard. Since this compound is the only one still commercialy available, the molar absorptivities of the others isomers could not be experimentally deter- mined. Thus, literature values were recorded at the λmax values for each isomer (RUBACH, 1969). Since λmax values fell within the range of 325 - 330 nm, analyses were developed at 325 nm, which led to only a very small analytical error, up to 2%

(PONTES et al., 2002).

Only in “Ginseng” and “Cavalinha” no CGA isomers have been detected (table 1). Among the remainder eighteen herbs, only “Quebra-pedra” didn’t show at least one of the CQA isomers. Therefore, the CQA group was considered the major one. The 4-CQA isomer was found in 55% of the twenty herbal infusions, followed by the 3-CQA that was present in 75% of them and, finally, by the 5-CQA that appeared in 80% of the herbs. Low and medium (18 - 950 mg.kg^-1 of herb) amounts of 3-CQA were found in herbal infusions, except for “Porangaba”, “Sene” and “Chá mate tostado” which showed the following high amounts of this isomer:

(4300 ± 260) mg.^kg^-1, (4300 ± 260) mg.^kg^-1 and (5200 ± 170) mg.^kg^-1, respectively.

The 4-CQA was found in low concentrations in “Cidreira” [(23 ± 2) mg.kg^-1], “Ale- crim” [(27 ± 13) mg.kg^-1] and “Boldo” [(36 ± 4) mg.kg^-1]. On the other hand, relative high amounts were detected in “Carqueja” [(1500 ± 200) mg.^kg^-1] and “Chapéu-de- couro” [(1600 ± 390) mg.^kg^-1]. The 5-CQA arose in high concentrations in “Sene”

[(1500 ± 290) mg.kg^-1], “Chá preto” [(1600 ± 50) mg.kg^-1], “Carqueja” [(2100 ± 120) mg.kg^-1], “Chapéu-de-couro” [(2500 ± 230) mg.kg^-1] and “Chá mate tostado”

[(9990 ± 410) mg.^kg^-1]. The comparision of these results with data available in literature showed that these five herbs were richer sources of 5-CQA than some vegetables and fruits (DE MARIA et al., 1999, PONTES et al., 2002). It is relevant to remember that the 5-CQA concentration could increase analysing the “Chá mate” in its green form instead of its roasted form (called “Chá mate tostado”), since this isomer could decompose during the roasting process. It could happens with others CGA isomers too. Excluding “Cavalinha” and “Ginseng”, that dind’t have none of the CGA isomers as above mentioned, no FQA was found in “Alecrim”, “Boldo”, “Chá mate tostado”,

“Ginkgobiloba”, “Guaco” and “Sene”. Comparing the remainder herbs, “Camomila”

[(1800 ± 400) mg.^kg^-1] and “Chá preto” [(1200 ± 50) mg.^kg^-1] were the major sources of the 4-FQA (table 1). This isomer was found in 35% of the herbal infusions analysed, while the 5-FQA was detected in 45% of them. “Chapéu-de-couro” showed a significative larger concentration of 5-FQA [(1300 ± 420) mg.^kg^-1] than the others herbs (p < 0.05). There was no informations about the 3-FQA content in the herbal infusions, since precisely quantitative data could not be obtained due to chromato- graphic resolution problems. Besides “Cavalinha” and “Ginseng”, no di-CQA isomers were found in the following samples: “Alecrim”, “Chá preto”, “Erva-doce”,

“Guaco” and “Sene”. Only 20% of the twenty herbs comprised the 3,4-diCQA. The highest concentrations were found in “Camomila” [(1700 ± 400) mg.kg^-1] and

“Chapéu-de-couro” [(1200 ± 400) mg.kg^-1]. The 3,5-diCQA was found in 50% of the samples with its content ranging from [(30 ± 3) mg.^kg^-1] in “Boldo” to [(560 ± 25) mg.^kg^-1] in “Chá mate tostado” (table 1). The 4,5-diCQA was detected in 15% of the herbal infusions, but considerable amounts were found just in “Sálvia” [(1100 ±

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270) mg.kg^-1] (table 1). According to the total CGA content, the herbs could be divided into five distinct groups. The first one comprised “Cavalinha” and “Ginseng”, where no CGA isomers were found. In the second group, formed by “Alecrim”,

“Quebra-pedra” and “Ginkgobiloba” the total CGA content was considered very low (79 - 135 mg.kg^-1 of herb). In the third group (low concentration) the total CGA content ranged from 612 - 2800 mg.^kg^-1 of herb. This group was formed by “Boldo”,

“Chá preto”, “Cidreira”, “Erva-doce”, “Espinheira-Santa”, “Guaco”, “Hortelã”, “Pata- de-vaca” and “Sálvia”. Medium concentrations of total CGA were observed in

“Camomila”, “Carqueja”, “Chapéu-de-couro”, “Porangaba” and “Sene” (5270 - 7550 mg.^kg^-1 of herb). These herbs constituted the fourth group. Finally, in the last group high concentrations of total CGA were achieved. This group comprised only

“Chá mate tostado” [(15750 ± 605) mg of total CGA.kg^-1 of herb].

As could be observed along the text, the CGA profile could be used to establish a identity pattern for the different kinds of commercial Brazilian herbal infusions. The use of this identity pattern could allow a better quality control of this herbs and the detection of frauds in these products.

4 – ACKNOWLEDGEMENTS

We acknowledge the financial support of UNESA, FAPERJ, CAPES and CNPq.

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