INVESTIGATION OF PHENOLIC ACIDS IN LEAVES OF BLACKCURRANT (RIBES NIGRUM L.) AND SOUR CHERRY (PRUNUS CERASUS L.)

Grzegorz Chrzanowski, Cezary Sempruch, Iwona Sprawka
Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce, Poland

ABSTRACT

Blackcurrant (Ribes nigrum L.) and sour cherry (Prunus cerasus L.) are good source of minerals, vitamins and flavonoids. Phytochemicals play protective roles against many human chronic diseases including cancer and cardiovascular disorder. Moreover, phenolic compounds participate in defence reactions of plants to biotic and abiotic stresses. The aim of this research was to examine phenolic acids content in R. nigrum and P. cerasus. The leaves of blackcurrant and sour cherry were collected in 2004. Eight phenolic acids were estimated in methanol extracts: gallic, chlorogenic, tannic, caffeic, salicylic, ferulic, p-coumaric and trans-cinnamic. Separations of analysed compounds were carried out with the use of VARIAN ProStar 210 pump and ProStar 325 UV-VIS detector on Microsorb-MV 100-5 C18 column. Leaves of blackcurrant contained 20.023 mg of phenolic acids per g of dry matter and cherry contained 30.702 mg of these compounds. This difference was statistically significant. It was found that salicylic acid was a dominant compound in leaves of sour cherry (17.723 mg·g^-1 dm). In blackcurrant leaves, tannic acid and salicylic acid had exhibited the same level of concentration (8.366 and 8.530 mg·g^-1 dm respectively). The content of p-coumaric acid was the lowest in both analysed plants (cherry – 0.046 and blackcurrant – 0.026 mg·g^-1 dm).

Key words: phenolic acids, blackcurrant, sour cherry, chromatography, HPLC.

INTRODUCTION

The increase of interest in possibilities of the use of naturally occurring anticancerogenic substances from plants is observed in the last decade. Plant phenolics, e.g. phenolic acids and flavonoids, are currently considered as one of the most promising groups of potential dietary anticarcinogens [13]. Nakamura et al. [7] and Tapiero et al. [12] showed that phenolics possess a wide spectrum of biochemical activities such as antioxidant, antimutagenic, anticarcinogenic as well as ability to modify the gene expression. Phenolic compounds play prominent roles in plant – herbivore and plant – pathogen interactions [1,2]. Moreover, these substances participate in defence reactions of plants to abiotic stresses [3,10].

In traditional folk medicine blackcurrant and sour cherry plants were recommended as antiinflamatory and antimicrobial factor. Sour cherry (Prunus cerasus L.) can be a shrub or tree representing Rosaceae family [14]. Blackcurrant (Ribes nigrum L.) is a deciduous shrub of Europe, Asia and America that belongs to Grossulariaceae family and ribes species [6,14].

The present studies were designed to evaluate the concentration of eight phenolic acids in methanol extracts from leaves of blackcurrant and sour cherry.

EXPERIMENTAL

Plant materials
The leaves from 15 shrubs (20 leaves from each shrub) of blackcurrant cv. “Bona” and leaves from 9 trees (20 leaves from each) of sour cherry cv. “Łutówka” were collected in June 2004. They were air dried in shadow at 37 °C and then ground in electric mill.

Extraction and identification of phenolic compounds
Isolation and purification of free phenolic acids were made on the basis of Kowalski and Wolski [4] work.
5g of dry leaves was successively extracted with 250 cm³ of petroleum ether and chloroform. Extraction was carried out in Soxhlet apparatus for 6 hours (boiling point at 50 – 55°C for petroleum ether and 65 – 70°C for chloroform). Residues of plant material after purification with non-polar solvent were extracted with boiling 80% methanol (250 cm³ for 6 hours). The methanol extracts were evaporated in the vacuum (to 10 cm³). 50 cm³ of 5% NaHCO₃ in water was added to residues and intensively mixed. Then the water solution was filtered and acidified with 36% HCl to pH = 3. Extraction with diethyl ether was carried out (3 x 50 cm³) after acidification. The residues after the ether evaporation were dissolved in methanol (100 cm³) in which phenolic acids (Fig. 1) were identified.

The HPLC method was used for identification and quantification of phenolic compounds from R. nigrum and P. cerasus leaves. Chromatographic analysis was performed with Varian Polaris System with ProStar 210 pump and ProStar 325 UV-Vis detector. Separation was carried out on analytical column Microsorb – MV 100-5 C18 (250 x 4.6 mm I.D. Varian) with ChromSep Guard Cartridge and 20 µL injection loop. The mobile phase used in separation was methanol – water (25-75) with 1% acetic acid (v/v) addition at flow rate 1 cm³ · min^-1. The column temperature was 27°C (adjusted with MetaTherm column heater). Detection was effected at 300 nm. Phenolic acids identification from chromatograms of examined plant extracts were conducted by comparison of retention times with respective standards purchased from Sigma Aldrich (St Louis, Mo, USA). Separated compounds were determined with the Star Chromatography Workstation 6.3 software.

Statistical analysis
All chemical analyses were performed in three replicates and differences between the mean values were calculated by analysis of variance (ANOVA) method and Duncan’s multiple-range test at 0.95 level of significance. Data are expressed as the mean ± standard deviation.

RESULTS AND DISCUSSION

The amount of the individual phenolic constituents (given in mg·g^-1 dry matter) in leaves of blackcurrant and sour cherry are shown in Table 1. Eight phenolic acids: chlorogenic, ferulic, gallic, caffeic, p-coumaric, salicylic, tannic and trans-cinnamic were identified. The average retention time values of phenolic acids from R. nigrum, P. cereasus and standards are shown in Table 2. A statistically significant greater content of phenolic acids was found in leaves of sour cherry in relation to blackcurrant (Fig. 2). One of the main phenolic acids isolated from leaves of P. cerasus was salicylic acid, and concentration of this compound was statistically the highest in relation to other analysed phenols. Moreover, salicylic acid and tannic acid were predominant compounds of free phenolic acids fraction from R. nigrum. In leaves of P. cerasus the lowest concentration of p-coumaric, ferulic, trans-cinnamic and gallic acid was found. Between these compounds no significant differences were ascertained. The p-coumaric acid in leaves of R. nigrum was in the lowest content. Taking into account however statistical calculations one ought to ascertain that p-coumaric and ferulic, as well as ferulic and trans-cinnamic acids showed the same statistical level of concentration. Moreover Duncan’s test did not show differences between content of chlorogenic and gallic, between chlorogenic and caffeic, and also between caffeic and trans-cinnamic acids.

Most of the identified phenolic acids show a certain pharmaceutical properties [11]. Puupponen-Pimiä et al. [8] showed antimicrobial activity of pure phenolic acids (cinnamic, 3-coumaric, caffeic, ferulic and chlorogenic) against Gram-negative bacteria. Moreover, they found that extracts from blackcurrant berries inhibited development of Escherichia coli CM 871 and Salmonella enterica SH-5014. However Rauha et al. [9] proved that caffeic and gallic acid possessed antimicrobial properties against Pseudomonas aeruginosa ATCC 9027. Moreover, these authors showed antimicrobial properties of R. nigrum extract to Micrococcus luteus YMBL. Lohachoompol et al. [5] have found that blueberry extract revealed antioxidant effect and this mixture contained antocyanins and other phenolic compounds.

CONCLUSIONS

The composition of the phenolic compounds in R. nigrum and P. cerasus suggests that not only fruits but also leaves of these plants can be used as a potential source of pharmaceutically active compounds and natural biopesticides.

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Accepted for print: 14.12.2007

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